Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153, USA
Correspondence
Steven Broyles
broyles{at}purdue.edu
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ABSTRACT |
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Published ahead of print on 28 May 2003 as DOI 10.1099/vir.0.18942-0
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INTRODUCTION |
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Temporal regulation of gene expression
The majority of information on the regulation of gene expression in poxviruses has come from studies on the laboratory prototype poxvirus, vaccinia virus. There is ample reason to regard vaccinia virus as a model for transcriptional regulation in all poxviruses. Transcriptional mechanisms appear to be conserved across the entire family of poxviruses. The RNA polymerase and transcription factor genes have been found in all poxviruses for which genome sequences are available, which includes representatives from all poxvirus genera except entomopoxvirus C. Transcription factors and promoter function appear to be conserved, since promoters from one type of poxvirus are functional in a cell infected with a different poxvirus (Kumar & Boyle, 1990; Tripathy & Wittek, 1990
). Like most other classes of virus, poxviruses coordinate the processes of genome replication and virion assembly through regulation of the timing of expression of individual genes. Proteins participating in DNA replication (Jones & Moss, 1984
; Lee-Chen & Niles, 1988
; Smith et al., 1989a
), nucleotide biosynthesis (Hruby & Ball, 1982
; Smith et al., 1989b
) and intermediate gene transcription (Jones et al., 1987
; Lee-Chen & Niles, 1988
; Ahn et al., 1990
; Broyles & Pennington, 1990
; Sanz & Moss, 1999
) are synthesized as early class genes, and those participating in virion morphogenesis and assembly tend to be expressed as post-replicative intermediate and late class gene products (Rosel & Moss, 1985
). Apparently, it is advantageous to accumulate many copies of the genome before any virus assembly is to proceed. Proteins involved in the evasion of host defences tend also to be early class gene products (Kotwal et al., 1989
; Moore & Smith, 1992
; Ng et al., 2001
). The control of gene expression is exerted at the level of transcription initiation and occurs through a cascade mechanism. The transcription factors required for intermediate genes are expressed as early proteins, factors required for late genes are intermediate gene products and those required for transcription of early genes are late gene products packaged inside progeny virions for use in the next cycle of infection. One early vaccinia virus promoter was shown to reactivate late in the infectious cycle (Garces et al., 1993
). The significance of reactivation of early promoters is unclear. It should be noted that a number of vaccinia virus genes have been described as being continuously transcribed throughout the infectious cycle. Usually this is accomplished by a tandem arrangement of early and intermediate or late promoters preceding the open reading frame (for examples, see Wittek et al., 1980
; Broyles & Moss, 1986
; Ahn et al., 1990
; Broyles & Pennington, 1990
).
Virtually all viruses, whether containing DNA or RNA genomes, couple the switch from early to late gene expression to genome replication, and vaccinia virus is no exception. Inhibition of DNA synthesis, either with chemical inhibitors or with conditional lethal mutations that block DNA replication, results in the persistence of early gene transcription and the inhibition of intermediate (Vos & Stunnenberg, 1988) and subsequent late gene transcription. With no DNA synthesis, no transcriptional switch occurs. Curiously, intermediate promoters when transfected into a virus-infected cell override the block by inhibition of DNA synthesis and actually continue to be transcribed at levels higher than when DNA synthesis is allowed to proceed normally. The continued transcription of transfected intermediate genes in the absence of DNA synthesis presumably occurs because the onset of late transcription does not occur and this must somehow limit intermediate transcription under normal conditions. The resistance of transfected intermediate promoters to the inhibition of DNA synthesis has been attributed to a requirement for a naked DNA template for intermediate transcription (Keck et al., 1990
). This concept posits that vaccinia virus DNA is relatively free of proteins after the onset of DNA replication; however, we have little information on the proteins that associate with viral DNA in the cell cytoplasm.
Vaccinia virus RNA polymerase
All three classes of vaccinia virus genes are transcribed by the virus-encoded RNA polymerase. This enzyme is remarkably complex, being composed of nine subunits totalling more than 500 kDa in mass (Table 1) (Moss, 1994
). The 147 and 136 kDa subunits show a high degree of amino acid similarity to the two largest subunits of eukaryotic and prokaryotic cellular RNA polymerases (Broyles & Moss, 1986
; Patel & Pickup, 1989
). In the bacterial RNA polymerase and RNA polymerase II from yeast, these two subunits come together to form a crab claw-shaped structure with a cleft that is the site of template interaction and the active site for phosphodiester bond formation (Davis et al., 2002
; Murakami et al., 2002
). The other subunits interact with the opposite face of the protein, distant from the catalytic site of the enzyme and, thus, are proposed to interact with transcription factors. Other than a modest similarity between the smallest vaccinia virus RNA polymerase subunit (7 kDa) and the smallest subunit of yeast RNA polymerase II (RBP10) (Amegadzie et al., 1992a
), the smaller subunits of vaccinia virus RNA polymerase have no significant resemblance to the smaller subunits of cellular RNA polymerases, possibly owing to the lack of sequence similarity to any vaccinia virus transcription factors to known cellular transcription factors.
