Membrane fusion And the Alphavirus Life Cycle

ADVANCES IN VIRUS RESEARCH, VOL. 45

MEMBRANE FUSION AND THE ALPHAVIRUS LIFE CYCLE Margaret Kielian Deportment of Cell Biology Albert Einstein College of Medicine Bronx, New York 10461

I. Introduction 11. Entry Pathway of Alphaviruses into Animal Cells A. Host Range and Tissue Dopism B. Interactions of Virus with the Plasma Membrane Receptor C. Endocytic Uptake and Low-pH-Triggered Fusion D. Inhibitors of Entry 111. Alphavirus Structure and Biosynthesis A. Structure of the Virus Particle B. Structure of the Spike Protein C. Viral RNA and Protein Synthesis D. Virus Exit from the Cell IV. Alphavirus Membrane Fusion Activity A. Properties of Fusion B. Membrane Fusion Mutants V. Involvement of Specific Lipids in the Alphavirus Life Cycle A. Role of Cholesterol and Sphingomyelin in Virus Fusion and Infection B. Role of Cholesterol in the Virus Exit Pathway VI. Molecular Events during Alphavirus Entry and Fusion A. Early Events Following Receptor Binding B. Effects on Spike Protein Dimerization C. Alterations in the E2/p62 Subunit D. Alterations in the E l Subunit E. Summary of Events during Fusion F. Studies with Truncated E l VII. Comparison of Alphavirus Fusion with the Influenza Virus Fusion Reaction VIII. Future Directions of Research References

I. INTRODUCTION The explosion of information in modern cell biology has in many cases been fostered by the use of viruses as experimental paradigms Subjects as wide ranging as RNA splicing, DNA replication, on cogenes, cell polarity, and membrane protein biosynthesis have all taken advantage of the experimental manipulability, high signal-tonoise ratio, and defined components of virus systems. The usefulness of viruses is similarly apparent from studies of the entry pathway of 113 Copyright 8 1995 by Academic Preas, Inc. All rights of reproduction in any form reserved.

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enveloped animal viruses into host cells; here viruses have been critical to our understanding of cellular endocytic uptake and the molecular mechanisms of membrane fusion. This review will summarize our current understanding of the life cycle of alphaviruses, focusing in particular on their entry pathway and membrane fusion activity. Alphaviruses comprise a genus of the family Togaviridae, currently containing about 26 members, including the well-characterized prototype viruses Semliki Forest virus (SFV) and Sindbis virus (SV) (Schlesinger and Schlesinger, 1986a; Peters and Dalrymple, 1990; Strauss and Strauss, 1994). The endocytic virus infection pathway and the involvement of low pH in triggering virus-membrane fusion were first delineated using SFV (Helenius et al., 19801, and this virus has remained an important tool in the study of both endocytosis and membrane fusion. Results from both SFV and SV will be reviewed, with additional information from other alphaviruses when available. The extensive literature on alphavirus replication, structure, entry, and fusion has also been summarized in a number of reviews that will be cited under the appropriate section.

11. ENTRYPATHWAY OF ALPHAVIRUSES INTO ANIMAL CELLS The initial interactions of viruses with their host cells are key steps in determining whether a productive infection results. Studies with SFV were among the first to focus on these early interactions, and our overall understanding of the entry pathway of this virus, while by no means complete, is far more detailed than our grasp of the entry of most other viruses. Although SFV entry has been extensively reviewed (Kielian and Helenius, 1986; Koblet, 1990; Kielian, 1993; Brown and Edwards, 1992; Marsh and Helenius, 1989; White, 1990; Strauss and Strauss, 19941, it is nonetheless useful to examine the evidence for entry by the endocytic pathway, and contrast it with the evidence for alternative modes of entry.

A . Host Range and Tissue Tropism Alphaviruses infect a wide variety of host cells in uitro, including mosquito, avian, fish, reptile, Xenopus, and mammalian cells [see Peters and Dalrymple (1990) and Wang et al. (19921, and references therein]. This host range reflects the maintenance of alphaviruses in nature by alternating cycles of replication in mosquito and vertebrate hosts (Koblet, 1990; Brown and Condreay, 1986). Within mammalian hosts, alphaviruses can cause encephalitis, probably by spread of the

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initial infection of skeletal muscle cells and fibroblasts to neurons within the central nervous system (Griffin, 1986; Griffin et al., 1994). In tissue culture, infection can occur in cell lines derived from many different mammalian tissues, including fibroblasts, skeletal and smooth muscle cells, endocrine cells, fat cells, lymphocytes, hepatocytes, epithelial cells, and neuronal cells (Griffin, 1986; Peters and Dalrymple, 1990; Wang et al., 1992) (M. Kielian, unpublished observations).

B . Interactions of Virus with the Plasma Membrane Receptor The first step in infection of any host cell by alphaviruses is the binding of virus to receptors on the plasma membrane of the host cell (Fig. 1).Cell surface binding of alphaviruses has been studied extensively using radiolabeled virus added to cells at 4°C. Virus binds via the spike protein, which is composed of three subunits, E l , E2, and E3 (see Fig. 2 and Section 111).In general, binding occurs with high affinity to proteinaceous sites on the microvilli of the cell (reviewed in Strauss and Strauss, 1994; Kielian and Helenius, 1986). The wide range of hosts and tissues susceptible to alphavirus infection and the differential protease sensitivity of virus binding to different host cells (Marsh and Helenius, 1980; Schmid et al., 1989) suggest that a number of different protein molecules may serve as alphavirus receptors. Early studies implicated several molecules in alphavirus binding. The class I major histocompatibility antigens were found to bind SFV (Helenius et al., 19781, but cells not expressing these antigens could also be infected

Fusion in endosome

Viral entry

Viral exit

FIG. 1. The SFV life cycle. ER, Endoplasmic reticulum; TGN, trans-Golgi network.

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A Putative fusion domain

+j N

d B Sequence of pulalive fusion domain: a875 ~ s tyr p gln rys lys ual lyr lhr

a897 tyr

FIG.2. (A) Schematic diagram of one protomer of the SFV spike protein, showing the approximate positions of the N-linked carbohydrate (boxes), p62 cleavage site, and putative fusion domain. A virus spike protein consists of a trimer of three such protomers, (E1/E2/E3h3.Taken from Kielian, M. (1993). Membrane Fusion Activity of Alphaviruses. In “Viral Fusion Mechanisms” (J.Bentz, ed.), pp. 385-412. Reprinted by permission of CRC Press, Boca Raton, Florida. ( B ) The sequence of the putative fusion peptide of SFV (Garoff et al., 1980a). Residues conserved among SFV, SV, and five other alphaviruses are underlined [see references in Kielian (1993)l.

(Oldstone et al., 1980), again indicating that multiple receptors probably exist. Studies of lymphoblastoid cells identified a 90-kDa protein that could be cross-linked to SV, although its direct involvement in virus uptake was not demonstrated (Massen and Terhorst, 1981). Recently the alphavirus receptors have been the focus of research by several groups, and several molecules potentially involved in virus binding have been identified. By immunizing mice with baby hamster kidney (BHK) cells, a common host cell line, Wang et al. (1992) isolated a monoclonal antibody that blocks SV binding to mammalian cells. The blocking antibody specifically recognizes the high-affinity laminin receptor, a 67-kDa protein whose normal function is to bind basement membrane laminin. The level of expression of the laminin receptor clearly correlates with the ability of the host cells to bind and be infected with Sindbis virus. Direct binding of virus to the laminin receptor has not yet been demonstrated, however. The laminin receptor is highly conserved on BHK and other mammalian cells, and the anti-

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body also shows partial inhibition of SV binding to mosquito cells. Thus, part of the wide host and tissue range of SV may be due to the use of the ubiquitous and highly conserved laminin receptor as a major receptor for virus-cell binding. Multiple receptors must also contribute to the broad tissue and host range, however, because binding to chicken cells is not mediated by the laminin receptor, and could require the 63-kDa protein described below. It is also not yet clear if alphaviruses other than SV can utilize the laminin receptor. Two other studies used antiidiotype antibodies as a n approach to identifying cell surface molecules interacting with the virus spike protein. An antiidiotypic antibody was made against a neutralizing monoclonal antibody that binds to the E2ab site on the SV spike protein (Wang et al., 1991). The antiidiotype antibody interferes with virus binding and infection of chicken cells, but does not interfere with the infiction of BHK cells. The antibody precipitates a protein of 63 kDa from chicken cells, but not from BHK cells. Antiidiotypic antibodies were also generated against a monoclonal that recognizes the E2c site on the SV spike protein (Ubol and Griffin, 1991). This antibody interferes with SV binding to mouse neuroblastoma cells, but not BHK cells, and specifically immunoprecipitates 74- and 110-kDa proteins from rodent cells. Interestingly, expression of this antigen is downregulated during development of the nervous system in mice (Ubol and Griffin, 1991). This age-dependent decrease in expression of a potential virus receptor could contribute to the decrease in susceptibility to SV-induced encephalitis observed during mouse maturation. There is thus good evidence for multiple alphavirus-binding proteins on host cells, of which the laminin receptor appears to act as a major SV receptor. The domains of the alphavirus spike that potentially interact with the cellular receptors will be discussed in Section II1,B.

C . Endocytic Uptake and Low-pH-Triggered Fusion An extensive body of work has addressed the entry pathway of SFV into animal cells. Most of the data suggest that virus infects cells by endocytic uptake followed by a membrane fusion reaction that is triggered by the low pH in vacuoles of the endocytic pathway (Fig. 1) [reviewed in Kielian and Helenius (1986)l. I will summarize the features of this pathway and its experimental underpinnings, and also discuss some of the evidence for alternative routes of entry. The ease of propagation, efficient radiolabeling, defined structure, and high infectivity of SFV have contributed to its usefulness in virus entry studies. Importantly, these properties have also allowed the establishment of assays for the various steps of SFV entry into host cells.

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From these studies, the following picture of the SFV pathway has emerged. After binding to receptors on the plasma membrane, SFV is taken into the cell by the constitutive cellular pathway of receptormediated endocytosis (Helenius et al., 1980; Marsh and Helenius, 1980). Virus is translocated along the plane of the bilayer to clathrincoated pit regions of the membrane. These membrane specializations invaginate and pinch off to form coated vesicles. From work on physiological ligands and receptors, it is clear that initial receptor-mediated endocytic uptake is independent of actin, microtubules, and acidic vacuolar pH (Silverstein et al., 1977; Goldstein et al., 1979). The endocytic uptake by SFV is similarly unaffected by agents that disrupt these cellular components (Marsh and Helenius, 1980; Marsh et al., 1982). The initial coated vesicle has a relatively neutral pH, is rapidly uncoated, and delivers its contents to early endosomes (Schmid et al., 1989). The pH within the endosome becomes mildly acidic due to the action of a proton-translocating ATPase in the endosome membrane (Mellman et al., 1986).This acidification is gradual, with early, peripheral endosomes having a pH of 5 6 . 2 and 25.3, and later, perinuclear endosomes having a pH of 5 5 . 3 (Tanasugarn et al., 1984; Schmid et al., 1989; Kielian et al., 1986). On exposure to a pH lower than the fusion threshold (about pH 6.2 for wild-type SFV), the virus spike is converted to a form that is active in membrane fusion. The spike protein conformational changes involved in the fusion reaction will be discussed in detail in Section VI. The virus then fuses its membrane with the endosome membrane, and the viral nucleocapsid is released into the cytoplasm. Nonfused virus and spike proteins remaining in the endosome membrane after fusion are further transported along the endocytic pathway to the lysosome compartment, where they are rapidly degraded. The released nucleocapsid is uncoated, and the viral replication pathway takes place as summarized in Section 111. The rates of the uptake, acidification, fusion, and degradation reactions are a function of the overall kinetics of the endocytic pathway and vary with the host cell type employed. The half-time of SFV internalization in BHK cells is -10 min, and virtually all of the bound virus is internalized by 30 min (Kielian et al., 1986). Acidification of endosomes to -pH 6.2 and fusion of virus occur with similar half-times of -15 min in this cell type. Subsequent degradation of virus proteins in lysosomes has a half-time of -90 min. Transit through the endocytic pathway and delivery to lysosomes in particular are markedly asynchronous. In contrast to these results in BHK cells, traffic through the endocytic pathway is more rapid and somewhat more synchronous in Chinese hamster ovary (CHO) cells (Schmid et al., 1988, 1989). The half-time of SFV internalization in CHO cells is -3-5 min, and deliv-

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ery to iysosomes occurs with a half-time of -35 min (Schmid et al., 1988). Acidification of endosomes to the wild-type virus fusion threshold of pH 6.2 occurs with a half-time of -5 min (Schmid et al., 1989). Fusion is dependent on the pH threshold of the SFV strain used. fus-1 is a n SFV mutant with a more acidic threshold for fusion, requiring a pH of about 5.3 or below to trigger fusion (Kielian et al., 1984).In keeping with the gradual acidification of endosomes described above, this mutant fuses later in the endocytic pathway than does the wildtype virus (Kielian et al., 1986). Fusion occurs within the late endosome compartment but prior to lysosomes (Kielian et al., 1984, 1986; Schmid et al., 1989). Revertants of fus-1 selected for fusion thresholds more like wild-type virus fuse earlier in the pathway with kinetics similar to wild-type virus (Kielian et al., 1986).Thus, there is a strong correlation between the in uitro pH dependence of virus fusion and the kinetics with which virus fuses within the cell. The detailed kinetic analysis of SFV infection thus strongly supports the entry of SFV via endocytosis and low-pH-triggered fusion, rather than by fusion directly at the plasma membrane. In addition, immunological studies demonstrate that SFV spike proteins cannot be detected at the cell plasma membrane after initial virus entry, while spike proteins from the pH-independent Sendai virus can readily be detected (Fan and Sefton, 1978). Electron microscopy of SFV bound to host cells showed that virus did not fuse with the plasma membrane under neutral pH culture conditions, but was observed to fuse efficiently with the plasma membrane after 2-10 sec of treatment at pH 5.5. (White et al., 1980). Importantly, endocytosed SFV was shown to infect efficiently from within its intracellular location after removal of plasma membrane-bound, extracellular virus (Helenius et al., 1982). Other key evidence for the role of low pH in uivo has come from the use of inhibitors of vacuolar acidification, as detailed below.

