Regulation of viral transcription elongation and termination during vaccinia virus infection

Biochimica et Biophysica Acta 1577 (2002) 325 – 336 www.bba-direct.com

Review

Regulation of viral transcription elongation and termination during vaccinia virus infection Richard C. Condit a,*, Edward G. Niles b a

Department of Molecular Genetics and Microbiology, P.O. Box 100266, University of Florida, Gainesville, FL 32610, USA b Department of Microbiology, 138 Farber Hall, SUNY School of Medicine, 3435 Main St., Buffalo, NY 14214-3000, USA Received 21 June 2002; accepted 21 June 2002

Abstract Vaccinia virus provides a useful genetic and biochemical tool for studies of the basic mechanisms of eukaryotic transcription. Vaccinia genes are transcribed in three successive gene classes during infection, early, intermediate, and late. Vaccinia transcription is regulated primarily by virus gene products not only during initiation, but also during elongation and termination. The factors and mechanisms regulating early elongation and termination differ from those regulating intermediate and late gene expression. Control of transcription elongation and termination in vaccinia virus bears some similarity to the same process in other prokaryotic and eukaryotic systems, yet features some novel mechanisms as well. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Poxvirus; Vaccinia virus; Transcription elongation; Transcription termination; RNA polymerase; DNA helicase; Poly(A) polymerase; mRNA cap methyltransferase; Nucleoside phosphodrolase

1. Introduction Vaccinia virus, the prototypic poxvirus, is a doublestranded DNA containing virus that replicates exclusively in the cytoplasm of infected cells. The cytoplasmic site of replication requires that the virus encodes all of the machinery necessary for synthesis of mRNA suitable for translation in a eukaryotic environment. Virus-coded enzymes required for synthesis and modification of the earliest class of viral RNA are packaged in mature virions. Therefore, because an entire transcriptional program is encoded in a relatively small ( f 200 genes) viral genome, and because significant portions of the mRNA synthetic machinery can be partially purified simply from isolated virions, vaccinia has long served as a model system for studies of fundamental aspects of eukaryotic transcription and mRNA modification. Over the past 15 years, it has it has become clear that during both the early and late transcriptional phases of vaccinia virus infection, both elongation and termination are regulated by virus gene products. This has offered a unique opportunity to understand the regulation of post*

Corresponding author. Tel.: +1-352-392-3128; fax: +1-352-392-3133. E-mail address: [email protected] (R.C. Condit).

initiation events in transcription in a simple eukaryotic system using both biochemical and genetic approaches. This article reviews the history and status of research in transcription elongation and termination in vaccinia virus. To establish an appropriate context, we first summarize relevant aspects of vaccinia virus biology. Then, because the early (pre-replicative) and late (postreplicative) phases of vaccinia transcription are distinctly different, we review each of these replication phases separately. 1.1. Vaccinia virus biology and history Vaccinia is the prototypic member of the virus family Poxviridae [95]. The most notorious of the poxviruses is variola, the cause of smallpox, a devastating disease until its eradication in 1979 [44]. There exist two subfamilies, seven genera, and dozens of species of poxviruses infecting a variety of host species including mammals, birds, and insects. Members of the family Poxviridae are characterized by a large dsDNA genome, a complex virion morphology, and a cytoplasmic site of replication. Vaccinia was used as a live vaccine in the successful two-century battle to eradicate smallpox. The precise origins of vaccinia are somewhat obscure. It is related at least historically to an agent isolated from a lesion on the hand of a milkmaid in 1796 during the

0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 4 6 1 - X

326

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

first vaccination experiments. However, the virus can no longer be found in nature, except in the context of escaped vaccine [34]. Nevertheless, vaccinia has the important properties of being serologically cross-reactive, and therefore protective, against smallpox while at the same time causing only limited pathology in humans. Vaccinia has an extraordinarily rich scientific history [95]. The concept of vaccination was pioneered with vaccinia, hence its name (the term vaccinia is derived from vacca, meaning ‘‘cow’’ in Latin). Vaccinia was the first virus to be visualized with a light microscope and the first virus to be purified. Vaccinia was the first virus characterized as containing an active RNA polymerase, and studies of the vaccinia virion transcription system played a central role in characterization of both polyadenylation and mRNA capping. More recently, interest in vaccinia and other poxviruses has centered on their utility as recombinant vaccines and tools for heterologous protein expression. Furthermore, vaccinia possesses a rich cache of genes designed to combat host immune defenses against virus infection, providing novel insights into the host response to virus infection. Most relevant to this review are studies of transcriptional regulation of gene expression during vaccinia infection. 1.2. Vaccinia genome structure and organization The vaccinia genome is a 200-kb, linear, double-stranded DNA molecule with 10-kb inverted terminal repeats and covalently closed ends [95]. The genome is packaged in a distinct core structure contained within a brick-shaped, enveloped virion particle. The genome has been sequenced and contains approximately 200 genes [51]. Roughly 25 virus-coded proteins have known or presumed roles in viral transcription and mRNA processing. Vaccinia genes lack introns, and each gene contains its own transcriptional promoter. Most genes within each terminal third of the genome are oriented such that transcription proceeds outward towards the termini of the genome, that is, leftward on the left end and rightward on the right end. In the middle third of the genome, genes are interspersed in either transcriptional orientation. There is no obvious logic to the distribution of genes on the genome with respect to function, however, genes often seem to be arranged in a fashion that would minimize transcriptional interference. Specifically, an early transcription termination signal for a given gene may often be embedded in an adjacent late gene where it will be ignored, thus economizing on genome usage. Furthermore, genes often seem to be arranged in a fashion that minimizes divergent and convergent transcription.

transcribed in three temporal classes, early, intermediate, and late. Each gene class is characterized functionally by distinct promoters and cognate trans-acting factors, mostly virus-coded. The factors required for initiation of transcription of each gene class are encoded primarily by genes of the preceding gene class, and thus the regulation can be described as a ‘‘cascade’’. Promoters in each gene class are relatively simple, comprising only about 30 base pairs of sequence upstream of the initiating nucleotide. Early, intermediate and late gene promoters differ in their sequence yet each contains a consensus core centered at about 12 to 15 [8,35,36] and an initiator region. Presumably, class-specific initiation proteins recognize each promoter contributing to the temporal specificity. Although different promoters within each gene class may have different intrinsic quantitative behavior, the levels of expression of a given gene are otherwise not regulated in response to external factors, and thus the virus does not encode enhancers, upstream activating elements, or specific cognate regulatory trans-acting transcription factors. Nascent transcripts are all capped at the 5Vend and polyadenylated at the 3Vend using virus-coded enzymes, but none of the mRNAs is spliced, consistent with the cytoplasmic site of synthesis. Viral RNA synthesis is carried out by a virus coded, multi-subunit, eukaryotic-like RNA polymerase [10,95,132]. RNA polymerase subunits are synthesized throughout infection and the assembled RNA polymerase is packaged into nascent virions late in infection. The RNA polymerase exists in two different forms, one specific for early genes and one specific for late genes. Both forms of the RNA polymerase have in common eight subunits, ranging in size from 147 to 7 kDa; Table 1. The largest two subunits

Table 1 Virus-coded RNA polymerase subunitsa Subunit

Gene

Molecular weight (kDa)

Comment

RPO 147 RPO 132

J6R A24R

147 132

nuclear pol homologue nuclear pol homologue, IBT resistance

RPO 35 RPO 30 RPO 22 RPO 19 RPO 18 RPO 7 RAP94

A29R E4L J4R A5R D7R G5.5R H4L

a

1.3. Transcriptional regulation of vaccinia gene expression: an overview Gene expression during vaccinia infection is regulated primarily at the level of transcription [95,96]. Genes are

35 30 22 19 18 7 93.5

TFIIS homologue

nuclear pol homologue virion RNA polymerase, early gene initiation and termination co-factor, binds to NPH I

Nomenclature: Vaccinia genes are identified by their location in the genome, including the letter of the HindIII restriction fragment, the number of the gene from one end of the fragment, and the transcriptional orientation. Thus J6R is the 6th gene from the left end of the HindIII J restriction fragment, and is transcribed in a rightward orientation. In many (but not all) cases, once a function is described for a gene, the gene is given another designation, in this case for example RPO 147 or RAP94.

