Nuclear Import of Human Immunodeficiency Virus Type-1 Preintegration Complexes

ADVANCES IN VIRUS RESEARCH, VOL.52

NUCLEAR IMPORT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 PREINTEGRATION COMPLEXES Ron A.

M. Fouchier* and Michael H. Malim*,t

*Howard Hughes Medical Institute and tDepartments of Microbiology and Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 191 044148

I. Introduction Pathways of Cellular Nuclear Import Nuclear Import in Nonretroviral Systems HIV-1 Infection of Nondividing Cells Isolation and Composition of HIV-1 Preintegration Complexes VI. NLSs in HIV-1 Reintegration Complex Proteins VII. Models for HIV-1 Reintegration Complex Nuclear Import VIII. Some Future Questions IX. Relationships between Oncoretrovirus and Foamy Virus Infections and the Cell Cycle X. Summary References 11. 111. IV. V.

I. INTRODUCTION The mechanism used by retroviruses to replicate their positive-sense RNA genomes is remarkable for two main reasons (1,2).First, replication proceeds through a DNA intermediate which is synthesized by the viral enzyme reverse transcriptase (RT).Indeed, it was the ability of retroviruses to reverse the flow of genetic information from RNA back to DNA that gave this family of viruses their name. Second, the viral DNA covalently integrates into the chromosomal DNA of the infected cell to establish what is known as the “provirud’; it is this form of retroviral genetic material that then serves as the template for future viral genome synthesis. Importantly, integration is mediated by a virally encoded enzyme, integrase (IN), that acts in the context of a large subviral nucleoprotein complex known as the “preintegration complex” (PIC), (defined below) (3,4). Because integration into host DNA is an obligate step of the retroviral life cycle, it is essential for the PIC to enter the nucleus. It has now been established that the mechanism by which this is accomplished differs among different retroviruses. In particular, the PIC of the oncoretrovirus murine leukemia virus (MLV) relies on mitotic dissolution 275

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of the nuclear envelope to gain access to host chromatin (5), a finding which explains why cell division is a prerequisite for productive MLV infection (6). Conversely, lentiviruses (7) and, most notably, human immunodeficiencyvirus type-1 (HIV-1) are able to infect both dividing and nondividing cells (8-13). In the case of HIV-1, the ability to infect cells productively in the absence of mitosis is directly attributable to the signal-mediated and energy-dependent nuclear import of PICs during interphase (14). To put HIV-1 PIC import into perspective, the nuclear translocation of viral nucleic acids (genomes) is critical for the productive infection of non-cycling cells by many viruses. In fact, the majority of DNA viruses (the exceptions being the poxviruses and the iridoviruses), as well as one family of RNA viruses, the orthomyxoviruses, replicate their genomes in the nucleus. Accordingly, before discussing HIV-1 in depth, we will provide a brief overview of the pathways and mechanisms of cellular nuclear import and relate this to what is currently understood concerning import in three other viral systems: adenovirus, simian virus 40 (SV40),and influenza virus.

11. PATHWAYS OF CELLULAR NUCLEAR IMPORT Transport into and out of the nucleus occurs via the nuclear pore complexes (NPCs) (15-17). These -125-MDa gated structures span both the inner and outer membranes of the nuclear envelope and contain multiple copies of at least 50 M e r e n t proteins termed “nucleoporins.” Particles with diameters in excess of -28 nm are too large to be accommodated by the central pore of the NPC,whereas certain molecules of less than -9 nm (which correspond to a -40-kDa protein) are able to diffuse passively through its outer channels. The transport of molecules and macromolecular complexes between these limits is mediated by specific energy-dependent pathways which are accessed by proteins that contain cis-acting targeting sequences known a s “nuclear localization signals” (NLSs) or “nuclear export signals” (NESs). Many such sequences have now been defined on the basis of their ability to impart nuclear import or export activity to coupled heterologous substrate molecules. In terms of import, numerous analyses have revealed that NLSs are frequently, although not always, characterized by one or two stretches of basic amino acids (basic-type or classical NLSs) (16,18). The signal-mediated import of proteins into the nucleus takes place in a number of discrete steps (Fig. 6.1, color plate). Initially, the NLS-

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bearing substrates are recognized in the cytoplasm by a member of an expanding family of -100-kDa shuttling transport receptors (17,19). The first of these receptors to be described mediates the import of basictype NLSs by the classical pathway and is known as “importin-P” (or “karyopherin-P”). In contrast to more recently identified import receptors such as transportidkaryopherin-&or karyopherin-&, which bind directly to their respective substrates, importin-/3 recognizes its substrates via members of the importin-a family of adapter proteins. Once formed, receptor-substrate complexes dock at the NPC via interactions between the import receptors and cytoplasmic filament proteins, and are then translocated through the pore by a process that is thought to be facilitated by sequential direct interactions between the receptors and various nucleoporins. Finally, the complexes are disassembled in the nucleus and the receptors (including importin-a) are recycled back to the cytoplasm. The Ran GTPase is also essential for nuclear import and may function in at least two ways (17,20,21). First, the asymmetric distribution of RanGTP (nucleus)-RanGDP (cytoplasm) across the nuclear envelope provides directionality to nuclear transport; in particular, binding of nuclear RanGTP to import receptors terminates nuclear import by inducing the dissociation of substrates (or importin-a) from the receptors. Second, &-mediated GTP hydrolysis may be thermodynamically coupled to translocation through the pore.