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Approximately half of the vaccinia virus genes belong to the early class (Oda & Joklik, 1967). A single early promoter, that of the 7·5K gene, has been characterized in detail (Davison & Moss, 1989b
). Early promoters can be studied in vivo only in the context of being resident in the viral genome. Transfected early promoter/reporter gene constructs are not functional (unpublished observations), presumably because they cannot access the interior of the core particle where the salient proteins reside. Analysis of the 7·5K promoter identified a single essential element upstream of the transcription start site spanning nt -12 to -29. Inspection of a number of early promoters reveals that each has a nearly universal G residue at -21 or -22 that is flanked by a sequence that is variable but highly AT rich (Fig. 1
) (Davison & Moss, 1989b
).
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The initiation of early mRNA synthesis requires ATP as an energy source that is distinct from the adenosine nucleotide incorporated into RNA. ATP analogues with a non-hydrolysable
bond can be incorporated into RNA chains by the viral RNA polymerase on artificial single-stranded templates, yet do not support transcription from vaccinia virus early promoters (Gershowitz et al., 1978
). An explanation for the ATP requirement emerged with the discovery of an ATPase activity associated with ETF (Broyles & Moss, 1988
). The ATPase activity is DNA dependent, with little regard for the form or sequence of DNA. Mutations in the conserved ATPase motifs, such as the P loop and DEAH box in ETF, inactivate its transcription factor activity in vitro (Li & Broyles, 1995
). ATP hydrolysis induces the accelerated dissociation of the ETFpromoter complex (Broyles, 1991
). Taken together, these results suggest a model in which ETF recruits the RNA polymerase to the transcriptional start site but simultaneously presents a steric hindrance to the RNA polymerase because of ETF's DNA contacts on the downstream side of the RNA polymerase (Fig. 2
). The release of ETF concurrent with ATP hydrolysis removes the impediment and RNA polymerase could then begin to traverse the template for RNA polymerization.
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Several lines of evidence indicate that the early transcription machinery, complete with RNA modification enzymes, may assemble on early promoters during morphogenesis and assembly into progeny virions. Virion extracts yield a RNA polymerase holoenzyme capable of transcription of early gene templates in vitro (Broyles & Moss, 1987). This complex contained ETF, capping enzyme, poly(A) polymerase and the transcription termination factor NPH I (nucleoside phosphohydrolase I). Inhibition of synthesis of the RNA polymerase H4L subunit, thought to dock with ETF, resulted in progeny virions that had normal ETF content but were deficient in RNA polymerase, poly(A) polymerase and capping enzyme (Zhang et al., 1993
). Similarly, viruses with ETF having impaired promoter-binding activity packaged reduced levels of ETF, RNA polymerase, capping enzyme, poly(A) polymerase and NPH I within their virion particles (Li et al., 1994
). Finally, inhibition of expression of either subunit of ETF caused severe defects in morphogenesis (Hu et al., 1996
, 1998
). The simplest interpretation of these findings is that the complete early transcription complex is anchored at early promoters during virion assembly through ETF and its complex with the RNA polymerase, and assembly of the transcription complex is an early event in virion morphogenesis. A study by Cassetti et al. (1998)
seems to contradict the DNA-mediated assembly of vaccinia virus transcription factors. A virus in which the gene A32L product was repressed failed to package significant amounts of DNA (Cassetti et al., 1998
). The A32L-deficient virus was, nonetheless, capable of packaging proteins participating in early gene transcription, including ETF. Therefore, factors other than assembly on transcriptional promoters may contribute to the assembly of transcriptional proteins into the virion core.