D . Inhibitors of Entry 1 . Inhibitors of Vacuolar Acidification

Two types of agents have been used to inhibit the acidification of endosomes. The first class of inhibitors includes the weak bases such as ammonium chloride, chloroquine, and methyl amine. These compounds diffuse across membranes in their unprotonated form and become protonated within acidic organelles such as lysosomes, endosomes, and the trans-Golgi network (Mellman et al., 1986). The positively charged inhibitor is trapped and concentrated within these acidic compartments, and acts rapidly to neutralize their acidic pH

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(Ohkuma and Poole, 1978). These agents prevent SFV infection if present during the first interaction of the virus with the cell, but not if present only during the binding of the virus to the cell, or during later stages after entry (Helenius et al., 1980, 1982). They are readily reversible in both their effects on fusion and endosomal pH, and have been shown not to affect virus-receptor binding or virus transit through the endocytic pathway. The concentration required to inhibit SFV infection is highly dependent on the pH of the medium, because the protonated forms are inactive (Kielian and Helenius, 1986). The inhibitory concentration for virus infection correlates with the concentration required to raise the endosome pH above the SFV fusion threshold of -pH 6.2 or the fus-1 threshold of -pH 5.3 (Kielian et al., 1984, 1986). The second class of acidification inhibitors includes the carboxylic ionophores such as monensin and nigericin (Marsh et al., 1982). These ionophores exchange protons for potassium and sodium, and thus neutralize endosome pH via a different mechanism than that of the weak bases (Mellman et al., 1986). Neutralization of endosome pH by either mechanism has the same inhibitory effect on virus fusion and infection. Both types of inhibitors can be “bypassed’ by acid treatment of cell surface-bound virus to induce direct fusion with the plasma membrane (Helenius et al., 1980; White et al., 1980). These compounds have also been shown to inhibit acid-induced conformational changes in the SFV spike protein during cell entry, in keeping with their neutralization of endosome pH (Kielian et al., 1986; Wahlberg and Garoff, 1992).Taken together, these data strongly support the involvement of endocytosis and low-pH-induced fusion in the productive infection pathway of SFV. [For a n alternative view of the action of weak bases during SV infection, see Coombs et al. (1981) and Cassell et al. (1984).J Some of these agents have also been shown to affect later biosynthetic stages in the virus life cycle, perhaps by neutralization of acid pH gradients in the Golgi (Cassell et al., 1984). Evidence suggests that Golgi processing or transport may require acidification of a t least some compartments of the Golgi (Mellman et al., 1986). Bafilomycin A, a specific inhibitor of vacuolar acidification, has been described (Bowman et al., 1988).This reagent has recently been shown to specifically inhibit SFV infection when present at early times of infection, thus further corroborating the role of intravacuolar low pH in alphavirus fusion (Perez and Carrasco, 1994). An alternative to the use of inhibitors of endosome acidification is the use of cell lines that are temperature sensitive for acidification. A study of such a CHO cell acidification mutant found that the cells were infected equivalently with Sindbis virus a t the permissive and restric-

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tive temperatures (Edwards and Brown, 1991). Notably, however, detailed studies of two other cellular acidification mutants suggest that the wild-type alphavirus pH threshold is in a range basic enough to be little affected by moderate increases in endosomal pH (Schmid et al., 1989). It is also clear that the acidification defects of these mutant cell lines are partially expressed even at the permissive temperature (Marnell et al., 1984; Schmid et al., 19891, and thus it is important to compare results with the mutant cell line to those with the wild-type line. Further analysis of the entry of viruses of varying pH thresholds into wild-type and mutant cells should resolve these issues. 2 . Temperature

Formation of initial coated vesicles, and thus the uptake of virus, is efficiently inhibited by low temperature ( ~ 1 0 ° C (Silverstein ) et al., 1977; Goldstein et al., 1979). Incubation on ice, for example, thus blocks virus entry, penetration, and infection, and has been a useful experimental tool in entry studies. It is also known that endocytosis occurs efficiently a t 20°C but that delivery of endocytosed ligands to lysosomes is inhibited a t this temperature (Dunn et al., 1980). Incubation of cells at temperatures of 15-20°C was therefore used to demonstrate that endocytosed virus was penetrating from the acidic environment of the prelysosomal endosome (Marsh et al., 1983a). 3 . Other Inhibitors

Fusion of SFV with the endosome membrane in uiuo can be inhibited by altering the ratio of sodium and potassium in the extracellular medium (Helenius et al., 1985). Although the mechanism of this inhibition is not understood, it appears to correlate with the depolarization of the cell membrane. It is clear that treatment of Sindbis or SFV with reducing agents results in conformational changes in the spike proteins that are in some respects similar to those caused by treatment at low pH (Kielian et al., 1990; Meyer et al., 1992) (see discussion of conformational changes below). Prolonged reduction or acid treatment followed by reduction causes complete disruption of the virus particle (Anthony et al., 1992). During biosynthesis, the Sindbis E l spike subunit is found as several different disulfide-bonded intermediates (Mulvey and Brown, 1994). To assess the possible relevance of these findings to the virus infection pathway, a recent study examined the role of thioldisulfide exchange reactions in the entry and fusion of Sindbis virus (Abell and Brown, 1993). Low-pH-induced polykaryon formation was enhanced by the presence of the reducing agent 2-mercaptoethanol. As discussed below, however, this morphological assay for fusion can be

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problematic. Virus infection was partially inhibited by the presence of a thiol-alkylating agent during entry, but it is not clear which step in the entry pathway is affected by this agent. The role of reduction of virus disulfide bonds in alphavirus fusion and infection thus remains to be determined.

111. ALPHAVIRUS STRUCTURE AND BIOSYNTHESIS To understand the current level of molecular information on alphavirus fusion, it is useful to present some basic information on alphavirus structure and the assembly pathway of the alphavirus particle. Readers are referred to other reviews for further information and references (Schlesinger and Schlesinger, 1986b; Simons and Warren, 1984; Koblet, 1990; Strauss and Strauss, 1994).

A . Structure of the Virus Particle Alphaviruses are small enveloped icosahedral viruses of very defined structure and a diameter of -640-680 A (Harrison, 1986; Vogel et al., 1986; Paredes et al., 1993; Fuller, 1987; Venien-Bryan and Fuller, 1994). Three-dimensional reconstruction of SFV and Sindbis viruses has been performed using cryoelectron microscopy (Vogel et al., 1986; Paredes et al., 1993; Fuller, 1987; Venien-Bryan and Fuller, 1994).The overall virus structures a t this level of resolution (35-28 A) are similar, although the SFV particle appears slightly smaller. The virus shape appears to be organized by the interactions between the icosahedral virus nucleocapsid and the spike proteins (Paredes et al., 1993). The nucleocapsid consists of one positive-stranded RNA molecule of about 11,000 bases that is packaged with a basically charged capsid protein of M, 29,828 (Garoff et al., 1980b). The RNA probably lies within the interior -32-nm diameter of the nucleocapsid, and is surrounded by a shell of capsid protein to give the total -40-nm diameter of the nucleocapsid. This shell consists of 240 copies of the capsid protein arranged to form an icosahedral protein lattice of triangulation number T = 4 (Paredeset al., 1993).There are openings within the protein shell of the nucleocapsid, in agreement with the observed sensitivity of the RNA in isolated nucleocapsids to nuclease digestion (Soderlund et al., 1972). Complete or partial sequence information is available on the RNA genomes from a number of alphaviruses. The RNA encodes the four subunits of the RNA replication complex, and the structural proteins

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A

B

FIG.3. ( A ) Schematic diagram of the SFV RNA genome, showing the coding regions in boxes (Lopez et al., 1994). (B) Schematic diagram of the proposed topology of the SFV spike protein during biosynthesis and translocation into the RER (Redrawn from Liljestrom and Garoff, 1991).Signal sequences are shown in hatched boxes and stoptransfer sequences in black boxes. The positions of signal peptidase cleavage sites are shown by arrows. The sizes of the protein domains are not to scale.

of the virus (see Fig. 3, and below). It associates with the capsid via interactions between packaging signals within the 5' end of the RNA (Weiss et al., 19891, and a positively charged RNA-binding region in the amino-terminal half of the capsid protein (Geigenmuller-Gnirke et al., 1993). cDNA clones of the complete genomes of SV, SFV, Ross River, and Venezuelan equine encephalitis viruses have been constructed (Rice et al., 1987; Liljestrom et al., 1991; Kuhn et al., 1991; Davis et al., 1989). RNA derived either from virus particles or from the cDNA clones is infectious on delivery into the cytoplasm of a host cell. Surrounding the viral nucleocapsid is a lipid bilayer of 30-40 A in thickness (Harrison, 1986; Vogel et al., 1986; Paredes et al., 1993). The virus membrane is derived from the host cell plasma membrane during virus budding, as described below, and its lipid composition is simi-

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lar, but probably not identical, to that of the host cell plasma membrane. Virus propagated in BHK cells has a phospholipid composition of about 25% sphingomyelin, 27% phosphatidylcholine, 19% phosphatidylserine, and 26% phosphatidylethanolamine (van Meer et al., 19811, with a cholestero1:phospholipid ratio of about 1:l (Laine et al., 1973). Virus propagated in mosquito cell lines appears to contain a significantly higher proportion of ethanolamine-based phospholipids (Luukonen et al., 1976), and to have a cholestero1:phospholipid ratio of about 1:6 (Luukonen et al., 1977). These lipid differences do not appear to be important in virus structure or infectivity. Because the bilayer is curved, there is -40% greater surface area in the outer lipid leaflet (Harrison, 1986). The phospholipid composition of the two leaflets is also asymmetric (van Meer et al., 1981). The virus spike proteins are also organized in a T = 4 icosahedral lattice and extend about 75 A out from the virus membrane. The SFV spike protein consists of three noncovalently associated subunits, the transmembrane E l CM,50,786) and E2 polypeptides (M,51,855), and the peripheral polypeptide E3 (M,11,369) (Garoff et al., 1980a) (Fig. 2). E2 and E3 are synthesized as a precursor, termed p62 in SFV and PE2 in SV, which is cleaved posttranslationally a t a tetrabasic cleavage site (Garoff et al., 1980a).The E3 subunit remains noncovalently associated with the E l and D2 subunits in SFV, but is released and not associated with the mature infectious Sindbis virus particle (Welsh and Sefton, 1979; Mayne et al., 1984);240 copies of this spike protomer are inserted in the virus membrane, and interact with the 240 copies of the capsid protein, presumably in a 1:l ratio (Paredes et al., 1993). Structural analysis and cross-linking studies (Rice and Strauss, 1982; Anthony and Brown, 1991) demonstrate that these spike protomers are organized into a triangular-shaped trimer (El/E2/E3), on the surface of the virus particle. Thus, 80 of these trimeric spike proteins combine to form the spike protein surface lattice (Fuller, 1987; Harrison, 1986; Vogel et al., 1986; Paredes et al., 1993; Venien-Bryan and Fuller, 1994). The outer portions of the spikes are well separated, but they spread toward the base to form a protein layer covering a large proportion of the lipid bilayer (Vogel et al., 1986). Connections between the spike proteins can be discerned, and there are also openings in the spike protein lattice that expose the virus lipid bilayer (Paredes et al., 1993). Difference imaging between detergent-extracted SFV, SFV, and SV suggests that the E2 subunits are located a t the center and vertices of the triangular-shaped spike, and E3 at the distal end of the spike interacting mainly with E2 (Venien-Bryan and Fuller, 1994). E l in this model forms the edges of the triangle and interacts most closely with the adjacent spikes.