111. NUCLEAR IMPORT IN NONRETROVIRAL SYSTEMS Following viral penetration into susceptible cells, a complex series of programmed trafficking and disassembly events ensue which culminate in the initiation of genome replication. For most viruses these processes are collectively defined as “uncoating.” Because retroviral reverse transcription begins soon after (and, in some instances, prior to) entry into the cytoplasm, whereas integration occurs much later and in the nucleus (Fig. 6.2, color plate), it is less clear how the term “uncoating” should be applied to these infections. However, for the purposes of this discussion, we have used the term loosely to include all postentry steps leading up to integration. In any event, it is useful to compare HIV-1uncoating and PIC nuclear import with current models for how other viruses target their genomes to the nucleus (refer to Table 6.1) (22,231. With a diameter of -90 nm, adenovirus capsids are well beyond the size limit for transport through NPCs. However, because this virus naturally infects airway cells of the upper respiratory tract that are

TABLE 6.1 NUCLEAR IMPORT FOR ~ E N O V I R U S SV40, , INFLUENZA Vmus. AND HW-1 POSTENTRY virus

Adenovirus

SV40

Family (genus) Genome

Adenoviridae dsDNA

Papovaviridae dsDNA

Envelopedhonenveloped Capsid dimensions

Nonenveloped 90 nm

50 nm

Nucleoprotein complex that DNA Protein VII enters the nucleus Terminal protein Protein V (?), p (?) NLSs that facilitate Protein VII (?) genome nuclear import Terminal protein (?) Uncoating is multistep Additional comments Capsid dissociation is triggered by NPC binding Signals that target capsids to the NPC are unknown

Nonenveloped

DNA VPL VP2, VP3 Histones

Influenza Virus Orthomyxoviridae Negative-sense RNA (segmented) Enveloped 30-100 X 10-20 nm rods (each v F U W

RNA

NIJ

PA, PB1, PB2 (Pol trimer)

NF' (multiple NLSs) VP3 Pol proteins (?) Vpl (?) Conformational changes in v R " NLSs are unmasked following endosomal capsid are required for its transport through "acid bath" induced NPCs dissociation of M1

HIV-1 Retroviridae (Lentivirus) Positive-sense RNA (DNA intermediate) Enveloped 100 X 50 (wide end) nm cone (Stokes radius of PICs is -28 nm) RNADNA IN, RT, MA, Vpr, NC p6 (?), PR (?)

IN,Vpr, MA (7) Reverse transcription begins in the cytoplasm MA phosphorylation is important

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nonproliferating, nuclear import of viral DNA is essential for replication. Adenovirus enters cells via endosomes, where limited uncoating takes place, and capsid structures that still contain -80% of their starting mass are released into the cytoplasm (24). These travel to the cytoplasmic face of NPCs, where docking specifically triggers capsid disassembly (25). Viral DNA, potentially in association with protein VII and the covalently bound terminal protein, is then imported into the nucleus. It is not known which viral proteins mediate either capsid targeting to the NPC or translocation through the pore itself. Like adenovirus, SV40 is a nonenveloped DNA virus that enters the cytosol from the endosomes. Its capsid is much less complex and is composed of three structural proteins: Vpl, Vp2, and Vp3. However, at -50 nm, this structure would also appear to be too large to be imported through NPCs. Nevertheless, and in contrast to adenovirus, ultrastructural studies have indicated that capsid-like structures do, in fact, enter the nucleus (26);given the aforementioned size constraints of NPC-mediated transport, it has therefore been predicted that SV40 capsids undergo substantial conformational changes in the cytoplasm. Although Vpl, Vp2, and Vp3 all harbor NLSs (these are required for the nuclear uptake of newly synthesized proteins as a prerequisite to viral assembly), it is not yet clear which, if any, of these function during transport t o the NPC or translocation into the nucleus. Antibody microinjection experiments have indicated that Vpl and Vp3 are both important for the eventual import of viral DNA but that only Vp3 appears to be associated with DNA once in the nucleus (27).The potential roles of Vp2 in uncoating and nuclear import remain undescribed. In contrast to adenovirus and SV40, the molecular events that govern the nuclear import of the influenza virus genome are relatively well understood. The genome comprises eight RNA segments which are individually packaged into each enveloped virion in the form of ribonucleoprotein particles (vRNPs). Following viral uptake into endosomes, acidification induces the dissociation of the M1 protein from vRNPs and, as a result, exposes their NLS(s) (28). Further acidification in late endosomes triggers fusion between the viral and cellular membranes and thereby allows the vRNPs to enter the cytosol. Although all the protein components of vRNPs-multiple copies of nucleoprotein (NP)and single copies of the three polymerase subunits-contain NLSs (vRNPs, like adenovirus and SV40, assemble in the nucleus), NP, by utilizing the importin-dfl pathway, is able to mediate the efficient nuclear import of viral RNA (29). Importantly, the dimensions of influenza virus vRNPs (30-110 X 10-20 nm) are compatible with efficient nuclear transport.

