Vaccinia Virus Nucleoside Triphosphate ... - Journal of Virology

JOURNAL OF VIROLOGY, Dec. 1993, P. 7561-7572

Vol. 67, No. 12

0022-538X/93/127561-12$02.00/0 Copyright C) 1993, American Society for Microbiology

Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase I Controls Early and Late Gene Expression by Regulating the Rate of Transcription MARGARITA DIAZ-GUERRAt

AND

MARIANO ESTEBANt*

Departments of Biochemistry and Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, New York, New York 11203 Received 27 May 1993/Accepted 9 August 1993

We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the reporter gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way of an auxiliary function which regulates mRNA transcription during the virus growth cycle. a

Vaccinia virus gene expression has the unique property of being carried out in the cellular cytoplasm by virus-encoded, host-independent transcriptional machinery. The virus contains within its core all of the proteins necessary to produce mature early mRNA. Several virion enzymes, including the DNA-dependent RNA polymerase, mRNA capping enzyme, mRNA 2'-O-methyl-transferase, poly(A) polymerase, and a transcription factor for viral early promoters (VETF), have known functions in mRNA synthesis. The functions of other enzymes, such as nucleoside triphosphate phosphohydrolases I and II (NPH-I and NPH-II), DNase, topoisomerase, and protein kinase, are still unknown (reviewed in reference 35). The regulation of transcription of the three different classes of viral genes (early, intermediate, and late genes) is thought to follow a cascade model (28). After entry of the virus into the host cell, transcription of early genes is initiated by enzymes present in the viral core. Early transcripts are extruded into the cytoplasm and translated on host polysomes producing, among others, specific transcription factors required for transcription of intermediate genes and proteins needed for DNA replication. Early protein synthesis is also required for secondary uncoating, which releases the viral genome and makes it accessible for DNA replication. Only after initiation of DNA synthesis are the intermediate transcriptional factors, which are present before replication, able to direct transcription of the intermediate genes. Some of the products of these genes are transcription factors specific for late gene expression (28, *

Corresponding author.

Present address: Centro Nacional de Biotecnologia, C.S.I.C., Campus Universidad Aut6noma, Madrid 28049, Spain. t

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57). The products of the late genes include the two subunits of the early specific transcription factor VETF, D6R and A8L gene products (7, 20), which are packaged into virus particles together with other proteins required for early viral transcription. Insights into the mechanism of vaccinia virus transcription have mainly come from in vitro studies. Permeabilized virus particles or purified cores provided the first in vitro transcription systems (27, 36), and RNA species made using these systems structurally and functionally resemble early mRNAs obtained during infection. ATP hydrolysis has been shown to be required for initiation (21) and elongation (53) of early transcription as well as for extrusion of RNA from the virus core (56). Further knowledge of the regulation and requirements of early transcription has come from the development of a template-dependent transcription system from vaccinia virus cores. Two protein factors are necessary and sufficient for accurate initiation, effective elongation and precise termination of RNA by vaccinia virus RNA polymerase in vitro (45). The transcription termination factor VTF, shown to be the mRNA capping enzyme (32), is required to render purified RNA polymerase competent for termination (51). On the other hand, VETF renders purified RNA polymerase competent in transcription of duplex DNAs containing vaccinia virus early promoters (10). Purified VETF binds specifically to early promoters and has an intrinsic DNA-dependent ATPase activity (9) that presumably induces VETF to rapidly dissociate from its binding site in the promoter (6). These properties of VETF may account for (or at least contribute to) the requirement for hydrolyzable ATP in early transcription (8, 21, 53). Recently, it has been demonstrated that VETF activates