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B . Structure of the Spike Protein The alphavirus spike protein thus contains two type 1 membranebound polypeptides, E l and E2, and a peripheral polypeptide, E3 (Fig. 2). It is clear that the spike protein expressed or reconstituted alone, in the absence of a virus particle, is sufficient to carry out efficient lowpH-dependent membrane fusion activity (Kondor-Koch et al., 1983; Marsh et al., 1983b). Because E3 is not associated with Sindbis virions, these data indicate that the E l and/or E2 subunits are responsible for the virus fusion activity. The bulk of both E l and E2 face out from the virus membrane. The ectodomains of both subunits are glycosylated, E l with a complex-type carbohydrate at position 141 and E2 with both high-mannose and complex-type chains at positions 200 and 262 (Garoff et al., 1980a; Mattila, 1979).The peripheral E3 subunit is also glycosylated at either position 13 or 58 (Garoff et al., 1980a). The pattern of spike protein glycosylation varies somewhat with host cell type and confluency (Hubbard, 1988; Schlesinger and Schlesinger, 1986a; Koblet, 1990). Spike protein function appears unaffected by these minor differences, but the folding and transport of the spike protein are aberrant in the absence of any glycosylation (Marquardt and Helenius, 1992). E l has two arginine residues in the interior of the SFV particle following the transmembrane domain. E2 has a domain of 31 amino acids following the transmembrane domain. This region of E2 appears to interact with the nucleocapsid during virus budding and in the virus particle, as discussed below. Both E l and E2 are palmitoylated on cysteine residues in either the transmembrane domain or cytoplasmic tail (Schmidt et al., 1988; Schmidt and Lambrecht, 1985). Mutation of these cysteines in the E2 subunit alters the palmitoylation pattern, impairs virus budding, and produces virus particles with multiple nucleocapsids (Gaedigk-Nitschko and Schlesinger, 1991; Ivanova and Schlesinger, 1993). The domaids) of the spike protein involved in receptor binding are not yet clearly defined [see Dubuisson and Rice (1993) and references therein], but there are indications that the E2 subunit is directly involved. As discussed above, antiidiotypic antibodies against neutralizing anti-E2 antibodies have been used to isolate potential receptors. Furthermore, mutations on the E2 subunit have been shown to alter virus binding efficiency to cells (Dubuisson and Rice, 1993; Salminen et al., 1992; Tucker and Griffin, 1991). Future studies will doubtless clarify the role of E2 and potential involvement of E l in receptor binding. Fusion peptides are either N-terminal or internal apolar regions of

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the spike protein that are conserved within but not between virus families (White, 1990). These peptides tend to be rich in alanine and glycine residues. In many cases they can be modeled as sided helices with the bulky apolar amino acids on one face of the helix (White, 19901, although the generality and significance of this helical model are unclear at present (Gallaher et al., 1992). It is believed that the fusion peptide is the domain of the virus transmembrane spike protein that interacts with the target and/or virus membrane bilayers to trigger fusion. Such a membrane interaction has been directly demonstrated in the case of the influenza virus fusion peptide (Harter et al., 1989; Stegmann et al., 1991), but has yet to be shown for other potential fusion peptides. Sequence comparisons of spike protein genes from a number of alphaviruses reveal a region of virtually identical sequence located near the N terminus of the E l polypeptide between amino acids 75 and 97 (Fig. 2). Hydropathy analysis of amino acids 79-97 of SFV and SV indicates that this domain is significantly hydrophobic, with a hydrophobicity value of about 0.6 (Garoff et al., 1980a; White, 1990). Alphaviruses are RNA viruses whose relatively high mutation rate results in considerable sequence diversity (Strauss and Strauss, 1994). Other hydrophobic regions of the spike protein, such as the transmembrane domains and signal sequences, are not highly conserved. The sequence conservation and hydrophobicity of the E l region thus suggested a n important role in the alphavirus life cycle, and it was proposed as a candidate for the alphavirus fusion peptide (Garoff et al., 1980a). The 75-97 E l domain would therefore be a n example of an internal fusion peptide. As expected, it has little sequence conservation with other fusion peptides identified in other viruses, but it does have considerable identity with a sequence in the VP4 hemagglutinin protein of rotavirus (Mackow et al., 1988; Lopez et al., 1991). This sequence identity between alphaviruses and the pH-independent, nonenveloped rotaviruses is intriguing, but its biological significance is to date undetermined. Interestingly, the alphavirus E l domain contains a number of highly conserved glycine and proline residues. Because these amino acids tend to be strong helix breakers, the E l peptide is most probably not in a sided helix conformation (Gallaher et al., 1992). Experimental evidence for the role of this E l region in fusion comes from the mutagenesis studies described below.

C. Viral RNA and Protein Synthesis On entry of the viral nucleocapsid into the host cell cytoplasm, the RNA genome is uncoated by removal of capsid protein. This occurs by

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binding of capsid protein to the large ribosomal subunit, probably via interaction with the ribosomal 28s RNA, which has a sequence similar to the packaging sequence on the viral RNA (Wengler and Wengler, 1984; Singh and Helenius, 1992a,b). The RNA genome is thus made available for translation, resulting in the synthesis of the four subunits of the viral replication complex, nsP1-4 (Fig. 3). This replicase then uses the plus-stranded genomic RNA as a template to synthesize a minus-stranded RNA. The minus-strand copy is subsequently transcribed to give both full-length plus strand RNA and a 4-kb subgenomic RNA encoding the viral structural proteins (reviewed in Strauss and Strauss, 1986, 1994). RNA synthesis takes place on the cytoplasmic surface of intracellular membranes derived from endosomes and lysosomes (Froshauer et al., 1988). Translation of the subgenomic RNA occurs from one initiation site and produces a polyprotein that is cleaved co- and posttranslationally to the mature virus structural polypeptides [reviewed in Schlesinger and Schlesinger (198611. The order of the polypeptides is NH,-capsidp62-6K-El. The subgenomic RNA is first translated on free ribosomes to give the soluble capsid protein. Interestingly, capsid protein is a serine proteinase with a typical catalytic triad and structural homology to chymotrypsin (Choi et al., 1991; Tong et al., 1993). Following its synthesis, capsid is autocatalytically cleaved from the growing polypeptide chain (Aliperti and Schlesinger, 1978; Hahn, 19851, thereby both releasing itself from the growing polypeptide chain and also inhibiting its further protease activity (Choi et al., 1991). Newly synthesized capsid protein then assembles with progeny full-length plus strand RNA to form nucleocapsids (Fig. 1). Following cleavage of capsid protein, a signal sequence on the nascent polypeptide is exposed (Fig. 3B). It interacts with signal recognition particle to mediate the cotranslational translocation of the p62 polypeptide into the rough endoplasmic reticulum (RER) (Garoff et al., 1990). The p62 signal is not cleaved by signal peptidase, but is transferred through the membrane into the lumen of the RER. A stoptransfer sequence arrests p62 translocation and anchors it in the membrane. A second signal sequence reinitiates translocation of the p62 C terminus and the small polypeptide known as 6K (Melancon and Garoff, 1986; Liljestrom and Garoff, 1991). This signal sequence is cleaved by signal peptidase. Following cleavage, the C-terminal domain of p62 is translocated through the membrane to the cytoplasmic face, where it will be free to interact with nucleocapsids during virus budding (Liu and Brown, 1993b). Translocation of 6K is arrested by another membrane-spanning domain, and then reinitiated by the signal sequence for E l , which lies at the C terminus of the 6K protein (Melan-

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con and Garoff, 1986; Liljestrom and Garoff, 1991). This sequence is cleaved by signal peptidase, and E l is synthesized and anchored in the membrane by its C-terminal membrane-spanning domain. Thus, as is typical for membrane proteins, a combination of signal and stoptransfer sequences set up the topology of the viral proteins in the membrane. The E l and p62 subunits associate within the RER to form noncovalent heterodimers (Ziemiecki et al., 1980). Within the RER the polypeptides are core glycosylated. The protein complex then moves through the Golgi apparatus where it is fatty acylated (Bonatti et al., 1989) and most of the carbohydrate chains are further processed to complex type (Quinn et al., 1983).The spike protein is transferred from the trans-Golgi network to the plasma membrane, and during this step p62 is cleaved next to the arginine pair to form E3 and E2 (deCurtis and Simons, 1988). The C-terminal arginine of E3 is removed (Garoff et al., 1980a). The spike protein is then transported to the plasma membrane, where budding will occur. Recent evidence suggests that the small 6K polypeptide is also associated with the spike protein in the RER and during its transport to the plasma membrane (Lusa et al., 1991). However, E l and E2 still dimerize and are transported to the plasma membrane when expressed in the absence of 6K (Liljestrom et al., 1991). The spike protein thus exists as a p62/E2-E1 dimer from the RER to the plasma membrane. Evidence suggests that dimerization is required for proper transport and folding. SFV E l is transported out of the RER only when coexpressed with p62 (Melancon and Garoff, 19861, although some evidence suggests that SV E l can be transported to the plasma membrane in the absence of pE2 (Migliaccio et al., 1989). Expression of SFV p62 in the absence of dimerization with E l results in transport of p62 to the plasma membrane but in a n aberrantly processed form (Levy-Mintz and Kielian, 1991; Kondor-Koch et al., 1983). Mutagenesis studies show that a deletion of the E3 subunit prevents dimerization of E l and E2 and transport of both subunits (Lobigs et al., 1990a). A point mutation in the SFV tsl mutant, E3 Cys-58 to Q r , was similarly found to block transport of both p62 and E l a t the nonpermissive temperature (Syvaoja et al., 1990). These results suggest that the E3 portion of the p62 subunit may be involved in dimerization with E l or transport of the polypeptide complex.

D . Virus Exit from the Cell In mammalian cells, budding of SFV or Sindbis virus occurs from the plasma membrane. In some mosquito cell lines, budding into intra-

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cellular vacuoles is also observed (Miller and Brown, 1992). In polarized epithelial cells, budding usually occurs predominantly from the basolateral surface (Fuller et al., 1985). The localization of budding appears to be directed by the localization and density of the virus spike proteins. For example, the basolateral budding of SFV in polarized epithelial cells such as MDCK and CaCo-2 correlates with the targeting of the SFV spike protein to the basolateral domain of these cells (Roman and Garoff, 1986; Zurzolo et al., 1992). In contrast, thyroid epithelial cells that localize the SFV and SV spike proteins to the apical surface also bud virus predominantly from this domain (Zurzolo et al., 1992). Although budding usually occurs at the plasma membrane of the host cell, it is rerouted if spike protein transport out of the Golgi complex is blocked by monensin treatment (Griffiths et al., 1983). Under these conditions, the density of spike proteins within the Golgi complex greatly increases, association of nucleocapsids with the cytoplasmic face of the Golgi membrane is observed, and budding of virus into the Golgi lumen occurs. Thus, the indication is that altering spike protein localization can alter the location of budding. There may be other control mechanisms as well that act to direct the site of budding. Budding is clearly not dependent on p62 cleavage, because virus that is not cleaved can be efficiently released (Salminen et al., 1992; Presley and Brown, 1989). A study with protein kinase and phosphatase inhibitors suggests a possible role for phosphorylation and dephosphorylation in a late virus maturation step (Liu and Brown, 1993a). A variety of experimental approaches suggest that alphavirus budding is mediated by the interaction of the E2 cytoplasmic tail with the nucleocapsid. Cross-linking and biochemical studies indicate that the internal portion of the spike protein interacts with capsid protein (Garoff and Simons, 1974; Helenius and Kartenbeck, 1980). The internal domain of E l , consisting of two arginine residues, is not involved in this interaction, because mutagenesis studies show that it is not required for virus budding or infection (Barth et al., 1992). However, several approaches strongly indicate that the E2 internal domain binds to the nucleocapsid. Mutations in this 31-amino acid region inhibit budding and produce multicored virus particles (GaedigkNitschko and Schlesinger, 1991; Ivanova and Schlesinger, 1993). A synthetic peptide corresponding to the E2 tail binds to nucleocapsids, and binding is enhanced for the oligomeric form of the peptide (Metsikko and Garoff, 1990). (See note added in proof.) Chimeric viruses containing the Ross River virus genome with the Sindbis virus capsid protein do not assemble well, and the defect can be largely corrected by replacing the Ross River virus E2 cytoplasmic domain amino acids