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Relatively early studies of HIV-1 in culture demonstrated that efficient infection of cells that are essentially nondividing-for example, monocyte-derived macrophages (MDMs)-can take place (30-32). Other findings showing not only that HIV-1 infects tissue macrophages, microglia (brain macrophages), and mucosal dendritic cells in uiuo (8,9,12,13),but also that macrophage tropic viruses predominate in the early phases of disease (33-37) appear to highlight the importance of nondividing cell infections to the transmission, persistence, and pathogenicity of HIV-1. Prior to the emergence of HIV-1, however, retrovirologists believed that productive retroviral infections were restricted to proliferating cells (see below) (6,38). Because of the indications that HIV-1 might not adhere to this dogma, it was important to demonstrate that HIV-1 could infect cultured cells that were unequivocally nondividing and therefore not subject to mitotic nuclear envelope disassembly. Accordingly, challenges of MDMs whose low levels of proliferation (3941) were abrogated by y-irradiation (10) or immortalized cell lines that had been arrested in G2by irradiation (11) or at the GI-S boundary by treatment with the DNA polymerase (Y and 6 inhibitor aphidicolin (42,431 were each shown to be susceptible to productive infection by HIV-1. Building on earlier studies of MLV (31, researchers also showed that cells freshly infected with HIV-1 contain large nucleoprotein complexes, the PICs, that can mediate the in uitro integration of viral DNA into target DNA (4). To account for the ability of HIV-1 to infect non-dividing cells, it was hypothesized that HIV-1 PICs must have the potential to be transported across the nuclear envelope via the NPCs. Indeed, it was soon established that HIV-1 PICs are imported into the nucleus by an active process, and it was predicted that certain components of these complexes harbor NLSs (14). The identification and analysis of PIC NLSs has subsequently become an important area of HIV-1 research.

V. ISOLATION AND COMPOSITION OF HIV-1 PF~EINTEGRATION COMPLEXES Preintegration complexes have been defined as uncoated viral nucleoprotein complexes that can be isolated from infected cells and can mediate integration in uitro. However, for the purposes of this review, it is easier to use this term more loosely to describe postentry nucleoprotein complexes formed during the continuum that starts with penetrat-

FIG6.1 (Left). Pathways of protein nuclear import. The shuttling import receptors (blue) play a central role in nuclear import. In the cytoplasm, proteins containing classical basictype NLSs (green) interact with importin-p via the adapter importin-a(red), whereas nonbasictype NLSs (purple) bind directly to importin-plike receptors such as transportin. Following import, RanGTP binds to the receptors and induces dissociation from the respective substrates. The cycle is completed by receptor export to the cytoplasm and dissociation of Ran in its GDP-bound form. RanGAF', Ran GTPase activating protein.

FIG6.2 (Right). A model for the early steps of the HIV-1 life cycle. Virions bind and fuse to susceptible cells by the concerted action of the viral glycoproteins (gp120/4lEnV), CD4, and chemokine receptors. Preintegration complexes form in the cytoplasm, undergo reverse transcription, are transported through NPCs by the complementary action of multiple and diverse NLS-bearing proteins, and finally mediate provirus formation. Note that MA which is associated with these complexes is phosphorylated. See text for details.

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ing viral capsids, proceeds through the complexes that mediate reverse transcription, and finishes with the complexes that catalyze integration (Fig. 6.2). Indeed, many of the studies which have examined these assorted complexes have referred to them as PICs. A number of groups have analyzed the composition of HIV-1 PICs (refer to the article by F.D. Bushman, this volume). Because the ability to mediate viral DNA integration is often used to monitor PIC activity, these experiments were generally performed 4 or 5 h r postchallenge, namely, at a time when reverse transcription was completed. Extracts that are considered to be nuclear or predominantly cytoplasmic were then derived from such cells with the use of a variety of isotonic and hypotonic extraction protocols. Subsequent analyses involved additional purification steps followed by the examination of cofractionation between viral DNA and proteins or, alternatively, direct immunoprecipitation of complexes with the use of specific antibodies against viral proteins followed by the detection of viral DNA. Importantly, however, the results of these differing approaches are fairly uniform. In terms of HIV-1 PIC dimensions, the most extensively purified preparations have an estimated Stokes radius of -28 nm (44). The virion protein most stably associated with HIV-1 PICs appears to be IN itself (45). Reverse transcriptase, matrix (MA, ~ 1 7 ~ % and ) , the -15kDa accessory protein Vpr are also readily detected under a number of conditions (44,46-50), whereas nucleocapsid (NC, P ~ ~ W low ), amounts of capsid (CA, ~ 2 4 ~ ' )and even protease (PR) are detected only under more limited circumstances (44,47,49) (Fig. 6.2). Because Vpr is packaged into virions by virtue of its interaction with pGGag(51), it might be expected that PICs also contain ~ 6 ~Agnumber . of cellular proteins have been shown to be associated with PICs. These include importin-a (see below) (50,52); HMG I(Y) (531, which is important for PIC activity in uitro; and histones (47).