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

share significant homology to the two largest subunits of eukaryotic and prokaryotic RNA polymerases. Only two of the remaining six subunits have any significant homology with other non-eukaryotic enzymes including, interestingly, a limited homology between a 30-kDa RNA polymerase subunit (called RPO 30) and the eukaryotic transcription elongation factor TFIIS [1,22]. The vaccinia RNA polymerase does not possess the regulatory C-terminal domain found in cellular eukaryotic RNA polymerase II. The early gene-specific form of the RNA polymerase contains one additional 94 kDa subunit (called RAP94, the product of gene H4L), which is required for recognition of early promoters but stays tightly bound to the enzyme during transcription; Table 1 [5,40,66]. The intermediate and late gene form of the RNA polymerase lacks RAP94 and contains only the eight common subunits described above. Importantly, the early RAP94-containing form of the RNA polymerase can initiate transcription only on early promoters and cannot initiate transcription on late (and presumably intermediate) promoters, while the enzyme lacking RAP94 can initiate transcription only on late (and presumably intermediate) promoters and not on early promoters [2,5,40,138]. Thus, an RNA polymerase in the process of elongation retains a memory of the promoter class at which it initiated transcription. This concept has important consequences for distinctions between elongation and termination of early as compared to postreplicative genes. Transcription of early genes and modification of early RNAs are carried out entirely by enzymes that are packaged in virions and are thus present in partially uncoated virus core particles in the cytoplasm immediately following uptake of the infecting virus into the cell. In vitro, purified virus particles permeabilized with neutral detergent and incubated with nucleoside triphosphates will produce authentic fully modified early mRNA. Most of the enzymes involved in this process can be solubilized and isolated from purified virions. Early transcription is catalyzed by the early gene-specific, RAP94-containing form of the multi-subunit viral RNA polymerase described above. Initiation of early gene transcription also requires the Vaccinia Early Transcription Factor, VETF, Table 2 [23,145], composed of two subunits, the products of genes D6R and A7L [18,48]. VETF binds to the promoter exhibiting contacts in both the core and the initiator region [19,25]. Bound VETF permits RAP94-containing RNA polymerase to assemble a stable preinitiation complex (PIC) at an early promoter [56,82]. Initiation ensues yielding an elongating ternary complex. The mRNA cap is built on the nascent transcript by multiple virus-encoded enzymes [43,133,135]. The viral mRNA capping enzyme, which consists of two subunits, the products of genes D1R and D12L [94,99], catalyzes the first three steps in cap formation to yield a cap 0 structure. Cap I is synthesized from cap 0 by the virus-coded (nucleoside-2V-O-)-methyltransferase, the product of gene J3R [117]. Whereas

327

Table 2 Virus-coded trans-acting class specific transcription factors Factora

Classb

Gene

Comment

VETF

early

D6R, A7L

VTF

early

D1R, D12L

NPH I

early

D11L

VITF1

intermediate

E4L

promoter binding initiation factor, ATPase termination factor, mRNA capping enzyme, intermediate gene transcription factor elongation, termination, release factor, binds to H4L TFIIS, Rpo 30 homologue

VITF3 Capping enzyme J3

intermediate intermediate

A8R, A23R D1R, D12L

intermediate/late

J3R

A18

intermediate/late

A18R

G2

intermediate/late

G2R

VLTF1 VLTF2 VLTF3 VLTF4

late late late late

G8R A1L A2L H5R

same as VTF elongation factor, poly A polymerase processivity factor, 2V-O-methyltransferase termination, release factor, DNA helicase, ATPase elongation factor, binds to H5R binds to VLTF2 binds to VLTF1 binds to G2R

a

Some factors have not been given function related names, for example A18. In these cases, the protein has been designated with the gene name, without the L or R designating transcriptional orientation. b Designates the stage of infection in which the factor has activity.

RAP94 is an integral subunit of the early form of RNA polymerase and remains a component of the elongating enzyme [40], the capping enzyme is dissociable from the elongation complex, in vitro. Stable association of capping enzyme to the transcription elongation complex is not observed until the nascent RNA reaches 51 nucleotides in length [53]. These observations impact on the roles of RAP94 and capping enzyme in early gene transcription termination. Early transcription termination, a subject of this review and therefore described in detail later, is a sequence-specific event requiring several viral factors including the D1/D12 heterodimeric capping enzyme. Nascent mRNAs are polyadenylated by a heterodimeric poly(A) polymerase, encoded by genes E1L and J3R [47]; the latter subunit is identical to the cap (nucleoside-2V-O-)methyltransferase described above. Early RNAs are extruded from virus cores in an energy-dependent fashion [67]. Virions contain several additional enzymes that have activities possibly relevant to mRNA synthesis, for example an RNA helicase and a topoisomerase, but clear roles for these enzymes in early transcription have not been described [95]. Early genes encode enzymes required for DNA replication and for initiation of intermediate gene transcription, and thus following early gene expression, the stage is set for viral DNA replication.

328

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

Intermediate and late gene transcription have several features in common, which distinguish them from early gene transcription. First, transcription of both intermediate and late gene classes is prevented by inhibitors of DNA replication, either drugs or virus mutants, and thus the intermediate and late gene classes can be referred to collectively as ‘‘postreplicative’’. Second, during initiation of transcription, RNA polymerase slippage at a common TAAA element present in the initiator region of both intermediate and late promoters results in addition of 30 to 50 residues of non-templated adenylate residues at the mRNA 5Vend, referred to as a ‘‘ poly(A) head’’ [4,9,15,105,119]. (Slippage and poly(A) head formation is also seen in early genes that have TAAAT in the initiator region, e.g., D7R [3,65].) Third, intermediate and late mRNAs are extremely heterogeneous at their 3Vends, thus transcription from a given promoter gives rise to a family of 5V coterminal transcripts ranging in size from 2 to 5 kb [33,86]. Lastly, as detailed in this review, intermediate and late gene transcription elongation and termination seem to be controlled by a common set of virus-coded factors. Despite these similarities, intermediate and late genes are distinct with respect to promoters and initiation factors. Transcription of intermediate genes requires five virus early gene products; Table 2. These Vaccinia Intermediate Transcription Factors include VITF 1, which is identical to the TFIIS homologous 30-kDa RNA polymerase subunit RPO 30, VITF 2, which is a heterodimer of the A8 and A23 gene products, and the D1/D12 heterodimeric capping enzyme [58,113,116,134]. A cellular factor, VITF 2, has also been identified [114]. Intermediate genes encode a variety of functions involved in late transcription, immune defense, and virus morphogenesis. Initiation of late gene transcription requires at least four viral gene products called Vaccinia Late Transcription Factors; Table 2. Three of these factors, VLTF 1 (gene G8R), VLTF2 (gene A1L), and VLTF 3 (gene A2L), are encoded by intermediate genes while one, VLTF 4 (gene H5), is an early gene product [24,64,74,75,78,104,137,139,148]. In addition, one or more host proteins are employed in late gene transcription [20,52,140,149]. Late gene products include RNA polymerase, early gene transcription factors, poly(A) polymerase, and additional functions involved in virion morphogenesis and immune defense. Postreplicative mRNAs are capped and polyadenylated, presumably by the same enzymes that modify early mRNAs. The precise mechanism of action of intermediate and late gene initiation factors is incompletely understood.