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with sequences derived from Sindbis virus (Lopez et al., 1994). In agreement with the importance of the E2-capsid interaction, expression of nucleocapsids or spike proteins alone does not produce alphavirus subviral particles (Suomalainen et al., 1992). Thus alphaviruses, unlike retroviruses, require both the core and the spike proteins for budding to occur. The structure of the capsid protein suggests that surface residues in the carboxy-terminal half of the capsid protein would be involved in binding the internal domain of E2 (Choi et al., 1991; Tong et al., 1993). A double mutation at positions 180 and 183 of capsid does affect virus structure, producing larger virus particles under some culture conditions (Lee and Brown, 1994). Interestingly, when E l and E2 are expressed in the absence of nucleocapsids, they are transported to the plasma membrane normally but are rapidly proteolyzed, suggesting that spike protein may be somehow stabilized by interaction with capsid (Zhao and Garoff, 1992; but see also Ekstrom et al., 1994). Studies with temperature-sensitive viral mutants suggest that lateral spike interactions, perhaps involving protomer trimerization or further spike oligomerization, must occur for efficient budding. For example, the ts20 mutant of Sindbis virus transports the spike protein to the plasma membrane, and accumulates nucleocapsids under the plasma membrane, but is defective in budding a t the nonpermissive temperature (Smith and Brown, 1977). The E2 mutation responsible for this phenotype is a change from His-291 to Leu (Lindqvist et al., 1986). The E2 mutation in the ts103 mutant, Ala-344 to Val, produces impaired budding and virus particles containing multiple nucleocapsids (Hahn et al., 1989). Coexpression studies of nucleocapsid-binding spike proteins with spikes inactive for binding show that both spike proteins can coassemble into SFV particles (Ekstrom et al., 1994).This result suggests that the lateral spike interactions are able to rescue the nonbinding spike protein, and that mixed trimers of the spike protomers are formed before budding. The small hydrophobic 6K polypeptide is found associated with the spike protein during its transport through the secretory pathway, as described above. During transport there appears to be about one 6K molecule interacting per spike protomer (El/E2/E3), but in the released virus particle the amount of 6K is estimated to be from about 1 per 10 spike protomers to 1 per 30 spike protomers (Gaedigk-Nitschko and Schlesinger, 1990; Lusa et al., 1991). Although little 6K is therefore incorporated into virus, it appears to play an important role in the maturation of the virus particle. A mutant with a complete deletion of 6K synthesizes, dimerizes, and transports SFV spike proteins normally, but releases only about 2% of the wild-type level of virus particles (Liljestrom et al., 1991). The 6K-depleted virus that is released appears morphologically normal and of the same specific infectivity as

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wild type. An in-frame insertion mutation of 15 amino acids near the center of SV 6K also causes decreased budding, but the virus produced is morphologically normal and functions normally in early stages of viral replication (Schlesinger et al., 1993). The 6K protein contains a n estimated three to four fatty acyl groups per molecule (GaedigkNitschko and Schlesinger, 19901, and mutation of cysteine residues in the 6K protein decreases the acylation of the protein (GaedigkNitschko et al., 1990; Gaedigk-Nitschko and Schlesinger, 1991).SV mutants with the underacylated 6K show decreased budding, especially from mosquito cells. The virus produced with the altered 6K acylation is multicored. Taken together, these data implicate 6K in the alphavirus exit pathway, probably a t the level of virus budding from the cell. Recent data indicate that 6K can modify membrane permeability when expressed in E . coli (Sanz et al., 1994). It is not known if 6K modifies membrane permeability in virus-infected cells, and if such a n activity might be involved in its role in virus budding. IV. ALPHAVIRUS MEMBRANEFUSION ACTIVITY

A. Properties of Fusion A number of different systems have been developed to assay the lowpH-dependent membrane fusion activity of alphaviruses. These systems are based on the use of either cell plasma membranes or artificial liposomes as target membranes. An early and still widely used fusion assay quantitates the fusion of cells by either exogenous virus or virus spike proteins expressed in the plasma membrane of the cell [reviewed in Kielian (199311. Because this polykaryon formation is evaluated by observing the changes in cellular morphology that follow fusion, however, care must be taken to differentiate conditions affecting the morphological reorganization from those affecting the primary fusion event. For example, alphavirus polykaryon formation is scored by culturing mammalian cells in neutral pH medium following a brief treatment a t low pH (White et al., 1981). This has led to the proposal that fusion is a two-step process that requires a return to neutral pH (Edwards and Brown, 1986). Notably, however, mosquito cells that are tolerant of low pH show efficient SFV-induced polykaryon formation after simple treatment with medium a t pH 6.0 (Omar et al., 1986). As discussed below, direct assays of virus-cell or virus-liposome fusion show no requirement for a return to neutral pH. Virus-induced polykaryon formation can also be affected by host cell factors, and is very dependent on the amount of added virus or the density of expressed spike protein (White, 1990).

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Other assays using host cell membranes are based on evaluating the fusion of virus particles, either with the plasma membrane, following low-pH treatment (White et al., 1980), or within the cell, following endocytosis and endosome acidification (Kielian et al., 1986). In recent work, the mixing of virus and target lipid bilayers has been analyzed by the fluorescence dequenching of virus labeled with fluorescent probes such as pyrene or octadecylrhodamine (Wahlberg et al., 1992; Bron et al., 1993; Justman et al., 1993; Stegmann et al., 1993). This assay can be followed in real time, enabling detailed kinetic analysis (Bron et al., 1993). Assays of virus fusion with liposome target membranes have used fluorescence to follow lipid mixing, or labeled RNA or capsid protein to follow content mixing (White and Helenius, 1980). Data from these fusion systems indicates that SFV fusion is strikingly pH dependent, with a fusion threshold of -pH 6.5-6.2. Fusion is rapid, occurring within 5 sec of pH treatment of plasma membrane-bound virus (White et al., 1980) and within seconds of lowpH treatment in the presence of liposomes (Bron et al., 1993). The efficient fusion with protein-free liposomes indicates that SFV fusion is independent of the virus receptor, although the receptor is required in host cells to carry the virus into the acidic endosome compartment. Early experiments suggested that fusion was not leaky to proteins or ions unless the virus membrane was damaged (White and Helenius, 1980; Young et al., 1983). More recently, the physical changes in the virus and target membranes during fusion have been followed by electrophysiological methods. Results from whole-cell patch-clamp recording suggest that cells expressing the SFV spike protein show a n increased membrane conductance on low-pH treatment (Lanzrein et al., 1993b). Although this conductance is blocked by calcium or zinc, these cations do not inhibit cell-cell fusion (Lanzrein et al., 1993a,b).Studies of capsid sensitivity to low pH suggested that the SFV spike protein might form a proton channel (Schlegel et al., 1991). Other studies have not observed changes in capsid on low-pH treatment of alphaviruses, however, arguing that the virus membrane stays impermeable to protons during fusion (Singh and Helenius, 199213, Stubbs et al., 1991). The initial steps in fusion of individual cell pairs have been followed by monitoring intercellular current flow using dual voltage-clamp (Lanzrein et al., 1993a). These experiments showed that fusion begins after a lag of from 3 to 138 sec, is complete within 7-70 sec, and involves the initial formation of an aqueous fusion pore with a diameter of about 1 nm. Interestingly, this pore does not appear to flicker open and closed, as has been reported for the fusion pore of influenza virus (Spruce et al., 1991). The lack of flickering suggests that the fusion process may be irreversible for alphaviruses.

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Studies of virus fusion with liposomes and cell membranes have revealed several important properties of the fusion process. Fusion of SFV is very efficient, with a final extent of from -50 to 90% input virus fusion, depending on the system used (Bron et al., 1993; Justman et al., 1993; White and Helenius, 1980). Fusion appears independent of calcium (White and Helenius, 19801, and does not require an ion or pH gradient across the liposomal target membrane (Helenius et al., 1985). Specific lipids in the target membrane are required for alphavirus fusion, as discussed in Section V. Fusion is temperature dependent, being most efficient at -37"C, but showing some activity even at 0°C (White and Helenius, 1980; Justman et al., 19931, and a constant activation energy implying a uniform fusion mechanism across a broad temperature range (Bron et al., 1993). Detailed analysis of fusion with liposomes reveals a pH- and temperature-dependent lag phase that is brief at the optimal conditions of 37°C and pH 5.5, and longer at lower temperature and pH values near the fusion threshold (Bron et al., 1993). Studies of fusion with either cell membranes or liposomes show that low-pH-triggered fusion is halted when the virus-membrane complex is shifted to neutral pH (Bron et al., 1993; Justman et al., 1993). Neutralization thus can freeze virus-membrane fusion at an intermediate value. Membrane-free spike protein preparations such as spike protein rosettes are inactive in fusion (Marsh et al., 1983b1, although it is clear that they react to low pH (Justman et al., 1993). Reconstitution of purified spike proteins into liposomes restores their fusion activity, thus indicating that the spike protein must be membrane anchored, and that fusion is a topologically organized process, not simply a membranolytic process (Marsh et al., 1983b).

B . Membrane Fusion Mutants A number of mutations affecting alphavirus fusion activity have been described and will be summarized here [reviewed extensively in Kielian (1993)l. Analysis of these mutations has already contributed to our understanding of the fusion function of the spike protein. Now that we have more molecular information on the events occurring in the spike protein during fusion, mutants can be analyzed to pinpoint the step in the fusion process that is altered. Information on spike protein conformational changes for both wild-type virus and mutants will be discussed in Section VI. Several specific mutations in the p62 cleavage site have provided information on the role of cleavage in SFV fusion, assembly, and infectivity. A variety of other methods have also suggested that p62 cleav-

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age is important in alphavirus infectivity [reviewed in Kielian (1993) and Heidner et al. (199411. Mutation of the Arg at position -1 of the cleavage site to Leu, Glu, or Phe blocks cleavage completely and shifts the pH threshold of fusion from the wild-type pH 6.2 to values of pH 5.0-5.2 (Lobigs and Garoff, 1990; Lobigs et al., 1990b; Jain et al., 1991). Fusion efficiency comparable to that of wild type requires pH treatment as low as pH 4.7-4.5. Exogenous protease cleavage of p62 reverses this acid-shift phenotype. These mutations were originally assayed using the expressed spike protein. More recent studies have analyzed the effects of the Leu mutation when expressed in the SFV infectious clone (Salminen et al., 1992). Mutant spike proteins are efficiently assembled into virus particles, but these virions are about 500-fold less infectious than wild type. Loss of infectivity is due to both impaired virus binding and uptake, and to the inhibition of virus fusion activity a t the normal pH range found in the endosome. p62 virus is also inhibited from fusing with liposomes at the normally fusogenic pH of 5.5, and from binding to liposome membranes at this pH (Wahlberg et al., 1992). As with the expressed spike protein, the effects of the mutations on the virus are reversed by protease cleavage of p62 (Salminen et al., 1992; Wahlberg et al., 1992). Interestingly, it appears that cleavage of only a portion of the p62 subunits of a virion may be sufficient to yield an infectious particle (Salminen et al., 1992). The effects of mutations in the putative fusion peptide of the SFV E l subunit have been evaluated (Levy-Mintz and Kielian, 1991; Kielian, 1993) (see Fig. 2). These analyses followed the expressed spike protein and evaluated cell surface transport and low-pH-dependent polykaryon formation. Several mutations have no observed effect on the spike protein, whereas other less conserved mutations block transport of the E l subunit to the cell surface. The most interesting mutations from the standpoint of fusion are those that shift the pH threshold to a more acidic value (Asp75Ala, Gly83Ala, GlySlAla), and a Gly91Asp mutation that completely blocks fusion at any pH tested. Recent work has begun to analyze the effects of the G91D and G91A mutations in the SFV infectious clone (Duffus et al., 1994). Both of these mutations, although they were previously shown not to affect the cell surface transport of the spike protein, appeared to inhibit the assembly of the spike protein into virus a t 37°C. Interestingly, incubation of the infected cells a t 28°C permitted the assembly and release of morphologically normal virus particles. Thus, these mutations within the fusion peptide caused a reversible, temperature-sensitive assembly defect. The G91A virus produced a t the permissive temperature was capable of carrying out a secondary infection, whereas virus with the G91D mutation was not infectious. These results to date are in keeping with the previously documented fusion phenotype of the expressed