VI. NLSs IN HIV-1 PREINTEGRATION COMPLEX PF~OTEINS Three viral proteins-IN, Vpr, and MA-have been implicated as facilitators of HIV-1 PIC nuclear import (Fig. 6.3).The first of these to be assigned NLS activity was MA. In a report which served to galvanize this field, disruption of an amino-terminal basic region of MA (26GKKKYKLKH)that bears some sequence similarity to basic-type NLSs was shown to inhibit both virus infectivity and the accumulation of nuclear reverse transcription products (specifically,the 2-long terminal repeat circles that are considered to be formed only in the nucleus)

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FIG6.3. Provirus organizations and preintegration complex NLSs of HIV-1, MLV, ASV, and HFV. Open boxes, viral genes; solid boxes with highlighted lettering, proposed preintegration complex NLSs; gray boxes, long terminal repeats (LTRs).

in growth-arrested, but not in proliferating, T cells (42). Consistent with the hypothesis that this motif was an NLS that was important for infection of non-dividing cells but dispensable in dividing cells, cytoplasmic microinjection experiments showed that a conjugate between bovine serum albumin and a synthetic peptide spanning this region was specifically imported into the nucleus. A number of subsequent experiments corroborated these original findings. First, viruses that were similarly mutated in the MA basic domain (namely, 26KK-+TT)were found to be replication defective in macrophage cultures but not in rapidly dividing T cells (48,54,55). Second, a recombinant glutathione S-transferase-MA fusion protein was imported, albeit very inefficiently, into the nuclei of microinjected cells (50). Third, the cellular import factor importin-a was shown t o be associated with PICs and to be capable of interacting with MA in a manner that was dependent on the integrity of the basic domain (50,521.Taken together, these observations suggested a model for PIC import whereby MA provided the signal for engagement to the importin-dB nuclear import pathway.

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In contrast to these results, other reports have indicated that the basic region of MA is not directly involved in the nuclear import of PICs (56,571. First, a series of conventional nuclear import assays performed with fusion proteins, synthetic peptide conjugates, transfections, and microinjections were unable t o confirm that this region could function as an autonomous NLS (57). Second, using a primary macrophage tropic isolate of HIV-1, the extent of replication impairment resulting from the 26KK+TTmutation was similar in both nondividing and dividing cells-a finding which suggests that this mutation could be affecting an aspect ofreplication that is important in all cell types (57). Third, the %K+TT mutation was shown to reduce the rate of ~ 5 5 polyprotein ~" processing (57). Because proteolytic processing represents a crucial aspect ofretroviral maturation (11,it would be predicted that its perturbation could result in the formation of viral particles (and eventually PICs) with altered structures and characteristics (58). I t is possible, therefore, that at least one aspect of the phenotype of 26KK+TTviruses may be attributable to the formation of aberrant PICs following viral challenge. Should these be prone to premature dissociation, not only would viral infection be generically affected, but a reduction in nuclear import would also be an expected downstream consequence. Despite the uncertainties that remain concerning the presence of an NLS in MA, it is clear that MA is a component of HIV-1 PICs (44,46,47,49).Interestingly, this creates a situation whereby MA must harbor opposing targeting signals. During virus assembly and production, the MA segment of the ~ 5 5 ~and " g p160Gap-P0' polyproteins is targeted to the plasma membrane by virtue of a bipartite signal that comprises an amino-terminal myristate moiety and neighboring basic residues (59). On the other hand, following entry into susceptible cells, the association of mature MA with the uncoating capsids requires that MA disengage from the membrane. Although the specific details of how this occurs are controversial (40,55,60) and are likely to be complex, there is good evidence that phosphorylation together with a conformational switch that results in the myristrate being hidden from MA's surface (611, participates in the reversal of membrane binding. The 1% of MA that is present in virion cores and enters the nucleus as part of the PIC has been shown to be phosphorylated on tyrosine, serine, and threonine residues (55,60). Phosphorylation of the carboxy-terminal tyrosine residue (position 132) of MA is thought to facilitate incorporation into PICs by inducing binding to IN (491, whereas phosphorylation of multiple serine residues is thought to induce dissociation from the negatively charged membrane by electrostatic repulsion (60,62). Interestingly, a number of cellular tyrosine (55,631 and serinelthreonine