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transcription by directly recruiting the RNA polymerase to early promoters (31). Transcription of vaccinia virus in vivo is likely to be more complex than the in vitro systems show, and other proteins are expected to be involved in its modulation. In fact, it has been previously proposed that NPH-I could play such a role (26, 30). Vaccinia virus NPH-I is a monomeric enzyme (68 kDa) synthesized late during infection (40, 43). The purified protein is specific for ATP or dATP and requires DNA or synthetic polydeoxyribonucleotides as cofactors (39), but neither completely single-stranded nor completely double-stranded DNA is effective. These properties are clearly different from those of the two other DNA-dependent ATPase activities also present in the virions. D6R (the 77-kDa subunit of VETF) has the same substrate and divalent cation requirements as NPH-I, but for D6R the optimal cofactors are duplex polydeoxynucleotides (9). Purified NPH-II, however, hydrolyzes all four ribo- or deoxyribonucleoside triphosphates and uses DNA or RNA as cofactor (39, 40). Interestingly, NPH-I has been shown to contain the consensus sequence for a family of established and putative DNA and RNA helicases. Besides NPH-I, there are three other members of this family encoded by the genome of vaccinia virus (29): A18R, a protein proposed to mediate viral mRNA metabolism (3, 38), D6R, and NPH-II, suggested to be involved in RNA extrusion from the core and the only protein in this group actually shown to have helicase activity (50). Thus, it is tempting to suggest that all the proteins in this family have key roles in the control of transcriptional and posttranscriptional processes in the infected cell. NPH-I has been found to be weakly associated with early viral transcription complexes (8). Two different temperature-sensitive mutants (ts36 and tsSO) (12, 13) have been genetically characterized as containing single point mutations in the NPH-I gene responsible for the phenotype at the nonpermissive temperature (26, 30). In monkey BSC-40 cells, using a multiplicity of infection (MOI) of 10, tsSO and ts36 behave phenotypically like wild-type virus with respect to replication of viral DNA but are defective in late protein synthesis at the nonpermissive temperature. However, their phenotype in human HeLa cells and mouse L929 cells is more severely affected: viral DNA replication does not occur and late protein synthesis is absent. Analysis of steady-state viral mRNA levels in L cells (MOI, 15) has also revealed that tsSO and ts36 mutants are mainly defective in the accumulation of intermediate and late viral RNAs (30). However, inhibition of the accumulation of one of the early transcripts analyzed was also noticeable, although DNA replication was not affected. On the basis of the above observations, it was suggested that NPH-I plays a role in early gene expression (25, 26, 30). By analyzing the mutant ts36, we demonstrate in this report that vaccinia virus NPH-I is required for early virus gene expression which, together with its previously known role in intermediate and late gene expression, makes this enzyme an important regulatory protein during the entire virus life cycle. Moreover, by measuring viral transcriptional activity in permeabilized virions, we demonstrate that NPH-I is required for transcription of vaccinia virus.

MATERIALS AND METHODS Cells and viruses. Mouse Ltk(-) cells, human HeLa cells, and the African green monkey kidney cell lines Vero, cos-7, CV-1, BSC-1, and BSC-40 were grown in Dulbecco's modified medium supplemented with 10% newborn calf serum. Cells were cultivated at 37°C with 5% CO2. The recombinant virus ts36LUC was obtained after introduction of the plasmid