Intermediate gene transcription
Until recently, vaccinia virus intermediate genes were believed to be few in number. They were uncovered initially by the identification of promoters that required the onset of DNA synthesis but lacked the TAAATG motif at the start site for transcription previously regarded as diagnostic for late promoters (Vos & Stunnenberg, 1988). Intermediate promoters are more prevalent in the vaccinia virus genome than previously appreciated, because many have the TAAATG motif at the start of their open reading frames, which was previously attributed to late gene promoters (X. Liu and S. S. Broyles, unpublished results). The RNA polymerase initiates on this motif within the A triplet (actually on the T triplet on the template strand) and slips repeatedly while attempting to initiate transcription (Bertholet et al., 1987
; Schwer et al., 1987
). The result is mRNA with a 5' end bearing a heterogeneous oligo(A) tail, averaging about 30 nt in length that is not template encoded. The significance of the 5' oligo(A) tail for mRNA function is not known.
Intermediate promoters are bipartite, having an initiator element at the transcriptional start site and an AT-rich upstream element (Fig. 1) (Baldick et al., 1992
). The initiator element minimally has the sequence TAAAT/A at nt -1 to +4 relative to the first A in the motif (Baldick et al., 1992
). Many, but not all, intermediate promoters have the dinucleotide GG immediately 3' of the TAAAT motif (X. Liu and S. S. Broyles, unpublished results), constituting a binding site for the nuclear transcription factor YinYang1 (YY1) in the form of the sequence TAAATGG. YY1 binds this sequence in the initiator element of the intermediate I1L promoter (Broyles et al., 1999
). The I1L promoter was initially described as a late class promoter (Vos & Stunnenberg, 1988
) but is now know to be an intermediate class promoter (X. Liu and S. S. Broyles, unpublished results). Replacement of the GG dinucleotide with C residues impaired binding to YY1 in vitro and reduced the I1L promoter's activity by about 90 % in vivo. The co-crystal structure of the DNA-binding domain of YY1 and the sequence AAAATGG showed that the TTT motif, on which the vaccinia virus RNA polymerase must initiate transcription, faces away from the YY1 interface (Houbaviy et al., 1996
) and is thus available for engagement. YY1 accumulates in the cytoplasm of vaccinia virus-infected cells, consistent with a role in transcription of the viral genome (Broyles et al., 1999
).
Several virus-encoded proteins are required for intermediate gene transcription. De novo synthesis of viral RNA polymerase is probably required for vaccinia virus intermediate gene transcription. Temperature-sensitive RNA polymerase mutants are defective for late transcription, suggesting that new RNA polymerase is required for late transcription (Hooda-Dhingra et al., 1989). This observation was reported prior to the discovery of intermediate genes. All RNA polymerase subunit genes whose transcripts have been characterized have early promoters. Therefore, intermediate transcription is likely to require new RNA polymerase also. Either the RNA polymerase brought into the cell by the infecting virion is rendered inactive upon uncoating of the genome and/or is incapable of supporting the burden of RNA synthesis activity necessary for intermediate and late transcription. In addition, the form of RNA polymerase that is most efficient in late gene transcription in vitro is the form that lacks the H4L polypeptide (Wright & Coroneos, 1995
). The H4L gene is transcribed as a late class gene (Rosel et al., 1986
) (although this has not been verified since the discovery of intermediate genes) and, therefore, should not be present during intermediate gene transcription. This means that any transcriptional process that is H4L-dependent is not likely to be functional for intermediate or late gene transcription.
At least four other proteins have been reported from two laboratories to be required for transcription from the I3L intermediate promoter in vitro. Vos and co-workers described two factors, ITF-A and ITF-B, that had intermediate transcription factor activity (Vos et al., 1991b). ITF-B is the viral capping enzyme and a fraction containing ITF-A was shown to have promoter DNA-melting activity (Vos et al., 1991a
, b
). Moss and co-workers have identified the intermediate factors VITF-1, VITF-2 (Rosales et al., 1994a
) and VITF-3 (Sanz & Moss, 1998
) and confirmed a requirement for the viral capping enzyme in intermediate transcription (Harris et al., 1993
). VITF-1 is the 30 kDa subunit of the viral RNA polymerase (Rosales et al., 1994a
). VITF-3 is a heterodimer of the viral A8L and A23R gene products (Sanz & Moss, 1999
). VITF-2 was identified in nuclear extracts from uninfected HeLa cells (Rosales et al., 1994b
), documenting the first known vaccinia virus transcription factor that is not virus encoded. The identity of the nuclear protein is not known nor is a molecular function for any VITF proteins or capping enzyme. The latter protein likely has a tethering role, linking one of the other factors to the RNA polymerase. The capping enzyme has been reported to be complexed with RNA polymerase in solution (Broyles & Moss, 1987
). Whether any of these proteins targets either of the two elements in intermediate promoters is not known.