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spike protein. The pH-dependent conformational changes in the mutant virus spike proteins will be discussed below. Several alphavirus mutants with alterations in the pH dependence of fusion have been described. Alphaviruses show varying degrees of neurovirulence in a number of different animal models (Griffin, 1986; Griffin et al., 1994). Various Sindbis virus strains have been demonstrated to have a slower penetration rate into BHK cells and/or increased neurovirulence [reviewed in Kielian (1993)l. One strain of increased neurovirulence has a more acidic fusion threshold due to two mutations in the E l subunit (Val-72 to Ala, and Gly-313 to Asp) (Boggs et al., 1989).fus-1, an SFV mutant with a more acidic fusion threshold of pH 5.3, was selected via a liposome fusion system (Kielian et al., 1984). The sequence alterations and neurovirulence properties of this mutant are not yet defined. V. INVOLVEMENT OF SPECIFIC LIPIDSIN

THE

ALPHAVIRUS LIFECYCLE

A . Role of Cholesterol and Sphingomyelin in Virus Fusion and Infection The first report of an involvement of cholesterol in alphavirus membrane interactions was a study showing a cholesterol requirement for Sindbis virus liposome binding a t low pH (Mooney et al., 1975).Analysis of SFV fusion with liposomes then demonstrated that cholesterol is required for fusion (White and Helenius, 1980).Fusion is maximal a t a stero1:phospholipid ratio of 0.5, a value similar to the ratios found in eukaryotic cell plasma membranes (Dawidowicz, 1987).Cholesterol appears not to act simply by modulating membrane fluidity, because cholesterol analogs that do not affect membrane fluidity or phospholipid condensation still support SFV fusion (Kielian and Helenius, 1984).Such analog experiments implicate the sterol 3P-hydroxyl group as a critical part of the molecule. More recent studies of SFV binding and fusion with liposomes also demonstrate a clear cholesterol requirement, in agreement with these initial results (Bron et al., 1993; Wahlberg et al., 1992). The cholesterol requirement appears to be specific for the target membrane, because virus spike proteins are fusion active after reconstitution in pure phosphatidylcholine liposomes (Marsh et al., 1983b). The role of cholesterol in vivo during virus fusion and infection was examined using cholesterol-depleted mosquito cells (Phalen and Kielian, 19911. Insects are cholesterol auxotrophs, and insect cells can be depleted of cholesterol by growth in the absence of' low-density lipoproteins (Silberkang et al., 1983). Cholesterol depletion does not

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appear to cause synthesis of other “replacement” sterols, or changes in phospholipid composition (Silberkang et al., 1983). The depleted cells permit SFV-receptor binding, virus uptake, and acidification in endosomes, but are blocked in virus fusion (Phalen and Kielian, 1991). Depleted cells are about 1000-fold reduced in their ability to be infected by SFV (Marquardt et al., 19931, but can be readily infected by the cholesterol-independent vesicular stomatitis virus (VSV) (Phalen and Kielian, 1991). It thus appears that alphavirus fusion with complex biological membranes also depends on the presence of cholesterol in the bilayer. The cholesterol-depleted cell system has been used as a selection for SFV mutants with alterations in their cholesterol requirement (Phalen et al., 1995).Such srfmutants (for sterol requirement in fusion) can more readily infect and fuse with sterol-depleted cells, and have more rapid and extensive growth in the absence of cholesterol than does wild-type virus. Further characterization and sequence analysis of these mutants should add to our understanding of the molecular role of cholesterol in alphavirus fusion. Recent work on SFV-liposome fusion has revealed a previously unsuspected lipid requirement for fusion (Nieva et al., 1994). In addition to the well-documented cholesterol requirement, SFV fusion is dependent on the presence of sphingomyelin in the target membrane. Cholesterol is required a t fairly high concentrations in the target membrane, with half-maximal fusion at about 25 mol% cholesterol (White and Helenius, 1980). In contrast, very low concentrations of sphingomyelin are sufficient to mediate fusion, with half-maximal fusion resulting from about 1 mol% sphingomyelin (Nieva et al., 1994; Wilschut et al., 1994). Low-pH-dependent virus-liposome binding is somewhat decreased in the absence of sphingomyelin, but the major sphingomyelin-requiring step involves fusion. Analog studies show that a double-chain sphingosine-based lipid such as ceramide is effective in fusion, whereas the single-chain sphingosine base is insufficient. The low concentrations of sphingolipid required for fusion are intriguing, and it will be important to determine the step(s) in the fusion reaction that require this lipid. Interactions between cholesterol and sphingolipid in the target membrane do not appear important, since galactosyl-ceramide, a sphingolipid that does not appreciably interact with cholesterol, is active m SFV fusion.

B . Role

of

Cholesterol in the Virus Exit Pathway

Cholesterol-depleted mosquito cells were used to evaluate the role of cholesterol in postendosome fusion steps in the virus replication cycle (Marquardt et al., 1993). Cells were infected at very high multiplicity

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or transfected with virus RNA to bypass the fusion block and introduce the SFV genome into the cytoplasm. Under these conditions, the initial stages of virus infection occur in the absence of cellular cholesterol, including RNA synthesis, synthesis and dimerization of the spike polypeptides, cleavage of p62, and spike protein transport to the plasma membrane. However, wild-type SFV particles are not efficiently released from cholesterol-depleted cells, although either a n srf mutant or VSV is efficiently assembled and released. Thus, there appears to be a n involvement of cholesterol in the exit of viruses that require cholesterol for fusion. The reason for this block in SFV exit is not clear at present, but could include defects in virus subunit assembly, subunit interactions, or the final budding reaction to release the completed virion. Further work must address the mechanism of the cholesterol effect during exit, and its absence in the srfmutant and VSV. It is also not known if sphingomyelin plays a role in the virus exit pathway in addition to its recently described role in membrane fusion. Recent evidence suggests that mosquito cells grown under sterol-depleting conditions for extended periods of time may become more permissive for SFV infection and exit (M. T. Marquardt and M. Kielian, unpublished results). The mechanism of this cellular alteration is not yet clear. VI. MOLECULAR EVENTSDURING ALPHAVIRUS ENTRYAND FUSION Considerable progress has been made over the last several years in understanding the events that lead to fusion of the virus and target membranes (Table I). Much of this work has centered on defining the TABLE I SUMMARY OF EVENTS DURING SFV LOW-PH-INDUCED FUSION Order of eventsa 1. Acid treatment 2. E1/E2 dimer dissociation 3. E2 alterations E l alterations E l homotrimerization 4. Lipid bilayer interaction 5. Other unknown steps 6. Fusion 7. E2 alterations Virus inactivation

Assay Sucrose gradient sedimentation, coimmunoprecipitation Trypsin sensitivity Trypsin resistance, mAb epitope exposure Sucrose gradient sedimentation, SDS-PAGE Liposome cosedimentation Observed as lag time after virus-lipid binding mAb epitope exposure Loss of infectivity and fusion activity

aEvents are numbered in the sequence of their occurrence during the fusion reaction. The multiple events listed under some numbers have not yet been separated kinetically.

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roles played by the spike protein subunits E l and E2. Because the subunits are transported and processed as a complex, expression studies of isolated subunits have not been very useful in clarifying their fusion functions [see Kielian (1993) for review]. Studies of either biochemically isolated E l or El-containing viruses have shown that E l is responsible for the acid-dependent hemagglutination activity of SFV and SV (Dalrymple et al., 1976; Helenius et al., 1976), and that E2depleted viruses are still infectious and fusogenic (Omar and Koblet, 1988). Furthermore, antibodies to E l can block hemolysis or virusliposome fusion (Chanas et al., 1982; Wahlberg et al., 1992). These results and studies of the spike protein conformational changes summarized in the following section implicate E l as the fusogenic subunit. However, the results also suggest that the spike protein subunits act in a coordinated fashion a t least during the initial stages of the fusion reaction.

A . Early Events Following Receptor Binding When Sindbis virus is bound to the cellular receptor and the complex is warmed to 37”C, new epitopes for mAb binding are exposed on both the E2 and E l subunits (Flynn et al., 1990). This conformational change appears independent of endosomal acidification. The half-time for exposure of these “transitional epitopes” is 10-15 min, similar or slightly faster than the half-time for virus penetration in these experiments. Although an mAb to an E2 transitional epitope did not neutralize infectivity when present during viral entry, it did appear to retard the kinetics of virus penetration. It is not known if these conformationa1 changes are triggered by-virus interaction with the protein receptor, or by virus interaction with a lipid target membrane. More recent studies have examined other conditions leading to exposure of these transitional epitopes. Incubation at 51”C, treatment with dithiothrietol, or in vitro incubation a t pH 5.8-6.0 cause similar conformational changes in the spike protein (Meyer et al., 1992). The pH optimum of transitional epitope exposure is coordinately shifted in a virus strain with a n altered pH optimum for polykaryon formation, suggesting possible common features between these conformational changes and the fusion process. The transitional epitopes appear to comprise a t least three sites on E l , and site E2d on the E2 subunit (Meyer and Johnston, 1993). Virus mutants selected for loss of the E2 transitional epitope mapped to either amino acid 200 or 202 of E2. Loss of the E l epitopes correlated with mutations at either E l amino acid position 300, or positions 361 and 381 (Meyer and Johnston, 1993). It will be interesting to evaluate the functional role of these domains in alphavirus fusion and entry.

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B. Effects on Spike Protein Dimerization As described in Section 111, E2 or its precursor p62 is found as a tightly associated dimer with E l from a time shortly after biosynthesis through incorporation into the virus particle. This dimer, although noncovalent, is normally stable in the presence of nonionic detergents such as Triton X-100. Several studies suggest that one of the first steps in the expression of virus fusion activity is a n alteration in the E2-El dimer resulting in subunit separation in nonionic detergent. Kinetic studies demonstrate that the dimer dissociates with a half-time of -22 sec even a t O'C, and that essentially all of the dimer is disrupted prior to the onset of fusion (Justman et al., 1993). Dissociation of the E2-El dimer has a pH dependence more basic than that of fusion, with a pH threshold of 7.4 and maximal dissociation at pH 6.2 (Wahlberg et al., 1989). Dimer dissociation appears to be a n essential prerequisite for fusion. For example, the pH threshold for dissociation of the p62-El dimer is considerably more acidic than that of the mature E2-El dimer, with a pH threshold of 5.0 rather than 7.4 (Wahlberg et al., 1989; Lobigs et al., 1990b; Salminen et al., 1992). An SFV mutant blocked in p62 cleavage thus shows no dimer dissociation until treatment a t pH 5.0, and is also fusion inactive until treatment at pH 5.0 (Salminen et al., 1992).This block in fusion in the normal pH range appears to be due to a block in the critical acid-induced conformational changes in the E l subunit. Both fusion and these E l conformational changes are permitted a t the more acidic pH that triggers dimer dissociation. Thus, dimer dissociation appears to free the E l subunit for further low-pH-induced reactions. Under normal wild-type virus conditions, the more acidic pH threshold required for p62-El dissociation may serve to mask the virus fusion function during transit of the spike protein through the mildly acidic pH of the Golgi complex. In this model, fusion would be activated by cleavage of p62 at a point following the exit of the spike protein from the trans-Golgi network. Further analysis will be necessary to determine the exact intracellular site of p62 cleavage and the extent of acidification of this compartment.

C . Alterations in the E2lp62 Subunit At least two different irreversible conformational changes in the E2 subunit are induced by low-pH treatment. First, within 10 sec of incubation a t pH 5.6 a t 37'C, E2 converts to a conformation that is very sensitive to trypsin digestion (Kielian and Helenius, 1985; Edwards et al., 1983). This alteration in E2 has a pH dependence similar to that of fusion, and results in the trypsin sensitivity of essentially all of the

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virus E2. A monomeric, proteolytically truncated form of E2 is trypsin resistant in its neutral conformation and trypsin sensitive following low-pH treatment, suggesting that the E2 conformational change does not simply reflect dissociation of the spike protein dimer (Kielian and Helenius, 1985). The pH threshold for E2 trypsin sensitivity is shifted to a more acidic value in fus-1, an SFV mutant with a lower pH threshold for fusion (Kielian and Helenius, 1985). A second E2 conformational change is detected by the exposure of new sites for monoclonal antibody binding (Kielian et al., 1990; Justman et al., 1993). This E2 alteration occurs with a pH threshold similar to fusion in either the wild-type or fus-1 SFV strain (Kielian et al., 1990). The kinetics of this conformational change are considerably slower than fusion, however, and conversion is not reproducibly observed during virus entry into the cell (Kielian et al., 1990; Justman et al., 1993). Thus this E2 alteration is not involved in fusion but may instead reflect the acid inactivation of the spike protein, which also occurs more slowly (see Section V1,E). The p62 precursor protein also reacts to acid treatment by exposure of these mAb epitopes, but the efficiency of this reaction is less than that of E2 (Kielian et al., 1990).