can be directed to form a paused ternary complex, which can be isolated [56,82]. Ternary complexes are relatively stable to salt and detergent treatment and are fully functional in vitro, permitting their characterization. The active RNA polymerase in the ternary complex protects 41 bases of template DNA, 24 bases 3Vto the catalytic site and 17 bases 5V[55]. Mapping analyses indicate that the transcription bubble encompasses about 17 bases including a template/ product hybrid of approximately 10 base pairs. An 18-base segment of the nascent RNA is protected from nuclease digestion in the ternary complex [54]. The vaccinia virus RNA polymerase possesses a 3VRNA hydrolytic activity, which can be observed in gel purified ternary complexes [57]. Cleavage is stimulated by divalent cations and CTP. However, cleavage does not require pyrophosphate, demonstrating that the mechanism does not employ a reversal of the elongation reaction. The resulting product is capable of being extended in the presence of four nucleoside triphosphates, demonstrating both that the RNA product is retained in the ternary complex and that it possesses a 3Vhydroxyl group. This hydrolytic activity is similar to those exhibited by both the bacterial and nuclear RNA polymerases with subtle differences in cleavage sites (see Fish and Kane, this volume). It is not clear which RNA polymerase subunit is required for hydrolysis or whether additional RNA polymerase accessory factors are employed. However, RPO 30 exhibits sequence homology to SII, a protein shown to be involved in RNA pol II catalyzed hydrolysis [1]. Whether RPO 30 is required for RNA hydrolysis remains to be determined. Only one putative early transcription elongation factor has been identified to date, the nucleoside triphosphate phosphohydrolase I (NPH I). NPH I, the product of gene D11L [21,112], is a single-stranded-DNA-dependent ATPase that is packaged into nascent virions; Table 2 [102]. As described below, NPH I, a component of the elongation complex, provides the energy required to dissemble the transcription termination complex [27,41]. In addition, evidence indicates that NPH I is required to stimulate elongation through an oligo T stretch at low UTP levels [41], indicating that NPH I might also serve as an early gene transcription elongation factor. However, it is clear that NPH I is not essential since elongation proceeds normally in vitro in the absence of NPH I [27]. Further studies will be required to determine the role of NPH I in elongation. Additional early gene transcription elongation factors remain to be identified, yet since in vitro studies to date have employed impure RNA polymerase, their existence has not been disproved.

2. Early gene transcription elongation and termination

2.2. Cis-acting termination sequences

2.1. Elongation

A cis-acting early gene specific termination sequence has been identified. Sequence analysis of cDNA derived from early mRNA permitted the identification of one or more stretches of the sequence TTTTTNT, which was derived

Relatively little is known about the details of early gene transcription elongation. The elongating RNA polymerase

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

from UUUUUNU in the UTR of the nascent early mRNA [146]. Mutagenesis studies showed that TTTTTNT was required for early gene transcription termination, in vitro [147]. Further analyses demonstrated that the relevant signal is UUUUUNU in the nascent mRNA [127,128]. UUUUUNU must reside in cis and must be at least 30 nucleotides from the 3Vend of the nascent mRNA [38,53], indicating that it serves as a recognition sequence required for termination. UUUUUNU is likely to bind to an essential transcription termination factor but so far, the identity of the binding factor has eluded detection. Analyses of the relationship between transcription elongation rate and transcription termination efficiency in vitro showed that the rate of elongation is inversely proportional to the extent of termination observed [53]. Furthermore, termination site selection is also related to the concentration of ATP with UUUUUNU proximal 3Vends predominating at elevated ATP levels. Logically, the choice of termination site is kinetically coupled to the rate of RNA polymerase movement on the template. 2.3. Trans-acting termination factors VTF, the vaccinia virus early gene transcription termination factor, was isolated from virion extracts [125]. VTF is a heterodimeric protein containing the D1R and D12L subunits and is identical to the viral mRNA capping enzyme; Table 2. Both subunits are required for transcription termination, in vitro [84]. However, the loss of cap formation activities has no affect on transcription termination factor activity demonstrating that cap formation is not linked to transcription termination [84,144]. The specific function of VTF in termination remains to be defined. Since VTF is an RNA binding protein, which exhibits a modest preference for U-rich sequences [85], it is suspected to be the UUUUUNU binding factor. However, since it binds RNA at multiple sites as the mRNA capping enzyme [60,98], specific recognition of UUUUUNU has not been demonstrated. VTF interacts with RNA polymerase [54] but the site of interaction is not known. It is active in termination when added at any time during the synthesis of the nascent mRNA. In addition, it can be added to isolated transcription ternary complexes to stimulate transcript release [83]. Transcription termination requires ATP hydrolysis [53,126]. Since VTF possesses ATPase activity in the mRNA triphosphatase active site [97,129], VTF was initially suspected to be the ATPase employed in termination. Mutation of the ATPase in VTF [144] demonstrated that an additional enzyme provided the energy employed in transcription termination. Subsequent in vitro complementation analyses demonstrated that the ATPase employed in termination is NPH I [27,41]. NPH I is the single-stranded-DNAdependent ATPase described above, which has been implicated in early gene transcription elongation. RNA does not stimulate the NPH I ATPase activity and competitively

329

inhibits single-stranded-DNA activation, demonstrating that RNA and DNA bind to the same site on NPH I [26]. Singlestranded-DNA binding shifts NPH I from an inactive state to an active conformation as measured both by changes in protease sensitivity and CD spectrum upon binding to single-stranded DNA (Hinckley and Niles, unpublished). Since single-stranded DNA is required to induce the active conformation of NPH I, and NPH I is required for termination, NPH I must be able to access single-stranded DNA at the termination site. NPH I added at any time during transcription or to the isolated ternary complex will support transcript release [41,92]. NPH I possesses motifs that belong to Superfamily II helicases, indicating that it might provide such a function in termination. Extensive studies have failed to reveal such a helicase activity in vitro. Perhaps NPH I cooperates with one or more additional proteins to generate an active helicase. Mutagenesis studies demonstrate that each helicase motif is essential for ATPase activity and for transcription termination in vitro [27,87]. In addition, each mutation exhibits a dominant negative phenotype in vitro [27], indicating that NPH I interacts with one or more essential termination factors. Deletion of as few as 28 amino acids from the C-terminal end of NPH I yields an enzyme that retains normal single-stranded-DNA binding and ATPase activity, yet fails to stimulate transcription termination in vitro. In addition, these short deletion mutations do not act as dominant negatives in vitro, indicating that these mutants fail to bind to the additional termination factors. It appears that the C-terminal end of NPH I is required for a step in the termination pathway other than simply binding DNA and providing energy. Several candidate viral proteins were tested for their ability to bind to the C-terminal region of NPH I. Importantly, the RAP94 subunit of the virion RNA polymerase exhibited strong binding to NPH I [92]. Mutations in NPH I that failed to support transcription termination or transcript release from an isolated ternary complex in vitro also failed to bind to RAP94, providing strong evidence that the interaction between NPH I and RAP94 is essential for transcription termination. Additional studies were conducted to directly evaluate the requirement for the Nterminal end of RAP94 in termination [93]. Polyclonal antibodies raised against RAP94 amino acids 1 –256 were shown to specifically inhibit termination in vitro, while antibodies raised against the C-terminal 2/3 of RAP94 had no effect on termination. Antibody inhibition was blocked by preincubation of the antibody with RAP94 fragments containing amino acids 1– 99 [90]. Further mapping studies demonstrated that the strong epitopes recognized by this polyclonal antibody reside in the first 99 amino acids of RAP94. Importantly, antibody inhibition was observed only when the antibody was added in the absence of NPH I. Additional studies showed that if NPH I was added first, the inhibition due to antibody binding was dramatically reduced, indicating that the antibody and NPH I are in