J. VIROL.

pSCLUC (44) into the tk region of vaccinia virus ts36 mutant DNA by homologous recombination. The procedure for construction and selection of the recombinant virus ts36LUC was as previously described for WRLUC, a recombinant derived from wild-type vaccinia virus (44). Rescued virus R36 was obtained by marker rescue of the point mutation in ts36, C to T at position 556 of the NPH-I gene, using the plasmid pCMK714 (26) containing positions 12,390 to 13,104 of fragment HindIII-D. Virus plaques appearing at 39.5°C were plaque purified three times. The wild-type strain of vaccinia virus (WR) and the rescued virus (R36) were propagated in BSC-40 cells at 37°C. The temperature-sensitive mutant was kindly provided by R. C. Condit (12, 13). This virus, as well as the recombinant ts36LUC, was propagated in BSC-40 cells at the permissive temperature (31°C) as described previously (26). Temperature-sensitive phenotype was routinely tested in virus preparations by comparison of plaque-forming ability at both 31 and 39.5°C. For in vitro transcription experiments, virions were purified by sedimentation through a sucrose cushion and subsequent sucrose gradient sedimentation as previously described (24). Where indicated, cycloheximide (CH) (100 pLg/ml) was added to the cultures 30 min before infection; hydroxyurea (HU) (5 mM) was added after viral adsorption. Unless noted, these drugs were maintained in the culture medium for the duration of the experiment. Plasmids. Plasmid pSCLUC (44) contains the Escherichia coli 3-galactosidase gene (lacZ) under the control of a late viral promoter (11-kDa promoter) and the firefly luciferase gene under the control of an early and late viral promoter (7.5-kDa promoter). Plasmid pBS(+)14k (22) contains the complete 14-kDa gene (RsaI-RsaI fragment) in the SmaI site of pBS(+) (Stratagene). Plasmid pBS39k was generated by inserting an internal BamHI-SpeI fragment of the 39-kDa gene (15) in the polylinker of pBluescriptlISK (Stratagene). Plasmids containing the DNA polymerase and D5 genes were kindly provided by P. Traktman. The former carries a ScaIEcoRI fragment of the DNA polymerase gene in pBR322, and the latter carries a PstI-EcoRI fragment of the D5 gene in the same vector. Measurement of luciferase activity. Infected cells were washed three times in phosphate-buffered saline (PBS) and lysed directly on the plate by the addition of 1% Triton, 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, and 1 mM dithiothreitol containing 1 mM phenylmethylsulfonyl fluoride as described previously (5). Luciferase activity in these extracts was measured by using a luminometer apparatus from Analytical Luminescence Laboratory (Monolight 2010). All experiments were independently repeated two to four times, and mean values are given. Immunoblot analysis. Protein samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose paper in a semi-dry blotting apparatus for 45 min at 200 mA, and analyzed by immunoperoxidase staining after reactivity with different sera. The coding sequence for NPH-I, lacking only the nucleotides coding for the first nine amino acids at the N terminus, was cloned in the E. coli expression vector pT7-7 (55). Exponentially growing E. coli BL21 (DE3) cells carrying the recombinant plasmid and induced with 400 ,uM isopropyl-p-D-thiogalactopyranoside produced a protein containing the first 12 amino acids encoded by the vector pT7-7 short open reading frame followed by amino acids 10 to 631 of NPH-I. The protein band containing NPH-I was excised from the gel, electroeluted, concentrated in a desiccator, resuspended in deionized water, and precipitated with methanol-acetone (50:50, vol/vol). The pellet was suspended in water and used as the antigen for

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injection into lymph nodes of New Zealand White rabbits. Polyclonal antibodies specific for the 39- and 32-kDa proteins were obtained using a similar protocol as described elsewhere (15, 33). Rabbit polyclonal anti-vaccinia virus serum was raised in rabbits immunized with live virus as described previously (15). Monoclonal antibodies specific for the 14-kDa protein were prepared as previously described (42). Rabbit polyclonal sera specific for vaccinia virus DNA polymerase (34) and D5 proteins (18) were kindly provided by P. Traktman. Immunoprecipitation. DNA polymerase immunoprecipitation was performed as previously described (34). For NPH-I pulse-chase experiments, BSC-40 and Ltk(-) confluent monolayers were infected with ts36 (MOI, 10) at 31°C for 6 h. Cultures were labeled at this temperature by incubation in methionine-free Dulbecco's modified medium supplemented with [135S]methionine at 100 iLCi/ml, and after 30 min, labeling was stopped by addition of unlabeled methionine. One plate was harvested immediately, and the others were chased for 1, 3, or 25 h either at the permissive temperature (31°C) or after being shifted to the nonpermissive temperature (39.5°C). Similarly, BSC-40 cells infected with WR virus at 37°C were labeled for 30 min after 6, 8, or 18 h of infection, but cells were collected immediately. Cells were washed and collected in PBS, and the pellets were resuspended in lysis buffer (20 mM Tris-HCI [pH 8], 80 mM NaCl, 20 mM EDTA, 1% Nonidet P-40 [NP-40]) containing protease inhibitors (2 pLg of trypsin inhibitor per ml, 1 mM phenylmethylsulfonyl fluoride, 10 ,ug of leupeptin per ml, 2 p.g of bacitracin per ml) and left on ice for 30 min. The cell extracts were sonicated and pelleted by centrifugation (10,000 x g for 5 min at 4°C), and the supernatant was incubated at room temperature for 4 h with preimmune rabbit sera (1:50 dilution) coupled to protein A-Sepharose beads. After centrifugation, the supernatants were incubated at 4°C for 12 to 16 h with rabbit anti-NPH-I serum (1:50 dilution) coupled to protein A-Sepharose beads. The immunoprecipitates were washed three times with lysis buffer and three times with PBS. The beads were then resuspended in sample buffer and analyzed by SDS-PAGE. RNA isolation and Northern blot analysis. Total RNA from mock-infected or infected cells was isolated by the guanidinium thiocyanate-CsCl method as described previously (47). For Northern blotting, denatured RNA (5 p.g) was fractionated on 1 % formaldehyde-agarose gels, transferred to nitrocellulose membranes, and hybridized with labeled probes as described previously (47). DNA polymerase (34) and D5 mRNAs (18) were detected using, respectively, a 1.5-kb fragment (BglII-EcoRI) and a 1.35-kb fragment (BglII-BglII), both within the coding sequences. RNase protection analysis. Total RNA (1 pLg) was hybridized overnight at 45°C with an experimentally determined excess of riboprobe (10 to 20 ng, specific activity about 2 x 108 cpm/pg). The conditions for hybridization and subsequent RNase treatment were according to standard protocols (47). The samples were analyzed by electrophoresis on 5% polyacrylamide-7 M urea gels, and the protected fragments were detected by autoradiography of dried gels. Uniformly 32p_ labeled, antisense RNAs were generated by in vitro transcription with bacteriophage T3 RNA polymerase as described previously (47). Plasmid pBS(+)14k was linearized with EcoRI and transcribed to generate 425-base-long RNA in which 361 bases correspond to the complete coding region plus additional 5' and 3' sequences. Plasmid pBS39k was linearized with HincIl and transcribed to generate a 449-base-long RNA that contains 409 bases corresponding to an internal region of the 39-kDa gene. Transcription by permeabilized virions. In vitro transcrip-