Protein phosphorylation has been implicated in intermediate transcription through a characterization of vaccinia virus mutants defective for the B1R protein kinase (Kovacs et al., 2001). B1R is a serine/threonine protein kinase (Traktman et al., 1989
; Banham & Smith, 1992
; Lin et al., 1992
) previously characterized as being required for DNA replication (Condit & Motyczka, 1981
; Condit et al., 1983
). The recent study found that B1R mutants are also defective for intermediate gene transcription but not late gene transcription. These findings suggest that one or more proteins functioning in intermediate transcription may require phosphorylation for function, but the substrate for the kinase that functions in intermediate transcription has not been reported.
Late transcription
Vaccinia virus late promoters also have a bipartite structure with an initiator-like element at the start site for transcription and an AT-rich upstream element (Fig. 1) (Davison & Moss, 1989a
). The initiator element has the nearly invariant sequence TAAAT. Nucleotides downstream of this sequence do not have a role in transcription. The upstream element is closer to the initiator than the intermediate promoters are, being located at about nt -16 to -11 (X. Liu and S. S. Broyles, unpublished results). Like the upstream element of intermediate promoters, the late element seems to tolerate considerable variation in sequence.
As described above, late transcription requires newly synthesized RNA polymerase (Hooda-Dhingra et al., 1989). Three other virus-encoded transcription factors were identified by Moss and colleagues by asking which viral genes must be co-transfected with a reporter gene driven by a vaccinia virus late promoter under conditions where DNA synthesis was inhibited (Keck et al., 1990
). G8R, A1L and A2L constituted the minimal set of genes required for late promoter activity. All three are intermediate class genes. No function has been ascribed to any of the three. A yeast two-hybrid screen suggested that the G8R and A1L proteins are interaction partners (McCraith et al., 2000
). A fourth factor, the product of the H5L gene, was identified through cell fractionation studies as having transcription stimulatory activity (Kovacs et al., 1994
; Kovacs & Moss, 1996
). The H5L gene belongs to the early class of vaccinia virus genes and, hence, would have escaped attention in transfection studies. The H5L protein is a substrate for the B1R protein kinase (Beaud et al., 1995
) and an interaction between H5L and B1R was detected in a yeast two-hybrid screen (McCraith et al., 2000
), suggesting that protein phosphorylation may have a role in regulating the function of this transcription factor. An effect of phosphorylation on the protein function of H5R has not been reported, nor has its phosphorylation status in vivo.
A fifth late transcription factor has been identified by Wright and colleagues, also through cell fractionation (Wright et al., 1998). This factor, called VLTF-X, was initially reported as being virus-induced (Wright & Coroneos, 1993
) but, subsequently, was identified in cytoplasmic and nuclear extracts from uninfected HeLa cells (Gunasinghe et al., 1998
) and is, therefore, a host protein implicated in vaccinia virus late transcription. Interestingly, this factor co-purified with a DNA-binding activity that demonstrated some specificity for oligo(T)-tract sequences. This is of interest because it has been reported that oligo(T) tracts are functional as an upstream element in a late vaccinia virus promoter (Davison & Moss, 1989a
). Thus, it is possible that VLTF-X is responsible for targeting late vaccinia virus promoters as sites of initiation. The transcriptional stimulation activity associated with VLTF-X can be fulfilled by either heterogeneous nuclear riboproteins A2/B1 or RBM3 (Wright et al., 2001
). The failure to identify any vaccinia virus-encoded factors with promoter-binding activity prompts speculation that host factors may target the viral promoters, forming a nucleation site for virus-encoded factors that eventually recruit the RNA polymerase to the site of initiation (Fig. 3
).
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Two proteins have been implicated in the elongation of transcription on vaccinia virus intermediate and late genes. The G2R and J3R proteins have been suggested to enhance rates of transcription elongation in a manner that likely impacts the ability of the RNA polymerase to terminate and release post-replicative transcripts (see below).