330

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

competition. If NPH I is bound first, the antibody does not inhibit NPH I action. It is clear that UUUUUNU-dependent transcription termination is restricted to early genes. At intermediate and late times in infection, RNA polymerase elongates through the TTTTTNT sequence without terminating. Only the form of RNA polymerase that recognizes early promoters is capable of terminating in response to TTTTTNT [30], indicating that recognition resides in the RNA polymerase itself. The RNA polymerase that is active in early gene transcription harbors the unique RAP94 subunit. The essential interaction between the C-terminal end of NPH I and the N-terminal end of RAP94 provides an adequate explanation for the restriction of signal-dependent termination to early genes. 2.4. A model for early termination Any model that attempts to describe the mechanism of early gene transcription termination must account for the following observations; Fig. 1. (1) The sequence UUUUUNU in the nascent mRNA must be in cis and be at least 30 nucleotides 5Vto the 3Vend. This location upstream from the 3Vend moves the sequence beyond the surface of the RNA polymerase in the ternary complex, permitting access to the signal. Single base substitutions result in reduced termination, consistent with UUUUUNU serving as a binding site for a required termination factor. (2) VTF, the viral mRNA capping enzyme, is essential. Although VTF was the first factor identified, the role for VTF in termination remains undefined. However, it is attractive to speculate that VTF recognizes and binds to the UUUUUNU sequence in the nascent mRNA. (3) NPH I, a single-stranded-DNA-dependent ATPase activity, must be present. NPH I provides the energy employed in the dissolution of the ternary complex. Since single-strandedDNA binding is required for ATPase activity, NPH I must have access to single-stranded DNA in the termination complex. The displaced noncoding strand in the transcrip-

Fig. 1. Early gene transcription termination. See text for details.

tion bubble is a likely source for the required singlestranded DNA. This begs the question as to why the ATPase is not always active if NPH I resides on the elongation complex. Perhaps the oligonucleotide binding site on NPH I is occupied by the nascent mRNA during elongation and the mRNA is removed from the site at termination, providing access to the single-stranded DNA and activation of NPH I. Binding of a protein to the UUUUUNU sequence in the nascent RNA might function to strip the blocking RNA from NPH I. (4) RNA polymerase must possess the RAP94 subunit. Minimally, the N-terminal end of RAP94 serves as a docking site for NPH I. However, RAP94 may very well carry on an active role in termination. Indeed, antibodies that prevent transcription termination also prevent active PIC formation [90]. Unlike transcription termination, antibody inhibition of PIC formation is not affected by the presence or absence of NPH I, demonstrating that the inhibitory antibody and NPH I can bind simultaneously to the N-terminal region of RAP94. This means that the antiRAP94 antibody inhibits NPH I function at a step downstream from NPH I binding to RAP94.

3. Postreplicative gene transcription elongation and termination 3.1. Cis-acting sequences? As described in the Introduction, postreplicative vaccinia virus mRNAs possess unique features distinct from early mRNAs; specifically, they are 5Vpolyadenylated and extremely heterogeneous at the 3Vends. Importantly, despite these unusual features, the 5Vpoly(A) heads are capped, and the 3Vends are polyadenylated. Evidence to be reviewed below strongly suggests that some, if not all, of these heterogeneous 3Vends are formed directly by transcription termination and are otherwise unprocessed before polyadenylation, though this concept currently lacks formal direct proof. The 3Vend heterogeneity of postreplicative mRNAs carries important implications for post-initiation events in their synthesis. First, it is apparent that the early termination cis-acting sequence, UUUUUNU, is not recognized by postreplicative transcription complexes [30]. In fact, many late genes contain one or more UUUUUNU sequences that are clearly ignored by transcribing RNA polymerase. Given the discussion presented above concerning early gene termination, the read-through of early termination sequences by postreplicative transcription complexes can be explained by the fact that RNA polymerase, which initiates at late promoters, lacks RAP94. Second, the 3Vend heterogeneity implies that any sequence which might be required for termination of postreplicative gene transcription must occur frequently and thus be either simple or degenerate, and it must be inefficient, since the polymerase must read through a given sequence with high frequency. In any case, no effect

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

of sequence on postreplicative gene transcription termination in vivo or in vitro has yet been described. 3.2. Trans-acting factors To date, three viral gene products have been identified as regulators of postreplicative vaccinia transcription elongation; Table 2. These are A18, a negative transcription elongation factor in vivo with DNA helicase and transcript release activity in vitro, and G2 and J3, both positive transcription elongation factors in vivo. One additional gene product, H5, is implicated in postreplicative elongation or termination by virtue of its physical association with G2. In addition, the antipoxviral drug isatin-h-thiosemicarbazone (IBT) promotes read-through transcription from intermediate (and presumably late) gene promoters, and has played a critical role in identifying postreplicative elongation factors. Lastly, the viral RNA polymerase itself is implicated in transcription elongation through mapping of an IBT-resistant mutation to one of its subunits. Each of these players is discussed separately in the following sections. 3.2.1. A18 Phenotypic analysis of A18R mutant infections shows that the A18R gene product behaves like a negative transcription elongation factor in vivo. As described above, during a wt virus infection, postreplicative mRNA 3Vends are extremely heterogeneous; nevertheless, the size of postreplicative mRNAs is limited to 2– 5 kb. RT-PCR analysis reveals that during infection with mutants defective in A18 function, read-through transcription occurs, generating a family of longer than normal transcripts, albeit still heterogeneous at the 3Vends [143]. Elucidation of this primary effect of A18R mutants was obscured for years because of several pleiotropic effects resulting from the read-through transcription. First, read-through transcription caused what was initially termed promiscuous or aberrant transcription, that is, some early genes that were normally silent late during wt virus infections became transcriptionally active late during A18R mutant infection [11]. Not all early genes became transcriptionally active, demonstrating that promiscuous transcription was not a result of either global reactivation of early promoters or generally random transcription of the genome [143]. In fact, genes that displayed promiscuous transcription were all positioned downstream from postreplicative genes, and genes that did not display promiscuous transcription had no postreplicative genes within 5– 10 kb in an upstream direction, consistent with the conclusion that promiscuous transcription represents read-through from postreplicative gene promoters. Second, analysis of RNA metabolism in A18R mutant virus-infected cells revealed that while early viral mRNA synthesis and structure were unaffected and RNA polymerization activity could still be detected late during infection, late mRNA and cellular rRNA were degraded [11,100]. Subsequent studies demonstrated that the observed RNA degradation resulted

331

from activation of the cellular ‘‘2 – 5A pathway’’, a cascade of events that is induced by dsRNA and results ultimately in activation of an otherwise latent ribonuclease, RNAse L [28]. Activation of the 2 –5A pathway probably results from elevated synthesis of dsRNA via read-through transcription from converging postreplicative promoters, which would result in an increase in the length of 3Voverlapping dsRNA hybrids [11]. Finally, activation the 2 – 5A pathway and global degradation of cellular mRNA and rRNA caused an abrupt cessation of viral protein synthesis late during an A18 mutant infection, termed an ‘‘abortive late’’ phenotype [100]. It is not clear why the elongation defect that exists during an A18R mutant infection ultimately inhibits virus growth, however, it is clear that induction of the 2 –5A pathway alone is not responsible. Two aspects of the A18R mutant phenotype are relevant to this issue. First, A18R mutants are defective in growth on cells that lack RNAse L, and neither rRNA nor mRNA breakdown occurs during A18R mutant infection of these cells [143]. On RNAse L-cells, Northern blot analysis confirms an increase in size of intermediate transcripts late during A18R mutant infection. In fact, A18R mutants are defective in growth on cells that lack both the RNAse L pathway, and the PKR pathway, the only other dsRNA-activated antiviral pathway known [141]. Second, on all cells tested, late gene transcription is diminished at late times in A18R mutant infections [141,143]. These results might suggest that the elongation defect in A18R mutants impacts directly on viral transcription, perhaps through interference with late transcription, or that A18R has some distinct direct involvement with initiation of late gene expression. However, it is also possible that the accumulation of dsRNA alone causes abortion of the infection through induction of apoptosis [76]. Biochemical studies on the A18 protein progressed concurrently with phenotypic analysis of A18R mutants. The A18R gene is transcribed at both early and late times during infection, and Western blot and immunoprecipitation analysis confirms that the 56 kDa protein is synthesized throughout infection [101,130]. The protein is packaged in mature virions, but a role for the protein in the early phase of infection has not been determined. Analysis of the A18R sequence reveals the presence of helicase motifs, and purified A18 protein expressed in bacteria possesses both DNA-dependent ATPase activity and DNA helicase activity [12,77,131]. The ATPase activity uses either rATP or dATP as a substrate, and is stimulated by ssDNA, dsDNA, and DNA/RNA hybrids, but not dsRNA. The helicase activity is weak in that it can melt only short dsDNA hybrids, 20 bp or less in length, it proceeds in a 3V-to-5Vdirection, and it is active only on dsDNA hybrids, not dsRNA or RNA/DNA hybrids. Thus, the A18 protein looks like a motor, a protein that binds to single-stranded DNA and moves in a 3V– 5V direction using ATP as fuel. A refined biochemical role for the A18 protein was revealed through the use of an in vitro transcription elonga-