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kinases [including the mitogen-activated protein ( M A P ) kinase ERK21 (62,64) that can mediate the phosphorylation of MA have been detected in purified virions; it is also possible that the viral accessory protein Nef may assist in the recruitment of kinases into viral particles (65). Vpr was the second HIV-1 protein to have been implicated in PIC nuclear import. Mutant viruses which lack an intact upr gene replicate less well than wild-type viruses, especially in non-dividing cells such as MDMs, where decreases in replication of -10-fold can be observed (48,56,66-70). Initial reports suggested that Vpr and the MA basic domain (the IN NLS had not been discovered at that time; see below) might function independently to access distinct nuclear import pathways and that disruption of both signals was required to observe a substantial inhibition of PIC import (48). Consistent with this hypothesis, productive infection by a upr-deficient virus, but not by a wild-type virus, was shown to be inhibited by a truncated version of importin-a that had retained the ability t o interact with MA but could no longer bind to importin-/.3.This indicated either that Vpr utilizes an importina-independent nuclear import pathway (52) or that Vpr accesses an importin-a-dependent pathway but in a nonconventional way. Analyses of Vpr in the context of fusion proteins have demonstrated that it contains a transferable NLS (69,70). Although the NLS appears to reside within the amino-terminal70 amino acids, the lack of sequence similarity between this region and previously defined NLSs suggested that there might be novel aspects to the mechanism(s) of Vpr nuclear import. Studies showing that cellular proteins involved in nuclear transport can interact with Vpr appear to bear out this hypothesis (Fig. 6.4). First, Vpr interacts specifically with yeast and vertebrate nucleoporins in uiuo, in uitro, and in the yeast two-hybrid system (69,701; these interactions are probably responsible for the striking accumulation of certain Vpr fusion proteins at the NPCs (69,70). It should be noted that classical basic-type NLSs do not usually associate with nucleoporins or localize to the NPCs. Second, Vpr can, in fact, interact with importin-a (70,711 but at a binding site which appears to be different from that used by basic-type NLSs (72); this finding may help to explain why the nuclear import of Vpr-containing PICs was not inhibited by a truncated form of importin-a (52). In addition, Vpr can stabilize the binding of importin-a to MA in uitro and can modestly stimulate the nuclear import of a basic-type NLS (Tantigen NLS coupled to bovine serum albumin at low valency) in permeabilized cell assays (71).

NUCLEAR IMPORT OF THE HIV-1 PIC

BASIC-TYPE NLS importin-a

importin$ - +

285

vpr

J

FIG6.4. Interactions involved in HW-1 preintegrationcomplex nuclear import. This scheme aims to summarizethe interactionsbetween componentsof the HIV-1preintegration complex and the cellular nuclear import machinery that have been proposed to be important for efficient NPC-mediated import. MA appears in parentheses to reflect the uncertainties that exist concerning its direct participation in nuclear import. See text for discussion.

The third HIV-1 PIC component shown to harbor an NLS was IN (50). On fractionation of cells challenged with a triply mutated virus that lacked IN, the basic domain of MA and upr (explained above), PIC componentswere found to reside in the cytoplasm. In contrast, infection with a counterpart virus in which the IN region of pol was intact resulted in the nuclear accumulation of PIC components. Consistent with this result, a glutathione S-transferase-IN fusion protein was imported into the nucleus following cytoplasmic microinjection of somatic cells and was shown to interact with importin-a! in uiuo and in vitro. A region of IN that spans residues 186 to 219 and contains was two stretches of basic amino acids (lS6KRKand 211KELKQKQITK) identified as being critical for this interaction and has accordingly been designated as the NLS (50). With respect to the crystal structure of the IN core domain (731, the '86KRKmotif forms part of an exposed loop between (Y helices 5 and 6, whereas the structure of the region containing the carboxy-terminal motif has yet to be solved. In a complementary series of experiments using fusions of IN to pyruvate kinase and transfection-based assays, we have confirmed the presence of strong NLS activity in this region (74). Of note, it has yet to be proven that the IN NLS(s) directly mediates PIC nuclear import. In particular, because all mutations that disrupt NLS activity have, t o date, also been found to abrogate infection of all cell types (50,74,75), it has