D . Alterations in the E l Subunit Low-pH treatment of SFV induces irreversible conformational changeb) in the E l subunit that result in alterations in its protease sensitivity, oligomerization, and reactivity with a series of conformationspecific mAbs, as detailed below. The low-pH conformation of E l is highly protease resistant, even when the virus is disrupted by nonionic detergent and the digestion performed at 37°C (Kielian and Helenius, 1985). Conversion to trypsin resistance occurs within 10 sec of pH 5.6 treatment at 37"C, is observed in uiuo during entry into host cells, and has a pH threshold similar to that of fusion for wild-type or fus-1 SFV (Kielian and Helenius, 1985; Kielian et al., 1986; Schmid et al., 1989). Reactivity with mAbs has been used extensively as a n assay for aciddependent conformational changes in the E l subunit. Four different acid-specific mAbs have been described, termed Ela-1, -2, and -3 (Kielian et al., 19901, and anti-El" (Wahlberg and Garoff, 1992). The acid conformational changeb) recognized by these antibodies occurs with kinetics and pH dependence similar to membrane fusion, and is efficiently generated during in uiuo acidification in endosomes. The epitopes bound by these four mAbs have not yet been mapped, and thus both the number of epitopes involved and the positions of the reactive amino acids in the E l sequence are unknown. The anti-El" mAb has

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been reported to inhibit low-pH-triggered virus-plasma membrane fusion, cell-cell fusion, and virus-liposome binding and fusion (Wahlberg and Garoff, 1992; Wahlberg et al., 19921, whereas the E l a mAb series does not appear to inhibit virus-liposome fusion (Kielian et al., 1990).This suggests that at least two new antibody-binding sites on E l may be exposed following low-pH treatment. A number of other E l antibodies show enhanced reactivity with E l from acid-treated virus due to its increased accessibility. Unlike the acid-conformation specific mAbs, however, these mAbs also bind well to detergent-solubilized E 1 from neutral virus (Schmaljohn et al., 1983). An increase in E l accessibility has also been observed by labeling acid-treated virus with radioactive sulfite (Omar and Koblet, 1989). Following endocytosis, E l forms an oligomeric species that can be detected by gradient centrifugation or by the resistance of the oligomer to dissociation by mild sodium dodecyl sulfate (SDS) treatment (Wahlberg and Garoff, 1992; Wahlberg et al., 1992). This oligomer is produced in a pH-dependent reaction within the endosome, and can also be efficiently formed in uitro by low-pH treatment of virus in the presence of a target membrane (Wahlberg et al., 1992; Justman et al., 1993).The oligomer appears to be a homotrimer of E l subunits, suggesting that it may assemble from the three adjacent E l molecules of the spike trimer (Wahlberg et al., 1992) [see also model of spike trimer in Venien-Bryan and Fuller (199411. Conversion to protease resistance, exposure of acid-specific epitopes, and E l trimerization all begin immediately following low pH treatment, without a significant lag time (Bron et al., 1993). All three alterations occur following acidification under conditions wherein virus fusion does not take place, such as in the presence of fusion-inactive sterol-free liposomes (Wahlberg et al., 1992; Justman et al., 1993). However, data to date suggest that these conformational changes may be more efficient or more rapid when a fusion-active bilayer, such as a cholesterol-containing target membrane, is present (Justman et al., 1993). The similar kinetics and pH requirements for all three of these assays suggested that they might reflect the same conformational change, a trimerization of E l (Wahlberg et al., 1992; Bron et al., 1993). Recent analysis of virus containing the G91D mutation, which blocks membrane fusion, suggests that in fact the acid-specific mAbs are not trimer specific (Kielian et al., 1994). Following acid treatment, E l from the G91D virus reacts efficiently with mAbs E l a-1 and anti-El", whereas the protein remains inactive in forming the E l homotrimer. This lack of homotrimer formation in a fusion-inactive mutant thus supports a critical role for E l oligomerization in membrane fusion.

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E . Summary of Events during Fusion The extensive data on the conformational changes in the spike protein, discussed above, may be summarized and ordered as follows (Table I): Following exposure of the spike protein to acid pH, a lag phase is observed prior to the onset of membrane fusion. The length of this period between acidification and fusion is both pH and temperature dependent (Bron et al., 1993). During this lag phase, the first observed change in the spike protein is the dissociation of the E2-El dimer. The E2 and E l subunits then undergo independent conformational changes resulting in the trypsin sensitivity of E2 and the trypsin resistance, acid-specific epitope exposure, and homotrimerization of E 1. The virus next associates with the target membrane lipid bilayer in a reaction that depends on cholesterol (Bron et al., 1993). An additional period of time after virus-membrane attachment then passes before fusion actually takes place. During this part of the fusion reaction it is presumed that E l trimers undergo further pH-dependent rearrangements or oligomerization within the target bilayer to a fusion-active complex (Bron et al., 1993). The final fusion event then occurs, resulting in bilayer mixing of the virus and target membranes, and content mixing of the viral nucleocapsid into the cell cytoplasm. Following fusion, and with much slower kinetics, E2 undergoes a n additional conformational change that exposes an epitope for mAb binding. If virus is acidified in the absence of a target membrane, the viral fusion capacity is inactivated (Edwards et al., 1983; Bron et al., 1993). The kinetics of inactivation are slower than those of fusion, and virus inactivation is preceded by a pH- and temperature-dependent lag phase slightly longer than the lag phase of fusion (Bron et al., 1993). Presumably inactivation is due to the generation of short-lived fusogenic conformational changes that are nonproductive in the absence of a target membrane. The E l conformational changes, virus-membrane binding, and fusion inactivation are all arrested by neutralization (Bron et al., 1993; Justman et al., 1993). Thus it appears that the spike protein can progress irreversibly through the stages of fusion triggered by low pH, and yet pause in an intermediate stage prior to fusion until reacidification induces irreversible progression to the next stage.

F. Studies with Truncated E l

A proteolytically truncated form of E l can be prepared by proteinase K digestion of SFV in nonionic detergent (Kielian and Helenius, 1985).

This E l fragment, termed El*, has an apparent molecular weight of 48,000, lacks the hydrophobic transmembrane domain, and behaves as

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a soluble monomer in aqueous solution. On low-pH treatment, E l * undergoes conformational changes similar to those of the intact E l subunit, resulting in trypsin resistance, reactivity with the acidspecific monoclonal antibodies, and self-association of E 1" monomers to form an SDS-resistant oligomer (Kielian and Helenius, 1985; Kielian et al., 1990; Klimjack et al., 1994). Unlike intact E l , however, all of these low-pH-induced conformational changes in E l * are dependent on the presence of 3P-hydroxysterols, and are greatly enhanced by sphingomyelin. The low-pH form of E l * also binds efficiently to membranes, again dependent on low pH, cholesterol, and sphingomyelin (Klimjack et al., 1994).This direct interaction with the target membrane is in keeping with a central role of E l and its fusion peptide in membrane mixing. Further analysis should define the specific domain of E l that mediates membrane binding, and the characteristics of this protein-lipid interaction. VII. COMPARISON OF ALPHAVIRUS FUSION WITH VIRUSFUSION REACTION

THE

INFLUENZA

The influenza hemagglutinin (HA) is currently the best understood fusion protein, and has therefore served as a general paradigm for protein-mediated membrane fusion [see Wiley and Skehel (1987), White (1992), and Bentz (1993) for review]. There is still much to be learned about the key events in both the influenza and the alphavirus fusion reactions, but already some strong similarities and key differences can be discerned. HA is synthesized as precursor, HAO, that is proteolytically cleaved to two disulfide-bonded subunits, HA1 and HA2. This cleavage is required for infectivity and fusion, and positions the hydrophobic fusion peptide a t the new amino terminus of the HA2 subunit. A bromelain fragment of the ectodomain of the spike protein, BHA, has been crystallized and its three-dimensional structure determined (Wilson et al., 1981). The HA spike protein is a homotrimer of -135 A in length, with the receptor-binding sites located in the globular head regions of the HA1 subunits, and the HA2 subunits anchored in the virus membrane by their transmembrane domains. The central region of HA2 contains a long a-helix that interacts with the helices of the two other HA2 subunits in the trimer to form a triple-stranded coiled coil. At neutral pH, the fusion peptide is buried in the interface between these trimeric a-helices about 100 A from the distal tip of HA. Influenza virus infects cells via endocytosis and low-pH-mediated fusion, and low pH has been shown to trigger a series of conformational changes in the HA mole-

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cule. On exposure to the fusogenic pH, the HA2 fusion peptide is released from its hidden state and inserts into the target membrane. This appears to be a fairly nonspecific hydrophobic interaction, because in the absence of a target membrane the fusion peptide will bind detergent or aggregate with adjacent fusion peptides. Insertion of a charge at the amino terminus of the fusion peptide (a mutation of Gly-1 to Glu) abolishes lipid mixing activity (Gething et al., 1986; Guy et al., 1992). Mutational and biochemical analyses indicate that rearrangements in the globular head region and the coiled-coil domains are involved in the pH-dependent release of the fusion peptide (Wiley and Skehel, 1987; Godley et al., 1992; Kemble et al., 1992). Interestingly, complete fusion of the viral and target bilayers requires anchoring of HA by a transmembrane domain, although “hemifusion,” or mixing of the two outer lipid leaflets, can occur with an HA that is anchored in only the outer leaflet by a glycosylphosphatidylinositol moiety (Kemble et al., 1994). Computer analysis (Lupas et al., 1991) of the loop region of HA2 located between the a-helical stem and the fusion peptide indicates that it contains heptad repeats characteristic of sequences that have a high propensity to form a coiled-coil structure (Chambers et al., 1990; Carr and Kim, 1993). Indeed, peptides containing this HA sequence do trimerize to form a triple-stranded coiled coil (Carr and Kim, 1993). The crystal structure of a proteolytic fragment of the low-pH conformation of HA has recently been determined (Bullough et al., 1994). A triple-stranded coiled coil is formed from the extended loop region found in the neutral pH structure. This conformational change may serve to translocate the fusion peptide from its location 35 8, from the virus membrane to a site closer to the target membrane bound a t the top of the molecule. Similar heptad repeats are located adjacent to the hydrophobic fusion peptides of paramyxovirus and retrovirus spike proteins (Chambers et al., 1990). Both HA and the alphavirus spike protein undergo a proteolytic cleavage that activates fusion. However, in the alphaviruses the cleavage occurs on a subunit other than the fusion polypeptide, and the uncleaved form is still fusogenic if treated at a significantly more acidic pH. Both proteins are trimers, composed of HA1 and HA2 subunits in the case of influenza, and of E l , E2, and E3 subunits in the case of SFV. The two proteins appear to act as trimers during fusion, and have a lag period before the onset of fusion that probably reflects further protein rearrangements or formation of higher order structures in the target membrane. Both fusion proteins undergo a series of conformational changes on exposure to low pH. The E l sequence was analyzed for heptad repeats (Lupas et al., 19911, and does not appear to

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have a high probability of forming a coiled-coil structure (C.M. Carr, personal communication). Thus, the trimerization of El subunits observed after low-pH treatment of either intact virus spike proteins or El* is presumably due to alternative interactions. An obvious and important difference between the two virus fusion mechanisms is that influenza virus does not have a specificlipid requirement for membrane fusion, acid-dependent conformational changes, or BHA membrane binding. In contrast, alphavirus fusion is dependent on cholesterol and sphingomyelin, as are both El*membrane binding and conformational changes. Finally, the fusion peptides show no sequence conservation between the two viruses, and are amino terminal in the case of HA and internal in the alphaviruses. Mutations in either fusion peptide can block membrane fusion activity, and mutations within this domain or other parts of the HA or alphavirus spike protein can alter the pH dependence of fusion. A hemifusion intermediate presumably occurs during alphavirus fusion, but has not yet been identified. Taken together, these results suggest that HA and the alphavirus spike protein both react at low pH to form trimeric assemblies involved in membrane insertion and fusion, but that their mechanisms of trimerization and membrane insertion are likely to be quite different. VIII. FUTUREDIRECTIONS OF RESEARCH X-Ray solution scattering analysis of Sindbis virus has shown that the spike proteins increase in length by about 40 A following pH 5.0 treatment (Stubbs et al., 1991).This lengthening presumably reflects the various spike protein conformational changes summarized in this review. The limited resolution of this analysis points to a need for more extensive structural information on the neutral pH spike protein and its alterations during fusion. It will be important to determine the protein domains that are exposed during fusion, defined by mAb binding and other techniques, as one approach to understanding how the protein reacts to low pH. The trimerization of the El subunit appears to be a key step in fusion, but the mechanism of trimerization is unknown, as well as whether the functional unit during fusion is an El trimer or a higher order oligomer. Although data point to the El hydrophobic domain as the fusion peptide, it will be important to demonstrate that it directly inserts into the target membrane, and then to analyze its orientation and interactions within the bilayer. A critical and unique feature of alphavirus fusion is its lipid dependence. The role of both cholesterol and sphingomyelin in spike protein conforma-

MARGARET KIELIAN

tional changes, fusion peptide insertion, and fusion are important questions, as well a s the potential synergy between these two lipids, which are known to interact in membranes. Further investigation will also reveal if other viral or cellular fusion proteins have similar lipid requirements, or use similar protein structures to carry out fusion. Finally, it will be important to correlate findings on the molecular mechanism of alphavirus fusion with the biology of the virus infection pathway in its wide variety of host cells, and its pathogenic phenotype in organisms.