332

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

tion, RNA release assay [80]. In vitro, release of nascent RNA from an elongation complex is ATP-dependent and requires both A18 and additional host factor(s). The accessory host factor(s) has so far eluded purification. The factor(s) is abundant in both the nucleus and cytoplasm of HeLa cells, it is heat-labile, and it binds to cation exchange resins but not phosphocellulose. However, it exhibits an extremely broad elution profile from a cation exchange column, suggesting a heterogeneous or highly modified substance [79]. 3.2.2. IBT The antipoxviral drug IBT induces an abortive late phenotype, which in all respects is identical to infection with A18R mutant virus [11,28,32,71,100,109,136]. Thus, IBT either stimulates elongation or inhibits termination of postreplicative gene transcription. IBT is highly specific for poxviruses, and displays little if any cytotoxicity on uninfected eukaryotic cells. Structurally, it bears provocative similarity to a purine nucleoside, but its precise mechanism of action is unknown [45,122]. Early work demonstrated that vaccinia mutants could be isolated, which were either dependent on or resistant to IBT [68 – 70,72,73]. This observation provides an opportunity to either identify the target of IBT action or additional virus genes involved in transcription elongation, specifically by mapping IBT resistant or dependent virus mutants. As described below, this approach uncovered two positive transcription elongation factors and implicated the second largest subunit of the RNA polymerase itself in the control of transcription elongation. 3.2.3. G2 The vaccinia gene G2R was first implicated in transcription elongation through mapping of an IBT-dependent mutant, IBTd-1 [89]. DNA sequence analysis revealed that IBTd-1 is a frameshift mutation in G2R, suggesting that IBT dependence results from inactivation of G2, a concept confirmed by the construction of three engineered IBTdependent deletions in the G2R gene. Interestingly, the preexisting temperature-sensitive mutant, Cts56, also maps to gene G2R, and displays an IBT-resistant phenotype at the permissive temperature and an IBT-dependent phenotype at the nonpermissive temperature. Several additional clustercharged to alanine scanning mutants of G2R were isolated, and these display a range of phenotypes between IBT resistance and IBT dependence [59]. The general conclusion from these mutants is that G2R is an essential gene and that growth of mutants lacking G2R function can be rescued by addition of IBT, hence the IBT-dependent phenotype. G2R mutants that are IBT-resistant probably encode a G2 protein that is crippled but not completely inactive; thus, resistance can be thought of as a phenotype intermediate between the wt, sensitive phenotype and the null, dependent phenotype. The fact that mutation of G2R alters sensitivity to IBT, combined with the similarity between the A18R mutant phenotype and effect of IBT on cellular RNA suggests a

connection between A18R and G2R, which is ultimately borne out by genetic experiments [31]. Specifically, logic dictates that, because IBT rescues growth of a G2R null mutant, and because the effects of IBT and A18R mutation are phenotypically identical, then mutation of A18R should also rescue growth of a G2R null mutant. This hypothesis was proven true by construction of a recombinant between a G2R deletion mutant and a temperature-sensitive mutant in A18R, which proved to be viable at high temperature in the absence of IBT, conditions nonpermissive for either of the two mutants alone. Thus, G2R mutants serve as extragenic suppressors of A18R mutants, or vice versa. Extrapolating on this observation, ts+ phenotypic revertants of A18R ts mutants were isolated and shown to be the result of extragenic suppression due to mutation of the G2R gene to an IBT-dependent phenotype. These genetic experiments suggest that A18 and G2 function in the same biochemical pathway. Phenotypic characterization of G2R mutants clarifies the relationship between the G2R and A18R genes [16]. Protein pulse labeling experiments reveal that, under nonpermissive conditions, mutations in the G2R gene result specifically in reduced synthesis of large late viral proteins, while all early proteins and small late proteins are synthesized in normal amounts. Northern blot analysis demonstrates that during G2R mutant infections, early mRNAs are synthesized in normal amounts and retain a normal structure, but intermediate and late mRNAs, while synthesized in normal amounts, are reduced in size, truncated specifically at their 3Vends. (The 2– 5A pathway is not induced in these mutant virus infections, and there is no evidence for mRNA degradation.) The simplest interpretation of these results, consistent with all other observations, is that G2 is a positive transcription elongation factor, and that when G2 is absent, elongation of postreplicative RNAs is impeded. Though still sufficiently long to encode small proteins, 3Vtruncated mRNAs are too short to encode large proteins, hence the diminished synthesis of large late proteins observed in G2R mutant infections. G2R mutants can be rescued by either treatment with IBT or by mutation of gene A18R because both of these treatments promote transcription elongation, thus compensating for the G2R defect. The 26-kDa G2 protein is synthesized throughout infection, it is not packaged in virions, and it shares no significant homology or functional motifs with any known protein. Attempts to demonstrate an elongation activity for the G2 protein in an in vitro elongation assay have so far been unsuccessful. 3.2.4. J3 Isolation of additional IBT dependent mutants and A18R extragenic suppressors revealed that gene J3R had genetic properties virtually identical to G2R [81,142]. Ts+ phenotypic revertants of an A18R mutant were screened for the presence of suppressor mutations by crossing the A18R phenotypic revertants with wt virus and screening for the appearance of IBT dependent virus, which theoretically

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

could arise by segregation of an IBT dependent suppressor mutation from the A18R ts allele. Any IBT-dependent mutants isolated from this test cross could be mapped by marker rescue, and one such mutant, J3x, proved ultimately to be a single-amino-acid substitution in the J3R gene. Knowing that mutation of J3R could result in IBT dependence, nine additional IBT-dependent vaccinia mutants were isolated in an attempt to reinforce a role for J3 in elongation and/or identify additional genes involved in elongation. Two of the nine mutants mapped to gene G2R, and seven proved to be null mutations of gene J3R. Thus, J3 behaves genetically like a functional homolog of G2, and the results to date suggest that there are no other vaccinia genes that have these genetic characteristics. Characterization of the J3R mutants reveals, as expected, a phenotype identical to G2R mutants with respect to transcription elongation, i.e., synthesis of 3Vtruncated intermediate and late viral mRNAs. J3 thus behaves like a positive transcription elongation factor in vivo. Interestingly, J3 possesses two additional previously characterized activities, one involved with 5Vcap formation and one involved with poly(A) tail formation [46]. The 39kDa J3 protein is synthesized throughout infection and is packaged in virions. The protein can be isolated from either infected cells or virions in two forms, as a monomer and as a heterodimer complexed with the 55-kDa E1L gene product [47]. Both the J3 monomer and the J3/E1 heterodimer possess (nucleoside-2V-O-)-methyltransferase activity, which converts a cap-0 structure to a cap-1 structure by methylation of the 2Vhydroxyl group of the first transcribed base in a cap-0 RNA [117]. The J3/E1 heterodimer is the viral poly(A) polymerase [47]. The catalytic site for polyadenylation resides in the E1 subunit, but is limited to addition of approximately 35 adenylate residues to the RNA 3Vend [49]. The J3 subunit confers processivity on the polymerase, such that the heterodimer is capable of synthesis of poly(A) tails 100 –150 nt in length [50]. Structural analysis of the J3 protein by X-ray crystallography combined with biochemical analysis of in vitro synthesized J3 mutant proteins reveals that the methyltransferase and poly(A) stimulation activities are genetically separable and occupy distinct surfaces on opposite sides of an oblate sphere [39,61,62,118,123,124]. Recent experiments have shown that the J3 elongation activity is distinct from the methyltransferase and poly(A) stimulation activities [150]. Specifically, several of the preexisting in vitro synthesized J3R mutations have been recombined into virus and assayed for all three activities in vivo. These experiments show that the A18R-suppressing J3x elongation mutant described above is active in both poly(A) stimulatory and methyltransferase activities in vivo, and that mutants which are specifically defective in either methyltransferase or poly(A) stimulatory activity are normal with respect to transcription elongation. Several of the mutants tested in these experiments had an IBT-resistant phenotype, and the locations of these mutations on the