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not been possible to show a selective replication defect for IN-mutant viruses in nondividing cells versus dividing cells. VII. MODELS FOR HIV-1 PREINTEGRATION COMPLEX NUCLEAR IMPORT Can all of the above information concerning multiple NLSs, interacting cellular proteins, and viral phenotypes be distilled into a model for NPC-mediated HIV-1 PIC nuclear import that could serve as a foundation for future analyses? In our view, current data indicate that IN provides the NLSs that are most important for the import of HIV1PICs (Figs. 6.2 and 6.4).At least two lines of evidence are consistent with this conclusion. First, IN clearly harbors an NLS (or NLSs) that mediates the efficient nuclear import of coupled marker proteins in conventional assay systems (50,741; in fact, using transfection-based analyses, we have found that the NLS of IN is markedly stronger than that of Vpr (74). Second, viruses which lack upr and/or carry the 26KK-+TTMA mutation are still able to replicate quite efficiently in nondividing cells, a finding which implies that the contributions of these sequences to such infections are nonessential and that the critical NLSs reside elsewhere. Indeed, in some targets, such as cell-cycle arrested HeLa cells or rat neurons, viruses with one or both of these mutations can be as infectious as wild-type viruses (11,50,76,77).Because IN readily forms a ternary complex with importin-a and $3, i t is presumed that these factors facilitate IN-mediated nuclear import. The finding that PIC import can be blocked in permeabilized cell assays by addition of importin-/3-specificantibodies is consistent with this import receptor playing an essential role in this process (71). Interestingly, the IN proteins of avian sarcoma virus (ASV, discussed below) (78) and the Saccharomyces cereuisiae retrotransposon Tyl (79,80) also contain NLSs. It has been suggested that the Tyl IN NLS is essential for transposition because it mediates the transport of cytoplasmically assembled nucleoprotein complexes across a nuclear envelope that is never subject to mitotic disassembly. In fact, we speculate that the obligate presence of IN proteins in PICs (or their equivalents) makes them logical choices as bearers of NLS activity. Because Vpr improves HIV-1 replication in many cell types, but most noticeably in nondividing macrophages, it is believed that Vpr increases the efficiency of PIC nuclear import (48,52,68-70). The demonstration that HIV-1 Vpr interacts with importin-a as well as with nucleoporins has provided insight into how this effect could be exerted (69-71). Specifically, it has been proposed that Vpr exploits its own distinct

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binding site on importin-a to augment PIC import by bridging PICbound importin-a, directly to the NPC. Although importin-/3 also binds to importin-a and nucleoporins (81-851, it seems improbable that Vpr actually functions as a bona fide transporter. In particular, it has not been determined whether Vpr can bind to RanGTP or whether it shuttles between the nucleus and the cytoplasm. In addition, it is possible that PIC-bound Vpr could interact with nucleoporins independent of the action of importin-a. Based on these considerations, we propose the following model for the complementary roles of IN and Vpr in the nuclear import of HIV1 PICs (Fig. 6.2). It seems most likely that IN acts as the principal mediator of importin-a, recruitment to the PIC. Once it has engaged the PIC, there appear to be at least two subsequent fates for importina. First, it could bind to its conventional target, importin-p, and target the PIC for import via the classical pathway. The fact that vpr-deficient viruses infect nondividing cells implies that this mechanism is sufficient to account for significant PIC nuclear import. Second, importina, could bind to Vpr and be directed to the NPC via an importin-pindependent process that is mediated by the ability of Vpr to interact with nucleoporins. Because we consider it as unlikely that Vpr functions as an import receptor, an interaction of this type would be expected to potentiate the activity of true receptors (importin-P)that are also bound to the PIC-perhaps by increasing the kinetics or affinity of interactions between PICs and NPCs. When considering cooperativity of this nature, it should also be remembered that PICs are presumed to contain multiple copies of each protein component. Thus, for a single PIC, some importin-a molecules could be directed t o the NPC via Vpr, whereas others, through binding to importin-& could be targeted for both docking and translocation. An additional layer of complexity arises when one considers the ability of Vpr to enhance the nuclear import of a basic-type NLS (71). Whether this effect is mediated through improved utilization of the importinCulp pathway, by “piggybacking”on Vpr that enters the nucleus, or via a novel mechanism remains to be established. Interestingly, the participation of multiple copies of different signals during PIC import is reminiscent of how multicomponent RNPs are thought to be exported out of the nucleus. Here it has been proposed that multiple export receptors must be bound to a single RNP for export to the cytoplasm to occur (17,861. A further analogy can be drawn from the nuclear import of the U1 small nuclear RNP; although it is much smaller than the HIV-1 PICs (main body diameter of -8 nm) (871, the efficient import of this ribonucleoprotein complex is also mediated by a composite NLS (88).