ACKNOWLEDGMENTS I thank Duncan Wilson, Dennis Shields, and Judy White for very useful comments on a draft of this review, the members of my lab for helpful discussions, and Marianne Marquardt for Fig. 1. 1 thank J a n Wilschut, Fred Hughson, and Chavela Carr for communicating and discussing results prior to publication. Work from my laboratory described in this review was supported by grants from the American Cancer Society (VM-41),the Hirschl Charitable Trust, and the Pew Scholars Program in the Biomedical Sciences, and by Cancer Center Core Support Grant NIHlNCI P30-CA13330.

REFERENCES Abell, B. A., and Brown, D. T.(1993).J. Virol. 67, 5496-5501. Aliperti, G., and Schlesinger, M. J. (1978).Virology 90, 366-369. Anthony, R. P., and Brown, D. T. (1991).J. Virol. 66, 1187-1194. Anthony, R. P., Paredes, A. M., and Brown, D. T.(1992). Virology 190, 330-336. Barth, B. U., Suomalainen, M., Liljestrom, P., and Garoff, H. (1992).J. Virol. 66,75607564.

Bentz, J. (1993). “Viral Fusion Mechanisms.” CRC Press, Boca Raton, Florida. Boggs, W. M., Hahn, C. S., Strauss. E. G., Strauss, J. H., and Griffin, D. E. (1989). Virology 169, 485-488. Bonatti, S., Migliaccio, G., and Simons, K. (1989).J . B i d . Chem. 264, 12590-12595. Bowman, E. J., Siebers, A., and Altendorf, K. (1988).Proc. Natl. Acad. Sci. U.S.A.85, 7972-7976.

Bron, R., Wahlberg, J. M., Garoff, H., and Wilschut, J. (1993).EMBO J. 12, 693-701. Brown, D. T., and Condreay, L. D. (1986). In “The Togaviridae and Flaviviridae” (S. Schlesinger and M. J. Schlesinger, eds.), pp. 171-207. Plenum, New York. Brown, D. T., and Edwards, J. 11992). Semin. Virol. 3, 519-527. Bullough, P. A., Hughson, F. M., Skekel, J. J., and Wiley, D. C. (1994).Nature 371, 3743.

Carr, C. M., and Kim, P. S. (1993). Cell (Cambridge, Massachusetts) 73, 823-832. Cassell, S., Edwards, J., and Brown, D. T.(1984).J . Virol. 52, 857-864. Chambers, P., Pringle, C. R., and Easton, A. J. (1990).J. Gen. Virol. 71, 3075-3080. Chanas, A. C., Gould, E. A., Clegg, J. C. S., and Varma, M. G. R. (1982).J . Gen. Virol. 58, 37-46.

Choi, H.-K., Tong, L., Minor, W., Dumas, P., Boege, U., Rossman, M. G., and Wengler, G. (1991).Nature (London) 354,37-43. Collier, N. C., Adams, S. P., Weingarten, H., and Schlesinger, M. J. (1992). Antiviral Chem. Chemother. 3, 31-36.

ALPHAVIRUS LIFE CYCLE

147

Coombs, K., Mann, E., Edwards, J., and Brown, D. T. (1981). J. Virol. 37, 1060-1065. Dalrymple, J. M., Schlesinger, S., and Russell, P. K. (1976).Virology 69, 93-103. Davis, N. L., Willis, L. V., Smith, J. F., and Johnston, R. E. (1989). Virology 171, 189204. Dawidowicz, E. A. (1987). Annu. Reu. Biochem. 56,43-61. deCurtis, I., and Simons, K. (1988).Proc. Natl. Acad. Sci. U.S.A.85, 8052-8056. Dubuisson, J., and Rice, C. M. (1993). J . Virol. 67, 3363-3374. Duffus, W., Levy-Mintz, P., Klimjack, M. R., and Kielian, M. (1994).Manuscript submitted. Dunn, W. A,, Hubbard, A. L., and Aronson, N. N. (1980).J . Biol. Chem. 255,5971-5978. Edwards, J., and Brown, D. T. (1986).J. Gen. Virol. 67, 377-380. Edwards, J., and Brown, D. T. (1991). Virology 182, 28-33. Edwards, J., Mann, E., and Brown, D. T. (1983). J . Virol. 45, 1090-1097. Ekstrom, M., Liljestrom, P., and Garoff, H. (1994).EMBO J. 13, 1058-1064. Fan, D. P., and Sefton, B. N. (1978).Cell (Cambridge, Massachusetts) 15, 985-992. Flynn, D. C., Meyer, W. J., Mackenzie, J. M., and Johnston, R. E. (1990). J. Virol. 64, 3643-3653. Froshauer, S., Kartenbeck, J., and Helenius, A. (1988).J. Cell B i d . 107, 2075-2086. Fuller, S. D. (1987).Cell (Cambridge, Massachusetts) 48, 923-934. Fuller, S. D., von Bonsdorff, C.-H., and Simons, K. (1985).EMBO J . 4, 2475-2485. Gaedigk-Nitschko, K., and Schlesinger, M. J. (1990). Virology 175, 274-281. Gaedigk-Nitschko, K., and Schlesinger, M. J. (1991). Virology 183, 206-214. Gaedigk-Nitschko, K., Ding, M., Levy, M. A., and Schlesinger, M. J . (1990).Virology 175, 282-291. Gallaher, W. R., Segrest, J . P., and Hunter, E. (1992).Cell (Cambridge, Massachusetts) 70, 531-532. Garoff, H., and Simons, K. (1974).Proc. Natl. Acad. Sci. U.S.A.71, 3988-3992. Garoff, H., Frischauf, A.-M., Simons, K., Lehrach, H., and Delius, H. (1980a).Nature (London) 288,236-241. Garoff, H., Frischauf, A.-M., Simons, K., Lehrach, H., and Delius, H. (1980b).Proc. Natl. Acad. Sci. U.S.A. 77, 6376-6380. Garoff, H., Huylebroeck, D., Robinson, A,, Tillman, U., and Liljestrom, P. (1990).J. Cell Biol. 111, 867-876. Geigenmuller-Gnirke, U., Nitschko, H., and Schlesinger, S. (1993).J. Virol. 67, 16201626. Gething, M J . , Doms, R. W., York, D., and White, J. (1986).J. Cell Biol. 102, 11-23. Godley, L., Pfeifer, J., Steinhauer, D., Ely, B., Shaw, G., Kaufmann, R., Suchanek, E., Pabo, C., Skehel, J. J., Wiley, D. C., and Wharton, S. (1992).Cell (Cambridge, Massachusetts) 68, 635-645. Goldstein, J . L., Anderson, R. G., and Brown, M. S. (1979).Nature fLondon) 279, 679685. Griffin, D. E. (1986). In “The Togaviridae and Flaviviridae” (S. Schlesinger and M. J . Schlesinger, eds.), pp. 209-249. Plenum, New York. Griffin, D. E., Levine, B., Ubol, S., and Hardwick, J. M. (1994).Ann. Neurol. 35, suppl., 523-527. Griffiths, G., Quinn, P., and Warren, G. (1983).J. Cell Biol. 96, 835-850. Guy, H. R., Dwell, S. R., Schoch, C., and Blumenthal, R. (1992).Riophys. J . 62, 95-97. Hahn, C. S. (1985).Proc. Natl. Acad. Sci. U.S.A.82, 4648-4652. Hahn, C. S., Rice, C. M., Strauss, E. G., Lenches, E. M., and Strauss, J . H. (1989).J. Virol. 63,3459-3465. Harrison, S. C. (1986).In “The Togaviridae and Flaviviridae” (S. Schlesinger and M. J. Schlesinger, eds.1, pp. 21-34. Plenum, New York.

148

MARGARET KIELIAN

Harter, C., James, P., Bachi, T., Semenza, G., and Brunner, J. (1989).J. Biol. Chem. 264, 6459-6464. Heidner, H. W., McKnight, K. L., Davis, N. L., and Johnston, R. E. (1994).J. Virol. 68, 2683-2692. Helenius, A,, and Kartenbeck, J. (1980).Eur. J. Biochem. 106, 613-618. Helenius, A,. Fries, E., Garoff, H., and Simons, K. (1976).Biochim. Biophys. Acta 436, 319-334. Helenius, A,, Morein, B., Fries, E., Simons, K., Robinson, P., Schirrmacher, V., Terhorst, C., and Strominger, J . L. (1978).Proc. Natl. Acad. Sci. U.S.A. 75, 3846-3850. Helenius, A., Kartenbeck, J., Simons, K., and Fries, E. (1980).J. Cell Biol. 84, 404420. Helenius, A., Marsh, M., and White, J . (1982).J . Gen. Virol. 58, 47-61. Helenius, A., Kielian. M., Wellsteed, J., Mellman I., and Rudnick, G. (1985).J. Biol. Chem. 260, 5691-5697. Hubbard, S. C. (1988).J. Biol. Chem. 263, 19303-19317. Ivanova, L., and Schlesinger, M. J. (1993).J. Virol. 67, 2546-2551. Jain, S. K., DeCandido, S., and Kielian, M. (1991).J. Biol. Chem. 266, 5756-5761. Justman, J., Klimjack, M. R., and Kielian, M. (1993).J. Virol. 67, 7597-7607. Kail, M., Hollinshead, M., Ansorge, W., Pepperkok, R., Frank, R., Griffiths, G., and Vaux, D. (1991).EMBO J. 10,2343-2351. Kemble, G. W., Bodian, D. L., Rose, J., Wilson, I. A., and White, J . M. (1992).J. Virol. 66, 4940-4950. Kemble, G. W., Danieli, T., and White, J. M. (1994).Cell (Cambridge,Massmhusettsl 76, 383-391. Kielian, M. 11993). In “Viral Fusion Mechanisms” (J. Bentz, ed.), pp. 385-412. CRC Press, Boca Raton, Florida. Kielian, M. C., and Helenius, A. (1984).J. Virol. 52, 281-283. Kielian, M., and Helenius, A. (1985).J . Cell Biol. 101, 2284-2291. Kielian, M., and Helenius, A. (1986).I n “The Togaviridae and Flaviviridae” (S. SchleKielian, M. C., Keranen, S., Kaariainen, L., and Helenius, A. (1984).J. Cell Biol. 98, 139-145. Kielian, M. C., Marsh, M., and Helenius, A. (1986).EMBO J. 5, 3103-3109. Kielian, M., Jungerwirth, S., Sayad, K. U., and DeCandido, S. (1990).J. Virol. 64,46144624. Kielian, M., Klimjack, M. R., and Duffus, W. (1994).Manuscript in preparation. Klimjack, M. R., Jeffrey, S., and Kielian, M. (1994).J. Virol. 68, 6940-6946. Koblet, H. (1990).Adu. Virus Res. 38, 343-402. Kondor-Koch, C., Burke, B., and Garoff, H. (1983).J. Cell Biol. 97, 644-651. Kuhn, R. J., Niesters, H. G. M., Hong, Z., and Strauss, J. H. (1991).Virology 182, 430441. Laine, R., Soderlund, H., and Renkonen, 0. (1973).Intervirology 1, 110-118. Lanzrein, M., Kasermann, N., Weingart, R., and Kempf, C. (1993a).Virology 196, 541547. Lanzrein, M., Weingart, R., and Kempf, C. (1993b). Virology 193, 296-302. Lee, H., and Brown, D. T. (1994).Virology 202, 390-400. Levy-Mintz, P., and Kielian, M. (1991).J. Virol. 65, 4292-4300. Liljestrom, P., and Garoff, H. (1991).J. Virol. 65, 147-154. Liljestrom, P., Lusa, S., Huylebroeck, D., and Garoff, H. (1991).J . Virol. 65,4107-4113. Lindqvist, B. H., DiSalvo, J., Rice, C. M., Strauss, J. H., and Strauss, E. G. (1986). Virology 151, 10-20. Liu, N., and Brown, D. T. (1993a). Virology 196, 703-711.