333

surface of the J3 protein suggest the outline of an elongation domain. Interestingly, the methyltransferase and poly(A) stimulatory mutants of the J3 protein are viable, suggesting that the only activity of this trifunctional protein that is absolutely essential in cell culture is the elongation activity. Attempts so far to demonstrate an elongation activity for the J3 protein in an in vitro elongation assay have been unsuccessful. 3.2.5. H5 The vaccinia H5R gene product is implicated in transcription elongation through a direct interaction with the G2 protein. When poly-histidine-tagged G2 is overexpressed using a vaccinia vector and purified by affinity chromatography on a nickel column, the H5 protein co-purifies with the G2 protein [17]. The H5 – G2 interaction was confirmed by two independent investigations in yeast 2 hybrid analysis [17,88]. The H5 gene product is an abundant 22-kDa phosphoprotein which is expressed throughout infection [14,115]. In infected cells, the protein localizes to ‘‘virosomes’’, cytoplasmic sites of active viral DNA replication and virion morphogenesis [13]. H5 has been identified biochemically as a stimulatory factor for in vitro transcription of late viral genes[78], however, ts mutants in H5R do not have a transcription phenotype [37]. Instead, H5R ts mutants are defective in virus morphogenesis, and this defect is characterized by accumulation of large dense inclusions within virosomes and an absence of viral membrane precursors [37]. If there is a common denominator to these observations, it is that the H5 protein may interact with transcribing or replicating viral DNA, a speculation not inconsistent with a presumed role in elongation. 3.2.6. A24 One IBT resistance mutation has been mapped to gene A24R, which encodes the second largest subunit of the viral RNA polymerase [29]. This finding is provocative for two reasons. First, this subunit of the polymerase is thought to play a role in nucleotide binding [110], and so the structural similarity between IBT and nucleoside makes A24 an interesting candidate as the primary target for IBT action. Second, mutation of the second largest subunit of yeast pol II or pol III can affect both elongation kinetics and termination, thus reinforcing the finding that mutation of A24R can compensate for the effects of a drug which promotes transcription read-through [111,120,121]. 3.2.7. Other genes? Recent experiments suggest that vaccinia may possess other genes which affect postreplicative transcription elongation (Cresawn and Condit, unpublished). Specifically, initial sequence analysis of the A24R, A18R, G2R, and J3R genes in 10 new IBT resistance mutations has shown that none contains mutations in either J3R or A18R, four contain mutations in G2R alone, one contains mutations in both G2R and A24R, and five do not contain mutations in

334

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

any of these four genes. Mapping of these latter five mutants may uncover additional vaccinia transcription elongation factors. 3.2.8. A postreplicative transcription elongation complex? Possible additional interactions between G2, A18, and H5 have been investigated [17]. As described above, G2 and H5 interact directly. While no direct interaction has been observed between A18 and H5 or G2 and A18, immunoprecipitation of A18 from an infected cell extract coimmunoprecipitates G2, and immunoprecipitation of H5 from an infected cell extract co-immunoprecipitates both G2 and A18. These results suggest that all three proteins may be components of a larger complex; an obvious candidate would be an elongation complex. The interaction of J3 with other postreplicative elongation factors has not been investigated; however, J3 interacts with both RAP94 and NPH I, suggesting that J3 may be a component of an early gene transcription elongation complex [91]. All of the genetic and phenotypic data are consistent with the hypothesis that J3 is a component of a postreplicative elongation complex: mutation of J3 might prevent formation of an elongation complex and thus impair elongation. 3.2.9. Posttranscriptional cleavage of late RNAs: a special case? Although the vast majority of postreplicative RNAs possess heterogeneous 3Vends, four genes have been identified which direct synthesis of mRNAs with defined, homogeneous 3Vends [6,103,105 –108] (D’Costa and Condit, unpublished). The mechanism of 3Vend formation has been investigated for two of these genes, and has been shown in both cases, the cowpox ATI gene [7,63] and F17R (D’Costa and Condit, unpublished), to result from posttranscriptional cleavage directed by virus-induced cleavage factor(s). The required cis-acting sequences for cleavage have been deciphered for the cowpox ATI gene transcripts, but the sequence bears no apparent similarity to sequence flanking the F17R cleavage site. The presumption has been that posttranscriptional cleavage of these RNAs represents a special case involving a minority of viral postreplicative transcripts; however, a broader role for cleavage in formation of postreplicative mRNA 3Vends has not been ruled out. These results are especially interesting in light of recent experiments that suggest coupling of RNA cleavage and transcription termination in metazoan cells [42]. 3.2.10. A model for postreplicative transcription elongation and termination Any model that describes postreplicative transcription elongation and termination in vaccinia virus must take into account the following facts: (1) postreplicative mRNA 3V ends are heterogeneous in length; (2) A18 is a DNA helicase that behaves like a negative transcription elongation factor in vivo and a transcript release factor in vitro; (3) G2 and J3

Fig. 2. Intermediate gene elongation complex. HF = host factor(s) required for A18-mediated RNA release. IBT = a hypothetical IBT binding site on the RNA polymerase. See text for additional details.

behave like positive transcription elongation factors in vivo; (4) G2 binds to H5; (5) G2 and A18 undergo indirect interactions; (6) one IBT resistance mutation maps to A24R, the second largest subunit of the RNA polymerase (Fig. 2). All of the factors involved are likely to be complexed with elongating RNA polymerase at least transiently. A18 is most likely a transcription termination factor. Its helicase activity suggests that it functions by binding DNA and moving. Like D11 in early gene transcription termination, the most likely site of action for A18 is a stretch of non-template strand DNA exposed on the surface of transcribing RNA polymerase. A18 could bind this DNA and translocate, destabilizing the transcription complex and causing termination. G2 and J3 are most likely either pause suppressors or antiterminators; pause suppression might be mediated by an interaction with RNA polymerase, while antitermination might be mediated by an interaction with A18. Alternatively, factors such as J3 may play a passive role in elongation: J3 could have evolved as an integral structural component of an elongation complex for the purpose of maintaining proximity between the J3 methyltransferase and polyadenylation activities and nascent RNA, so that alteration of J3 compromises the integrity of the complex and indirectly inhibits elongation.