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With an estimated Stokes radius of -28 nm (44), HIV-1 PICs could be too large to pass through NPCs. Even though the purity and monomeric nature of these preparations have yet to be fully defined, current data tend to suggest that the composition of nuclear and cytoplasmic PICs may be similar; it therefore seems unlikely that nuclear translocation would be preceded by a major “adenovirus-like” disassembly step. Importantly, other structures whose dimensions would also appear to preclude transport through NPCs have, in fact, been found to be capable of doing so. For example, it has been proposed that SV40 capsids undergo conformational changes in the cytoplasm that facilitate nuclear import (22) and that the -50-nm Balbiani ring mRNPs of Chironomus partially unfold in the nucleus to allow export to the cytoplasm to occur (89). Similarly, it is conceivable that the conformation (but not the composition)of HIV-1 PICs may alter prior to (andor during) translocation through the NPC. Additional support for the importance of efficient HIV-1 PIC import has been gleaned from studies of viruses of the sooty mangabey-derived simian immunodeficiency virus (SIVsM )/HIV type-2 (HIV-2) primate immunodeficiency virus lineage. These viruses contain two related genes, upr and upx; studies have shown that Vpx functions during PIC import (go), whereas the SIVsM/HIV-2 Vpr protein carries out the other major activity of HIV-1 Vpr, namely, cell cycle arrest (90-93). Importantly, up-deficient viruses display a substantial loss of replicative capacity in both cultured macrophages (90) and challenged rhesus macaques (94). The future analysis of SIV Vpr and Vpx proteins should be helpful in determining which HIV-1 Vpr interacting proteins are relevant for which function. Where does this leave MA, the original PIC NLS? This remains a controversial and unresolved issue. Arguing against a direct role for the MA amino-terminal basic domain in PIC import is the finding that this region did not display NLS function in a variety of nuclear import assays (57). On the other hand, and in light of the influence of Vpr on the import of the NLS of T antigen (71), it should not be discounted that MA, in the presence of Vpr, may assist in enhancing PIC import. Of note, even though our MA-import experiments were carried out in the absence of Vpr (571, many were performed in a cell line, HeLa, which has been proposed as harboring a Vpr-complementing factor (71). It should also be emphasized that the MA basic domain influences steps of the virus life cycle that are independent of PIC import, namely, membrane binding and virion maturation (57,95,96).Nevertheless, understanding the postentry function(s) of MA remains an area of consid-

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erable interest. In particular, not only is MA present in PICs, but its phosphorylation has been shown to modulate infectivity (55,60,62). To summarize, we are just beginning to appreciate and understand the complexity of HIV-1 PIC nuclear import (and, more broadly, the whole uncoating process). For HIV-1, as well as for the three other viral systems discussed earlier (Table 6.11, it is apparent that efficient import of postentry nucleoprotein complexes involves cooperativity between multiple and diverse targeting signals. Even so, an obvious question that arises is: why do HIV-1 PICs harbor more than one type of NLS-particularly given the multivalent nature of its various components? The evolutionary answer lies, presumably, in the fact that NPCmediated nuclear import of PICs is critical for the establishment and progression of HIV-1 infections in uiuo and that the complementary action of multiple signals ensures that this can occur with the required efficiency. Although signal-mediated PIC nuclear import is essential for the productive infection of nondividing cell targets, it is expected to increase viral replication in proliferating cell populations because mitosis-independent provirus formation would also tend to accelerate the onset of the next round of virion synthesis.

VIII. SOMEFUTURE QUESTIONS Our appreciation of HIV-1 PICs remains rudimentary and therefore offers many opportunities for future analyses. Understanding all aspects of these complexes will not only be fascinating from the cell biology and virology standpoints, but should also be informative with respect to the development and implementation of lentivirus-based gene delivery systems (75). There appear to be at least three postentry phases that culminate in the delivery of HIV-1 PICs to the site of integration: transport from the site of penetration to the NPC, translocation through the NPC, and intranuclear transport to chromatin. Here we have discussed signals and factors important for the completion of nuclear uptake and have not, therefore, attempted to differentiate between trafficking to the pore and translocation itself. Our view is, however, that these steps should also be considered individually, and that they likely involve both common and distinct factors; for instance, Vpr may play a role in NPC docking but not in translocation. Given the high protein concentration of the cytoplasm and the effects of macromolecular crowding (971,it is possible that diffusion alone would be a highly inefficient and undesirable mechanism for transporting complexes as large as PICs to the NPCs. A potential solution to

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this problem would be for directionality and force to be applied through interactions with the cytoskeleton. This scenario would be consistent with the microtubule-assisted targeting of incoming adenovirus (98) and herpes simplex virus 1(99)capsids to the nuclear envelope. Because import receptors dissociate from their substrates following nuclear entry, directional trafficking within the nucleus (if i t occurs) may be mediated by alternative mechanisms. One area that deserves attention appears to be detailed biochemical and biophysical descriptions of HIV-1 PICs (refer to our earlier working definition of this term) at the various stages of infection. In particular, do these complexes “mature” as an infection proceeds (other than by the progressive synthesis of DNA) as has been suggested for MLV (loo), are any of the components modified, and what are the relative stoichiometries of various components during this process? Answering these questions might be important for understanding possible points of postentry regulation (61,100-102). Although IN and Vpr have been shown to contain bona fide NLSs, these sequences have yet t o be fully characterized. Because the ultimate goal is to understand PIC import, it will be important to define these sequences both in isolation and in the context of PICs. In particular, it would be very helpful if inactivating mutations could be identified in the IN NLS that do not impact on the other functions of IN. It is also expected that the use of the permeabilized cell system (1031, together with recombinant import factors and depleted extracts, will assist in the further definition of the import pathways that are used by these NLSs, although in experiments examining the import of complexes, the utility of this approach will be heavily dependent on the purity of PIC preparations. IX. RELATIONSHIPS BETWEEN ONCORETROVIRUS AND FOAMY VIRUSINFECTIONS AND THE CELLCYCLE In addition to lentiviruses, there are two other subfamilies of retroviruses: oncoretroviruses (formerly known as “RNA tumor viruses”) and foamy viruses. It is well established that both murine and avian oncoretroviruses require target cell proliferation for productive infection (6,38). To date, the underlying nature of this restriction has been investigated in detail only for MLV. Here an elegant series of experiments that utilized a variety of methods to synchronize cycling cells without affecting viral DNA synthesis demonstrated that both provirus formation and the nuclear accumulation of viral DNAs require passage