Early gene transcriptional termination
Transcription of vaccinia virus early genes terminates just downstream of open reading frames in response to the sequence TTTTTNT (where N is any nucleotide) on the non-template strand of the DNA (Yuen & Moss, 1987). Termination occurs heterogeneously about 3050 nt downstream of the signal. At least two trans-acting factors are required to induce termination and transcript release by the RNA polymerase. The termination signal is actually sensed in the form of the sequence UUUUUNU in the nascent RNA, a conclusion derived from the observation that bromo-UTP specifically inhibits the termination of transcription in vitro (Shuman & Moss, 1988
). Presumably the RNA polymerase carries the capping enzyme along as it transverses the template as an elongation complex. As the termination signal in the RNA is extruded from the elongating RNA polymerase, the capping enzyme, by an as yet undefined mechanism, induces the RNA polymerase to cease transcription and release the template. The termination process has been proposed to be the result of a kinetic balance between transcription elongation rates and signalling through the capping enzyme (Deng & Shuman, 1997
). Reaction conditions that slow the rate of elongation by the RNA polymerase slow the rate of signalling, thereby shifting termination sites farther downstream. The second factor, NPH I, was identified following the demonstration of an ATP requirement for the termination process (Deng & Shuman, 1998
). NPH I is required for termination of transcription and transcript release in vitro and for termination of transcription using extracts from cells infected with a NPH I mutant virus (Christen et al., 1998
). As described above, NPH I is a ssDNA-dependent ATPase with nucleic acid helicase motifs in its amino acid sequence. Five of the six helicase motifs in NPH I are essential for termination factor activity (Christen et al., 1998
). It seems likely that NPH I is the motor that drives dissociation of the transcription elongation complex in response to the signal in RNA. A model for termination of early transcription has been proposed in which RNA polymerase carries capping enzyme and NPH I as an elongation complex (Deng & Shuman, 1998
). As the termination signal in the mRNA is extruded from the RNA polymerase, it contacts the capping enzyme, signalling NPH I to drive release of the transcript through hydrolysis of ATP (Fig. 4
). Contact between NPH I and the RNA polymerase is supported by evidence for a requirement for the H4L subunit of the RNA polymerase in termination of transcription (Mohamed & Niles, 2001
) and demonstration of direct interaction between NPH I and the H4L polypeptide (Mohamed & Niles, 2000
). Thus, the H4L polypeptide seems to be the key specificity factor for virtually all aspects of early gene transcription. It is required for initiation, elongation and termination of early transcripts. Therefore, the RNA polymerase molecules that lack H4L would not be expected to perform any of these processes.
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A role for the vaccinia virus G2R protein in transcription termination was initially revealed by the mapping of mutations conferring dependence on the anti-poxvirus drug isatin -thiosemicarbazone (IBT) (Meis & Condit, 1991
). G2R mutants display a phenotype in which intermediate and late transcripts are shorter than normal (Black & Condit, 1996
), just the opposite of that of A18R mutants. The reduced transcript lengths implied that the G2R protein may play a role in promoting transcription elongation by the RNA polymerase. An interrelationship between G2R and A18R was inferred by demonstrating that G2R mutants could act as extragenic suppressors of the A18R mutants (Condit et al., 1996
). An interaction between G2R with the late factor H5R was also detected in a yeast two-hybrid screen (McCraith et al., 2000
).
A third protein, the product of the J3R gene, has been implicated in intermediate and late transcription termination. Additional IBT-dependent mutants and extragenic suppressors of A18R mutations indicated that J3R mutants are identical in phenotype to those in G2R (Latner et al., 2000; Xiang et al., 2000
). Post-replicative transcripts from J3R mutants are truncated at their 3' ends, supporting the conclusion the J3R protein is also a positive elongation factor. A role for J3R in transcription elongation is somewhat surprising because this polypeptide is the viral mRNA 2'-O-methyltransferase (Schnierle et al., 1992
) and the stimulatory subunit of the mRNA poly(A) polymerase (Gershon et al., 1991
). Mutational analysis showed the mRNA methyltransferase and poly(A) polymerase stimulatory activities to be distinct from the elongation factors' properties (Xiang et al., 2000
; Latner et al., 2002
). An interaction between the J3R protein and the late transcription stimulatory factor H5R has been documented in two independent studies (Black et al., 1998
; McCraith et al., 2000
); however, the significance of this complex is not yet apparent. Thus, the termination of post-replicative transcripts appears to result from a dynamic balance between maintaining a transcription elongation complex and promotion of transcript release.
Transcript 3'-end processing
The cowpox virus A type inclusion body protein (ati) transcript is a late class mRNA and has the unusual property of terminating at a precise location after the open reading frame (Antczak et al., 1992). The coding sequence terminates at a specific nucleotide by a site-specific riboendonucleolytic cleavage and the 3' end is polyadenylated. The sequence immediately surrounding the cleavage site and another block of sequence 10 nt downstream are essential for the cleavage reaction (Howard et al., 1999
). The same 3' end is found on the transcript from the equivalent gene in vaccinia virus (Amegadzie et al., 1992b
), indicating that the gene structure is conserved in more than one poxvirus. It is not clear whether 3'-end processing is common in vaccinia virus. Because the first example of 3'-end processing was found in the highly abundant ati transcript, it is tempting to speculate that the processing enhances the expression of highly active genes.
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CONCLUSION |
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