4. Summary and conclusions Vaccinia virus provides an ideal model system for investigation of the basic mechanisms of mRNA synthesis and modification, using a combination of genetic and biochemical approaches. Investigations summarized here have shown that during vaccinia virus infection, prereplicative and postreplicative transcription elongation and termination occur by distinct mechanisms, and each is controlled by virus coded gene products. The reactions bear similarity to processes described in both prokaryotic and eukaryotic systems, including the involvement of

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

helicases in both early and late vaccinia virus termination, the utilization of a specific cis-acting termination sequence in prereplicative transcription termination, and the absence of a well defined termination sequence in postreplicative transcription termination. Analysis of some of the virus gene products which control vaccinia elongation and termination reveals an economy of function and implies a coupling of events in mRNA biogenesis. For example, the D1/D12 heterodimer serves not only as an mRNA capping enzyme, but also as an early gene termination factor and intermediate gene initiation factor; the H5 protein both stimulates late transcription in vitro and binds the elongation factor G2; and the J3 protein participates in mRNA capping, transcription elongation, and polyadenylation. Continuing research on vaccinia transcription will likely uncover additional factors regulating post-initiation events in transcription, and will also provide mechanistic insight into these processes. References [1] B.Y. Ahn, P.D. Gershon, E.V. Jones, B. Moss, Mol. Cell. Biol. 10 (1990) 5433. [2] B.Y. Ahn, P.D. Gershon, B. Moss, J. Biol. Chem. 269 (1994) 7552. [3] B.Y. Ahn, E.V. Jones, B. Moss, J. Virol. 64 (1990) 3019. [4] B.Y. Ahn, B. Moss, J. Virol. 63 (1989) 226. [5] B.Y. Ahn, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 3536. [6] B.Y. Amegadzie, J.R. Sisler, B. Moss, Virology 186 (1992) 777. [7] J.B. Antczak, D.D. Patel, C.A. Ray, B.S. Ink, D.J. Pickup, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 12033. [8] C.J. Baldick Jr., J.G. Keck, B. Moss, J. Virol. 66 (1992) 4710. [9] C.J. Baldick Jr., B. Moss, J. Virol. 67 (1993) 3515. [10] B.M. Baroudy, B. Moss, J. Biol. Chem. 255 (1980) 4372. [11] C.D. Bayliss, R.C. Condit, Virology 194 (1993) 254. [12] C.D. Bayliss, R.C. Condit, J. Biol. Chem. 270 (1995) 1550. [13] G. Beaud, R. Beaud, J. Gen. Virol. 78 (Pt. 12) (1997) 3297. [14] G. Beaud, R. Beaud, D.P. Leader, J. Virol. 69 (1995) 1819. [15] C. Bertholet, E. Van Meir, B. Heggeler-Bordier, R. Wittek, Cell 50 (1987) 153. [16] E.P. Black, R.C. Condit, J. Virol. 70 (1996) 47. [17] E.P. Black, N. Moussatche, R.C. Condit, Virology 245 (1998) 313. [18] S.S. Broyles, B.S. Fesler, J. Virol. 64 (1990) 1523. [19] S.S. Broyles, J. Li, J. Virol. 67 (1993) 5677. [20] S.S. Broyles, X. Liu, M. Zhu, M. Kremer, J. Biol. Chem. 274 (1999) 35662. [21] S.S. Broyles, B. Moss, J. Virol. 61 (1987) 1738. [22] S.S. Broyles, M.J. Pennington, J. Virol. 64 (1990) 5376. [23] S.S. Broyles, L. Yuen, S. Shuman, B. Moss, J. Biol. Chem. 263 (1988) 10754. [24] M.S. Carpenter, A.M. DeLange, Virology 188 (1992) 233. [25] M.A. Cassetti, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 7540. [26] L.A. Christen, M. Sanders, E.G. Niles, Biochemistry 38 (1999) 8072. [27] L.M. Christen, M. Sanders, C. Wiler, E.G. Niles, Virology 245 (1998) 360. [28] R.J. Cohrs, R.C. Condit, R.F. Pacha, C.L. Thompson, O.K. Sharma, J. Virol. 63 (1989) 948. [29] R.C. Condit, R. Easterly, R.F. Pacha, Z. Fathi, R.J. Meis, Virology 185 (1991) 857. [30] R.C. Condit, J.I. Lewis, M. Quinn, L.M. Christen, E.G. Niles, Virology 218 (1996) 169.

[31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44]

[45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78]

335

R.C. Condit, Y. Xiang, J.I. Lewis, Virology 220 (1996) 10. J.A. Cooper, B. Moss, E. Katz, Virology 96 (1979) 381. J.A. Cooper, R. Wittek, B. Moss, J. Virol. 39 (1981) 733. C.R. Damaso, J.J. Esposito, R.C. Condit, N. Moussatche, Virology 277 (2000) 439. A.J. Davison, B. Moss, J. Mol. Biol. 210 (1989) 749. A.J. Davison, B. Moss, J. Mol. Biol. 210 (1989) 771. J. Demasi, P. Traktman, J. Virol. 74 (2000) 2393. L. Deng, J. Hagler, S. Shuman, J. Biol. Chem. 271 (1996) 19556. L. Deng, L. Johnson, J.M. Neveu, S. Hardin, S.M. Wang, W.S. Lane, P.D. Gershon, J. Mol. Biol. 285 (1999) 1417. L. Deng, S. Shuman, J. Biol. Chem. 269 (1994) 14323. L. Deng, S. Shuman, Genes Dev. 12 (1998) 538. M.J. Dye, N.J. Proudfoot, Cell 105 (2001) 669. M.J. Ensinger, S.A. Martin, E. Paoletti, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 2525. J.J. Esposito, F. Fenner, in: D.M. Knipe, P.M. Howley (Eds.), Fields Virology, Lippincott, Williams & Wilkins, Philadelphia, 2001, pp. 2885 – 2922. A. Ferranti, L. Garuti, G. Giovanninetti, E. Katz, Arch. Pharm. (Weinh.) 318 (1985) 415. P.D. Gershon, Semin. Virol. 8 (1998) 343. P.D. Gershon, B.Y. Ahn, M. Garfield, B. Moss, Cell 66 (1991) 1269. P.D. Gershon, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 4401. P.D. Gershon, B. Moss, Genes Dev. 6 (1992) 1575. P.D. Gershon, B. Moss, J. Biol. Chem. 268 (1993) 2203. S.J. Goebel, G.P. Johnson, M.E. Perkus, S.W. Davis, J.P. Winslow, E. Paoletti, Virology 179 (1990) 247. S.K. Gunasinghe, A.E. Hubbs, C.F. Wright, J. Biol. Chem. 273 (1998) 27524. J. Hagler, Y. Luo, S. Shuman, J. Biol. Chem. 269 (1994) 10050. J. Hagler, S. Shuman, Science 255 (1992) 983. J. Hagler, S. Shuman, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 10099. J. Hagler, S. Shuman, J. Virol. 66 (1992) 2982. J. Hagler, S. Shuman, J. Biol. Chem. 268 (1993) 2166. N. Harris, R. Rosales, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 2860. D.E. Hassett, R.C. Condit, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4554. M.A. Higman, L.A. Christen, E.G. Niles, J. Biol. Chem. 269 (1994) 14974. A.E. Hodel, P.D. Gershon, F.A. Quiocho, Mol. Cell 1 (1998) 443. A.E. Hodel, P.D. Gershon, X. Shi, F.A. Quiocho, Cell 85 (1996) 247. S.T. Howard, C.A. Ray, D.D. Patel, J.B. Antczak, D.J. Pickup, Virology 255 (1999) 190. A.E. Hubbs, C.F. Wright, J. Virol. 70 (1996) 327. B.S. Ink, D.J. Pickup, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 1536. E.M. Kane, S. Shuman, J. Virol. 67 (1993) 2689. J. Kates, J. Beeson, J. Mol. Biol. 50 (1970) 1. E. Katz, Rev. Clin. Basic Pharmacol. 6 (1987) 119. E. Katz, E. Margalith, B. Winer, J. Gen. Virol. 21 (1973) 477. E. Katz, E. Margalith, B. Winer, J. Gen. Virol. 25 (1974) 239. E. Katz, E. Margalith, B. Winer, N. Goldblum, Antimicrob. Agents Chemother. 4 (1973) 44. E. Katz, E. Margalith, B. Winer, A. Lazar, J. Gen. Virol. 21 (1973) 469. E. Katz, B. Winer, E. Margalith, N. Goldblum, J. Gen. Virol. 19 (1973) 161. J.G. Keck , C.J. Baldick Jr., B. Moss, Cell 61 (1990) 801. J.G. Keck, G.R. Kovacs, B. Moss, J. Virol. 67 (1993) 5740. K.V. Kibler, T. Shors, K.B. Perkins, C.C. Zeman, M.P. Banaszak, J. Biesterfeldt, J.O. Langland, B.L. Jacobs, J. Virol. 71 (1997) 1992. E.V. Koonin, T.G. Senkevich, J. Gen. Virol. 73 (Pt. 4) (1992) 989. G.R. Kovacs, B. Moss, J. Virol. 70 (1996) 6796.