ALPHAVIRUS LIFE CYCLE

149

Liu, N., and Brown, D. T. (1993b). J . Cell Biol. 120,877-883. Lobigs, M., and Garoff, H. (1990). J . Virol. 64,1233-1240. Lobigs, M., Hongxing, Z., and Garoff, H. (1990a).J. Virol. 64,4346-4355. Lobigs, M., Wahlberg, J. M., and Garoff, H. (1990b). J. Virol. 64,5214-5218. Lopez, S., Lopez, I., Romero, P., Mendez, E., Soberon, X., and Arias, C. F. (1991).J. Virol. 65,3738-3745. Lopez, S., Yao, J.-S., Kuhn, R. J., Strauss, E. G., and Strauss, J. H. (1994). J. Virol. 68, 1316-1323. Lupas, A., Van Dyke, M., and Stock, J. (1991).Science 252, 1162-1164. Lusa, S., Garoff, H., and Liljestrom, P. (1991).Virology 185,843-846. Luukonen, A., Kaariainen, L., and Renkonen, 0. (1976).Biochirn. Biophys. Acta 450, 109-120. Luukonen, A., von Bonsdorff, C. H., and Renkonen, 0. (1977).Virology 78,331-335. Mackow, E. R., Shaw, R. D., Matsui, S. M., Vo, P. T., Dang, M.-N., and Greenberg, H. B. Proc. Natl. Acad. Sci. U.S.A. 85, 645-649. (1988). Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S. P., Stone, D. K., and Draper, R. K. J. Cell Biol. 99, 1907-1916. (1984). Marquardt, T., and Helenius, A. (1992).J. Cell Biol. 117,505-513. Marquardt, M. T., Phalen, T., and Kielian, M. (1993).J. Cell Biol. 123,57-65. Marsh, M., and Helenius, A. (1980). J. Mol. Biol. 142,439-454. Marsh, M., and Helenius, A. (1989). Adu. Virus Res. 36, 107-151. Marsh, M., Wellsteed, J., Kern, H., Harms, E., and Helenius, A. (1982). Proc. Natl. Acad. Sci. U.S.A.79, 5297-5301. Marsh, M., Bolzau, E., and Helenius, A. (1983a). Cell (Cambridge, Massachusetts) 32, 931-940. Marsh, M., Bolzau, E., White, J., and Helenius, A. (1983b). J. Cell Biol. 96, 455-461. Massen, J. A,, and Terhorst, C. (1981). Eur. J. Biochem. 115, 153-158. Mattila, K. (1979). Biochim. Biophys. Acta 579, 62-72. Mayne, J. T.,Rice, C. M., Strauss, E. G., Hunkapiller, M. W., and Strauss, J. H. (1984). Virology 134, 338-357 (abstract). Melancon, P., and Garoff, H. (1986). EMBO J. 5, 1551-1560. Mellman, I., Fuchs, R., and Helenius, A. (1986). Annu. Reu. Biochem. 55, 663-700. Metsikko, K., and Garoff, H. (1990). J. Virol. 64,4678-4683. Meyer, W. J., and Johnston, R. E. (1993). J . Virol. 67,5117-5125. Meyer, W. J., Gidwitz, S., Ayers, V. K., Schoepp, R. J., and Johnston, R. E. (1992). J . Virol. 66,3504-3513. Migliaccio, G., Pascale, M. C., Leone, A,, and Bonatti, S. (1989). Exp. Cell Res. 185,203216. Miller, M. L., and Brown, D. T. (1992). J . Virol. 66,4180-4190. Mooney, J. J., Dalrymple, J. M., Alving, C. R., and Russell, P. K. (1975). J. Virol. 15,225231. Mulvey, M., and Brown, D. T. (1994). J . Virol. 68,805-812. Nieva, J . L., Bron, R., Corver, J., and Wilschut, J. (1994). EMBO J. 13,2797-2804. Ohkuma, S.,and Poole, B. (1978). Proc. Natl. Acad. Sci. U.S.A. 75,3327-3331. Oldstone, M.B. A,, Tishon, A,, Dutko, K., Kennedy, S. I. T., Holland, J . J., and Lampert, P. W. (1980). J. Virol. 34, 256-265. Omar, A,, and Koblet, H. (1988). Virology 166, 1'7-23. Omar, A., and Koblet, H. (1989).Virology 168, 177-179. Omar, A,, Flaviano, A,, Kohler, U., and Koblet, H. (1986). Arch. Virol. 89, 145-159. Paredes, A. M., Brown, D. T., Rothnagel, R., Chiu, W., Schoepp, R. J., Johnston, R. E., and Prasad, B. V. V. (1993). Proc. Natl. Acad. Sci. U.S.A. 90,9095-9099. Pbrez, L., and Carrasco, L. (1994).J . Gen. Virol. 75, 2595-2606.

150

MARGARET KIELIAN

Peters, C. J., and Dalrymple, J. M. (1990). In “Virology” (B. N. Fields and D. M. Knipe, eds.), 2nd Ed., pp. 713-761. Raven Press, New York. Phalen, T., and Kielian, M. (1991). J . Cell Biol. 112, 615-623. Phalen, T., Marquardt, M. T., and Kielian, M. (1995). Manuscript in preparation. Presley, J. F., and Brown, D. T. (1989). J. Virol. 63, 1975-1980. Quinn, P., Griffiths, G., and Warren, G. (1983). J. Cell Biol. 96, 851-856. Rice, C. M., and Strauss, J. H. (1982). J. Mol. Biol. 154, 325-348. Rice, C. M., Levis, R., Strauss, J. H., and Huang, H. V. (1987). Virology 61, 3809-3819. Roman, L., and Garoff, H. (1986). J. Cell Biol. 103, 2607-2618. Salminen, A., Wahlberg, J. M., Lobigs, M., Liljestrom, P., and Garoff, H. (1992). J. Cell Biol. 116, 349-357. Sanz, M. A., Perez, L., and Carrasco, L. (1994). J . Biol. Chem. 269, 12106-12110. Schlegel, A., Omar, A., Jentsch, P., Morell, A., and Kempf, C. (1991). Biosci. Rep. 11, 243-255.

Schlesinger, M. J., and Schlesinger, S. (1986a). In “The Togaviridae and Flaviviridae” (S. Schlesinger and M. J. Schlesinger, eds.), pp. 121-148. Plenum, New York. Schlesinger, S., and Schlesinger, M. J. (1986b). “The Togaviridae and Flaviviridae.” Plenum, New York. Schlesinger, M. J., London, S. D., and Ryan, C. (1993). Virology 193, 424-432. Schmaljohn, A. L., Kokubun, K. M., and Cole, G. A. (1983). Virology 130, 144-154. Schmid, S. L., Fuchs, R., Male, P., and Mellman, I. (1988). Cell (Cambridge, Massachusetts) 52, 73-83. Schmid, S. L., Fuchs, R., Kielian, M., Helenius, A., and Mellman, I. (1989). J. Cell Biol. 108, 1291-1300.

Schmidt, M. F. G., and Lambrecht, B. (1985). J . Gen. Virol. 66, 2635-2647. Schmidt, M., Schmidt, M. F. G., and Rott, R. (1988). J. Biol. Chem. 263, 18635-18639. Silberkang, M., Havel, C. M., Friend, D. S., McCarthy, B. J., and Watson, J. A. (1983). J . Biol. Chem. 258,8303-8311. Silverstein, S. C., Steinman, R. M., and Cohn, Z. A. (1977).Annu. Rev. Biochem. 46,669722.

Simons, K., and Warren, G. (1984). Adu. Prot. Chem. 36, 79-132. Singh, I., and Helenius, A. (1992a). J. Virol. 66, 7049-7058. Singh, I., and Helenius, A. (1992b). Semin. Virol. 3, 511-518. Smith, J. F., and Brown, D. T. (1977).J. Virol. 22, 662-678. Soderlund, H., Kaariainen, L., von Bonsdorff, C.-H., and Weckstrom, P. (1972). Virology 47, 753-760.

Spruce, A. E., Iwata, A., and Almers, W. (1991). Proc. Natl. Acad. Sci. U.S.A.88, 36233627.

Stegmann, T., Delfino, J. M., Richards, F. M., and Helenius, A. (1991). J . Biol. Chem. 266, 18404- 18410.

Stegmann, T., Schoen, P., Bron, R., Wey, J., Bartoldus, I., Ortiz, A., Nieva, J.-L., and Wilschut, J. (1993). Biochemistry 32, 11330-11337. Strauss, E. G., and Strauss, J. H. (1986). In “The Togaviridae and Flaviviridae” tS. Schlesinger and M. J. Schlesinger, eds.), pp. 35-90. Plenum, New York. Strauss, J. H., and Strauss, E. G. (1994). Microbiol. Reu. 58, 491-562. Stubbs, M. J., Miller, A., Sizer, P. J. H., Stephenson, J. R., and Crooks, A. J. (1991). J . Mol. Biol. 221, 39-42. Suomalainen, M., Liljestrom, P., and Garoff, H. (1992). J. Virol. 66, 4737-4747. Syvaoja, P., Peranen, J., Suomalainen, M., Keranen, S., and Kaariainen, L. (1990). Virology 179,658-666. Tanasugarn, L., McNeil, P., Reynolds, G. T., and Taylor, D. L. (1984). J . Cell Biol. 98, 717-724.

ALPHAVIRUS LIFE CYCLE

151

Tong, L., Wengler, G., and Rossmann, M. G. (1993). J. Mol. Biol. 230, 228-247. Tucker, P. C., and Griffin, D. E. (1991).J . Virol. 65, 1551-1557. Ubol, S., and Griffin, D. E. (1991). J. Virol. 65, 1551-1557. van Meer, G., Simons, K., Op den Kamp, J. A. F., and van Deenen, L. L. M. (1981). Biochemistry 20, 1974-1981. Venien-Bryan, C., and Fuller, S. D. (1994).J. Mol. Biol. 236, 572-583. Vogel, R. H., Provencher, S.W., von Bonsdorff, C.-H., Adrian, M., and Dubochet, J. (1986).Nature (London) 320, 533-535. Wahlberg, J. M., and Garoff, H. (1992). J. Cell Biol. 116, 339-348. Wahlberg, J. M., Boere, W. A. M., and Garoff, H. (1989).J. Virol. 63, 4991-4997. Wahlberg, J. M., Bron, R., Wilschut, J., and Garoff, H. (1992).J . Virol. 66, 7309-7318. Wang, K.-S., Schmaljohn, A. L., Kuhn, R. J.,and Strauss, J. H. (1991). Virology 181,694702. Wang, K.-S., Kuhn, R. J., Strauss, E. G., Ou, S., and Strauss, J. H. (1992).J. Virol. 66, 4992-500 1. Weiss, B., Nitschko, H., Ghattas, I., Wright, R., and Schlesinger, S. (1989).J. Virol. 63, 5310-5318. Welsh, W. J., and Sefton, B. M. (1979).J. Virol. 29, 1186-1195. Wengler, G., and Wengler, G. X. (1984).Virol. 134, 435-442. White, J. M. (1990). Annu. Rev.Phys. 52, 675-697. White, J. M. (1992). Science 258, 917-924. White, J., and Helenius, A. (1980). Proc. Natl. Acad. Sci. U.S.A.77, 3273-3277. White, J., Kartenbeck, J., and Helenius, A. (1980). J. Cell Biol. 87, 264-272. White, J., Matlin, K., and Helenius, A. (1981).J. Cell Biol. 89, 674-679. Wiley, D. C., and Skehel, J. J. (1987). Annu. Rev.Biochem. 56,365-394. Wilschut, J., Corver, J., Nieva, L., Bron, R., Moesby, L., and Bittman, R. (1994). Mol. Memb. Biol. In press. Wilson, I. A,, Skehel, J. J., and Wiley, D. C. (1981).Nature (London) 289, 366-378. Young, J. D.-E., Young, G. P. H., Cohn, Z. A., and Lenard, J. (1983). Virology 128, 186194. Zhao, H., and Garoff, H. (1992).J. Virol. 66, 7089-7095. Zhao, H., Lindqvist, B., Garoff, H., von Bonsdorff, C.-H., and Liljestrom, P. ( 1994). EMBO J . 13,4204-4211. Ziemiecki, A., Garoff, H., and Simons, K. (1980).J. Gen. Virol. 50, 111-123. Zurzolo, C., Polistine, C., Saini, M., Gentile, R., Alof, L., Migliaccia, G., Bonatti, S., and Nitsch, L. (1992). J. Cell B i d . 117, 551-564. NOTEADDEDIN PROOF.One study found that peptides derived from the E2 tail inhibit budding when microinjected into infected cells, and the carboxy-terminal 8 amino acids appear responsible for inhibition (Kail et al., 1991). In contrast, Collier et al. (1992) reported that peptides derived from the N-terminal region of the E2 tail are most active in inhibiting virus release. A recent mutagenesis study found the single tyrosine in the amino-terminal half of the E2 tail was critical for budding (Zhao et al., 19941, in addition to the previously identified requirement for the cysteine residues in the carboxyterminal portion of the tail.