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through mitosis (5). It was therefore concluded that MLV PICs, unlike HIV-1 PICs, cannot traverse the nuclear envelope during interphase and that access to cellular chromatin occurs only following the temporary dissolution of the nuclear envelope during mitosis ( 5 ) . One interesting issue that arises from this model concerns the mechanism(s) by which MLV PICs are localized to the nucleoplasm followingreestablishment of the nuclear envelope. A prediction would be that interactions between PICs and nuclear factors are likely to be involved. In contrast to these ideas, one ultrastructural analysis of cells challenged with MLV has suggested that some PIC components, namely, NC and IN, can enter the nucleus via NPCs (104). However, the lack of information concerning the transport of accompanying viral nucleic acids and the apparent absence of measurable NLSs in components of these PICs (Fig. 6.3) render these studies difficult to evaluate. A number of observations made with ASV have suggested that the resistance of nondividing cells to productive oncoretrovirus infection may not be imparted by a uniform mechanism. Although one group has shown that aphidicolin treatment prevents provirus formation (1051, another has suggested that the integration of ASV DNA could occur prior to the onset of mitosis in cultures that had been synchronized and then challenged in S phase and stimulated to divide (106).Further interest in this area developed when it was shown that the IN protein of ASV harbors a transferable NLS (Fig. 6.3) (78). It has also been demonstrated that mutations which disrupt this element’s nuclear import activity do not appear to affect IN function adversely in uitro. Because these mutations do retard ASV replication in cultures of dividing cells, it has been proposed that the NLS of IN may enhance viral infectivity by stimulating PIC nuclear import during interphase (107). Future testing of this hypothesis using methodologies that have been applied to HIV-1 or MLV, and in particular the use of protocols which interfere with the cell cycle in the absence of serum deprivation, are awaited with interest. Should it turn out that ASV PICs are subject to NPC-mediated nuclear import, it will be intriguing to determine why productive ASV infection of noncycling cells does not occur. Whether productive foamy virus infections are dependent on cell division has yet t o be definitively established. One group has demonstrated that productive infection is inhibited in cells that are arrested either by irradiation or by aphidicolin treatment (108). In contrast, other groups have shown, first, that human foamy virus (HFV) can infect stationary phase fibroblasts more efficiently than MLV (although the infection efficiencies were still relatively low) (109);second, that HFV infection of aphidicolin-arrested cells results in the nuclear accu-

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mulation of viral DNA (2-LTR circles) (110); and, third, that foamy viruses infect nonproliferating cells of the central nervous system in uiuo (111).Taken together, it appears that foamy viruses may be able to infect nondividing cells to a limited degree and that their PICs may therefore have nuclear import capabilities. Consistent with this notion, the HFV Gag protein has been shown to contain an NLS in its NC region (Fig. 6.3) (112,113) and to traffic to the nucleus in a microtubule dependent manner soon after viral challenge (110). As with ASV, the future analysis of the composition and fate of PIC in defined culture systems should help to elucidate the relationship between foamy virus infections and the cell cycle.

x. SUMMARY Following infection-mediated entry into the cytoplasm, retroviral cores form large nucleoprotein complexes (PICs)which undergo reverse transcription and, ultimately, catalyze provirus formation. The ability of these complexes to be specifically imported into the nucleus via NPCs explains why nondividing cells can be productively infected with lentiviruses such as HIV-1, whereas productive infection by the oncoretrovirus MLV is restricted to proliferating cells. Current evidence suggests that virally encoded protein components of the HW-1 PIC, in particular IN and Vpr, act in concert to target these complexes for nuclear import by recruiting cellular import factors and interacting with the NPCs. Here have we reviewed recent advances made in this complex and fascinating area of HIV-1 biology and have discussed them in relation to models for postentry nuclear import in other retroviral and nonretroviral systems.

ACKNOWLEDGMENTS We thank Vicki Pollard and James Simon for many helpful discussions, Anna Marie Skalka and Mario Stevenson for sharing unpublished results, and Laurie Zimmerman for outstanding secretarial assistance.

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