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tion was carried out in NP-40-permeabilized purified virions prepared as described previously (52). Reaction mixtures containing 60 mM Tris-HCI (pH 8.0), 10 mM MgCl,, 10 mM dithiothreitol, 5 mM ATP, 1 mM CTP, 1 mM GTP, 0.2 mM [(x-5,6-3H]UTP, 10 FM S-adenosyl-L-methionine, 0.05Cc NP40, and purified virions (8.4 x 10'9 particles per ml) were separated in two identical aliquots immediately before incubation at 31 or 39.5°C. Samples were removed at different times, and after addition of trichloroacetic acid, were assayed by filtration for incorporation of labeled nucleotides into insoluble material. Where indicated, virions were preincubated for 30 min at the indicated temperatures in reaction mix lacking

nucleotides. RESULTS Vaccinia virus NPH-I is required for early expression of the reporter gene luciferase in cell lines of different origins. A role for NPH-I in early viral gene expression has been suggested in some cell lines (26). To prove that NPH-I acts during this phase of the virus life cycle, we infected mammalian cell lines of different origins with the recombinant viruses WRLUC and ts36LUC (MOI, 5) in the presence of HU, a reversible inhibitor of initiation of viral DNA synthesis. Under these conditions only early viral genes are expressed (17). Luciferase production, driven by the constitutive promoter p7.5, was measured in cells infected for 18 h at the permissive (31°C) and nonpermissive (39.5°C) temperatures in the presence of the drug. Moderate reduction in luciferase levels was observed in cells infected with the control virus WRLUC at the nonpermissive temperature (Fig. 1), one possibility being the lower stability of this protein in these particular conditions of infection. However, luciferase production was severely inhibited in all different cell lines infected with ts36LUC at the nonpermissive temperature. Inhibition, calculated as the percentage of luciferase expression for ts36LUC relative to WRLUC infections performed at the same temperature, is 97% in mouse Ltk(-) cells, 98.7% in human HeLa cells, and 73.8% both in monkey cos-7 and BSC-40 cells. Analysis of additional monkey cell lines (inset in Fig. 1) showed similar results. Although moderate inhibition is also observed in infections with ts36LUC at the permissive temperature, the results shown in Fig. 1 demonstrate that luciferase expression is temperature sensitive in different cells infected with NPH-1 mutant virus and functional NPH-I is required for early expression of a reporter gene under the control of the constitutive p7.5 promoter. Early viral protein synthesis is inhibited in ts36 infections at the nonpermissive temperature. In order to establish that NPH-I plays a general role in early viral protein synthesis, we analyzed the accumulation of several early polypeptides in cells infected with ts36 at the permissive and nonpermissive temperatures in the presence of HU. We analyzed Ltk( -) cells and BSC-40 cells as representative cell lines showing different levels of inhibition of viral gene expression. Cell extracts obtained from mock-infected cells or from cells infected for 18 h (MOI, 5) were analyzed by immunoblotting with a rabbit polyclonal anti-vaccinia virus serum. In Ltk(-) cells (Fig. 2A), the early polypeptides recognized by this serum are completely absent when infected with ts36 at the nonpermissive temperature (lane 6) compared with those infected at the permissive temperature (lane 5). The results obtained with the rescued virus R36 (lane 8) demonstrate that the temperature-sensitive phenotype of ts36 is only due to the point mutation in the NPH-I gene. The levels of early polypeptides at 39.5°C were the same in cells infected with the wild-type virus (lane 4) as in