336

R.C. Condit, E.G. Niles / Biochimica et Biophysica Acta 1577 (2002) 325–336

[79] C.A. Lackner, PhD thesis (2000). [80] C.A. Lackner, R.C. Condit, J. Biol. Chem. 275 (2000) 1485. [81] D.R. Latner, Y. Xiang, J.I. Lewis, J. Condit, R.C. Condit, Virology 269 (2000) 345. [82] J. Li, S.S. Broyles, J. Biol. Chem. 268 (1993) 2773. [83] Y. Luo, J. Hagler, S. Shuman, J. Biol. Chem. 266 (1991) 13303. [84] Y. Luo, X. Mao, L. Deng, P. Cong, S. Shuman, J. Virol. 69 (1995) 3852. [85] Y. Luo, S. Shuman, J. Biol. Chem. 268 (1993) 21253. [86] A. Mahr, B.E. Roberts, J. Virol. 49 (1984) 510. [87] A. Martins, C.H. Gross, S. Shuman, J. Virol. 73 (1999) 1302. [88] S. McCraith, T. Holtzman, B. Moss, S. Fields, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4879. [89] R.J. Meis, R.C. Condit, Virology 182 (1991) 442. [90] M.R. Mohamed, L. Christen, E.G. Niles, Virology (2002), (in press). [91] M.R. Mohamed, D.R. Latner, R.C. Condit, E.G. Niles, Virology 280 (2001) 143. [92] M.R. Mohamed, E.G. Niles, J. Biol. Chem. 275 (2000) 25798. [93] M.R. Mohamed, E.G. Niles, J. Biol. Chem. 276 (2001) 20758. [94] J.R. Morgan, L.K. Cohen, B.E. Roberts, J. Virol. 52 (1984) 206. [95] B. Moss, in: D.M. Knipe, P.M. Howley (Eds.), Fields Virology, Lippincott, Williams & Wilkins, Philadelphia, 2001, pp. 2849 – 2884. [96] B. Moss, B.Y. Ahn, B. Amegadzie, P.D. Gershon, J.G. Keck, J. Biol. Chem. 266 (1991) 1355. [97] J.R. Myette, E.G. Niles, J. Biol. Chem. 271 (1996) 11945. [98] J.R. Myette, E.G. Niles, J. Biol. Chem. 271 (1996) 11936. [99] E.G. Niles, G.J. Lee-Chen, S. Shuman, B. Moss, S.S. Broyles, Virology 172 (1989) 513. [100] R.F. Pacha, R.C. Condit, J. Virol. 56 (1985) 395. [101] R.F. Pacha, R.J. Meis, R.C. Condit, J. Virol. 64 (1990) 3853. [102] E. Paoletti, B. Moss, J. Biol. Chem. 249 (1974) 3281. [103] B.L. Parsons, D.J. Pickup, Virology 175 (1990) 69. [104] A.L. Passarelli, G.R. Kovacs, B. Moss, J. Virol. 70 (1996) 4444. [105] D.D. Patel, D.J. Pickup, EMBO J. 6 (1987) 3787. [106] D.D. Patel, D.J. Pickup, J. Virol. 63 (1989) 1076. [107] D.D. Patel, D.J. Pickup, W.K. Joklik, Virology 149 (1986) 174. [108] D.D. Patel, C.A. Ray, R.P. Drucker, D.J. Pickup, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 9431. [109] T.H. Pennington, J. Gen. Virol. 35 (1977) 567. [110] A. Polyakov, V. Nikiforov, A. Goldfarb, FEBS Lett. 444 (1999) 189. [111] W. Powell, D. Reines, J. Biol. Chem. 271 (1996) 6866. [112] J.F. Rodriguez, J.S. Kahn, M. Esteban, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 9566. [113] R. Rosales, N. Harris, B.Y. Ahn, B. Moss, J. Biol. Chem. 269 (1994) 14260. [114] R. Rosales, G. Sutter, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 3794. [115] J.L. Rosel, P.L. Earl, J.P. Weir, B. Moss, J. Virol. 60 (1986) 436. [116] P. Sanz, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 2692.

[117] B.S. Schnierle, P.D. Gershon, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 2897. [118] B.S. Schnierle, P.D. Gershon, B. Moss, J. Biol. Chem. 269 (1994) 20700. [119] B. Schwer, P. Visca, J.C. Vos, H.G. Stunnenberg, Cell 50 (1987) 163. [120] S.A. Shaaban, E.V. Bobkova, D.M. Chudzik, B.D. Hall, Mol. Cell. Biol. 16 (1996) 6468. [121] S.A. Shaaban, B.M. Krupp, B.D. Hall, Mol. Cell. Biol. 15 (1995) 1467. [122] R.W. Sheffield, D.J. Bauer, S. Stephenson, Br. J. Exp. Pathol. 41 (1960) 638. [123] X. Shi, T.G. Bernhardt, S.M. Wang, P.D. Gershon, J. Biol. Chem. 272 (1997) 23292. [124] X. Shi, P. Yao, T. Jose, P. Gershon, RNA 2 (1996) 88. [125] S. Shuman, S.S. Broyles, B. Moss, J. Biol. Chem. 262 (1987) 12372. [126] S. Shuman, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 7478. [127] S. Shuman, B. Moss, J. Biol. Chem. 263 (1988) 6220. [128] S. Shuman, B. Moss, J. Biol. Chem. 264 (1989) 21356. [129] S. Shuman, M. Surks, H. Furneaux, J. Hurwitz, J. Biol. Chem. 255 (1980) 11588. [130] D.A. Simpson, R.C. Condit, J. Virol. 68 (1994) 3642. [131] D.A. Simpson, R.C. Condit, J. Virol. 69 (1995) 6131. [132] E. Spencer, S. Shuman, J. Hurwitz, J. Biol. Chem. 255 (1980) 5388. [133] S. Venkatesan, A. Gershowitz, B. Moss, J. Biol. Chem. 255 (1980) 903. [134] J.C. Vos, M. Sasker, H.G. Stunnenberg, EMBO J. 10 (1991) 2553. [135] C.M. Wei, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 3014. [136] B. Woodson, W.K. Joklik, Proc. Natl. Acad. Sci. U. S. A. 54 (1965) 946. [137] C.F. Wright, A.M. Coroneos, J. Virol. 67 (1993) 7264. [138] C.F. Wright, A.M. Coroneos, J. Virol. 69 (1995) 2602. [139] C.F. Wright, J.G. Keck, M.M. Tsai, B. Moss, J. Virol. 65 (1991) 3715. [140] C.F. Wright, B.W. Oswald, S. Dellis, J. Biol. Chem. 276 (2001) 40680. [141] Y. Xiang, PhD thesis (1998). [142] Y. Xiang, D.R. Latner, E.G. Niles, R.C. Condit, Virology 269 (2000) 356. [143] Y. Xiang, D.A. Simpson, J. Spiegel, A. Zhou, R.H. Silverman, R.C. Condit, J. Virol. 72 (1998) 7012. [144] L. Yu, S. Shuman, J. Virol. 70 (1996) 6162. [145] L. Yuen, A.J. Davison, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 6069. [146] L. Yuen, B. Moss, J. Virol. 60 (1986) 320. [147] L. Yuen, B. Moss, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 6417. [148] Y. Zhang, J.G. Keck, B. Moss, J. Virol. 66 (1992) 6470. [149] M. Zhu, T. Moore, S.S. Broyles, J. Virol. 72 (1998) 3893. [150] D.R. Latner, J.M. Thompson, P.D. Gershon, C. Starrs, R.C. Condit, Virology (2002), (in press).