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cells infected with R36 (lane 8). In BSC-40 cells (Fig. 2A), mutant ts36 shows a less severe phenotype. Using anti-vaccinia virus serum, we found no significant differences in the levels of early viral proteins between cells infected with ts36 at the permissive (Fig. 2A, lane 13) or nonpermissive (lane 14) temperature. There were, however, decreased levels in some early proteins, like DNA polymerase and D5 (lane 14, lower part), at the nonpermissive temperature. We next investigated the time course of protein synthesis in Ltk(-) cells. Monolayers were infected with either wild-type virus or ts36 virus (MOI, 5) and maintained at 31 or 39.5°C in the presence of HU. At various times postinfection, cells were radiolabeled with [35S]methionine for 30 min and collected immediately. Extracts were subjected to immunoprecipitation analysis, using anti-DNA polymerase serum. The results obtained with wild-type-infected cells (Fig. 2B, left) are in agreement with the transient pattern of expression described for this protein (34). That is, DNA polymerase synthesis peaks by 2 to 4 h postinfection and then declines, with changes in expression occurring at an accelerated rate at 39.5°C, changes which are not affected by blocking of viral DNA replication. The profile obtained for ts36 infections performed at 31°C (Fig. 2B, lanes 13 to 17) is similar to that of wild-type virus. However, at 39.5°C (lanes 18 to 22), very low levels of DNA polymerase synthesis are detected between 2 to 4 h postinfection with ts36 and, thereafter, DNA polymerase synthesis is completely inhibited. In the same experiment, analysis of the synthesis and accumulation of other viral polypeptides, as shown by immunoperoxidase staining after reaction with rabbit anti-vaccinia virus serum (Fig. 2C), produced similar results. Clearly, the results shown in Fig. 2 demonstrate that NPH-I is required for the synthesis of early viral proteins. Inhibition of early virus protein synthesis by mutated NPH-I correlates with a decrease in steady-state mRNA levels. Since synthesis of early viral polypeptides is inhibited in

Ltk(-) cells infected with ts36 at the nonpermissive temperature, we next investigated whether this is the result of a block of translation or resulted from a block at an earlier phase of gene expression. Thus, we measured the steady-state mRNA levels of DNA polymerase and D5 genes. These two early mRNAs have a transient pattern of expression that is not affected by blocking viral DNA replication, and RNA levels correlate with the pattern of protein synthesis (18, 34). Total RNA from Ltk( -) cells infected with ts36 or wild-type virus (MOI, 5), in the presence of HU or CH, was extracted at various times postinfection and was analyzed by Northern blot hybridization using specific probes for these two early genes (Fig. 3). The molecular weights of the RNAs detected were in agreement with previously published results (18, 34), and control experiments using mock-infected cells demonstrated their viral origins (data not shown). In Ltk(-) cells infected with ts36 in the presence of HU, similar levels of mRNA are found for DNA polymerase and D5 at 31 (lane 1) and 39.5°C (lane 2) by 2 h postinfection. However, as infection progresses, these mRNAs become undetectable at 39.5°C (lanes 4 and 6). As expected, in cells infected at the permissive temperature in the presence of CH, mRNA levels are elevated compared with results obtained at 6 h postinfection for HU (lane 9). This drug prevents both early protein synthesis and the secondary uncoating event and has been shown to cause prolonged and elevated levels of DNA polymerase mRNA transcription (34). On the contrary, a strong reduction in mRNA levels was observed 6 h after infection at the nonpermissive temperature in the presence of CH (lane 10). Control infections with wild-type virus in the presence of this drug did not show significant differences in mRNA levels at both temperatures (lanes 11 and 12). The differences in levels of mRNA were not the result of variations in amounts of RNA, as shown by ethidium bromide staining (Fig. 3, lower panels). The results shown in Fig. 2 and 3

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