Late Viral Protein Synthesis Research Articles

Inhibition by prostaglandin PGA1 on the multiplication of influenza virus is a dose-dependent effect

Cyclopentenone prostaglandins (PGs), are strong inhibitors of the multiplicative cycle of a wide variety of enveloped RNA and DNA viruses. Their antiviral activity is generally associated with alterations in the synthesis or maturation of specific virus proteins. In this report, we describe the effect of cyclopentenone PGA1 on the replication of influenza A virus Ulster 73 in LLC-MK2 cells. PGA1 was found to inhibit viral replication in a dose-dependent fashion and virus particle yield was reduced at a PGA1 concentration, which did not suppress protein synthesis in mock-infected cells. The kinetic of late viral protein synthesis was delayed in PGA1-treated cells till 10 h post-infection; after that period, viral polypeptide synthesis appeared to be similar in PGA1-treated as well as untreated cells both infected by Ulster 73 virus. This finding suggests that PGA1 might interfere with one or more events in the viral multiplicative cycle such as protein synthesis and assembly, correct insertion of virus polypeptides into the cell membrane and, or maturation of Ulster 73 virion particles. In particular, inhibition of viral replication in LLC-MK2 cells by PGA1 is accompanied by the induction of a cellular polypeptide of 70K molecular weight. We identified this cell protein as a heat shock protein (HSP) related to the inducible isoform of HSP 70, a polypeptide of 72K molecular weight. Induction of this polypeptide by PGA1 was found to be dose-dependent and a substantial accumulation could be seen at a PGA1 concentration that did not inhibit cell protein synthesis in uninfected cells. HSP 70 synthesis started after the beginning of PGA1 treatment and remained at the same level for at least 10 h, leading us to hypothesize that the delay of production of late Ulster 73 proteins could be the consequence of HSP 70 synthesis. These results suggest that HSP 70 could play a role in the antiviral activity of cyclopentenone PGA1 in LLC-MK2 cells.

A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation.

Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasmic DNA viruses for which substantial sequence information is available. Nevertheless, the roles of these proteins are imprecisely known. Multiple alignments of ERV1/ALR proteins indicated an invariant C-X-X-C motif, but no similarity to the thioredoxin fold was revealed by secondary structure predictions. We chose a virus model to investigate the role of these proteins as thiol oxidoreductases. When cells were infected with a mutant vaccinia virus in which the E10R gene encoding an ERV1/ALR family protein was repressed, the disulfide bonds of three other viral proteins-namely, the L1R and F9L proteins and the G4L glutaredoxin-were completely reduced. The same outcome occurred when Cys-43 or Cys-46, the putative redox cysteines of the E10R protein, was mutated to serine. These two cysteines were disulfide bonded during a normal virus infection but not if the synthesis of other viral late proteins was inhibited or the E10R protein was expressed by itself in uninfected cells, suggesting a requirement for an upstream viral thiol oxidoreductase. Remarkably, the cysteine-containing domains of the E10R and L1R viral membrane proteins and the glutaredoxin are in the cytoplasm, in which assembly of vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum. These data indicated a viral pathway of disulfide bond formation in which the E10R protein has a central role. By extension, the ERV1/ALR family may represent a ubiquitous class of cellular thiol oxidoreductases that interact with glutaredoxins or thioredoxins.

The vaccinia virus A9L gene encodes a membrane protein required for an early step in virion morphogenesis.

The A9L open reading frame of vaccinia virus was predicted to encode a membrane-associated protein. A transcriptional analysis of the A9L gene indicated that it was expressed at late times in vaccinia virus-infected cells. Late expression, as well as virion membrane association, was demonstrated by the construction and use of a recombinant vaccinia virus encoding an A9L protein with a C-terminal epitope tag. Immunoelectron microscopy revealed that the A9L protein was associated with both immature and mature virus particles and was oriented in the membrane with its C terminus exposed on the virion surface. To determine whether the A9L protein functions in viral assembly or infectivity, we made a conditional-lethal inducible recombinant vaccinia virus. In the absence of inducer, A9L expression and virus replication were undetectable. Under nonpermissive conditions, viral late protein synthesis occurred, but maturational proteolytic processing was inhibited, and there was an accumulation of membrane-coated electron-dense bodies, crescents, and immature virus particles, many of which appeared abnormal. We concluded that the product of the A9L gene is a viral membrane-associated protein and functions at an early stage in virion morphogenesis.

A glutaredoxin, encoded by the G4L gene of vaccinia virus, is essential for virion morphogenesis.

Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.

P53-Independent and -Dependent Requirements for E1B-55K in Adenovirus Type 5 Replication

The adenovirus type 5 mutant dl1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53(-) and some p53(+) tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639-645, 1997). It has been suggested that dl1520 might be used as a "magic bullet" that could selectively lyse tumor cells without harm to normal tissues. However, we report here that dl1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce dl1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653-8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109-124, 1982), dl1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.

P53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39 degreesC, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39 degreesC. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.

Does nitric oxide play a critical role in viral infections?

Does nitric oxide play a critical role in viral infections?

Apoptosis Induced by a Postbinding Step of Vaccinia Virus Entry into Chinese Hamster Ovary Cells

Unlike most cell types examined, nonpermissive Chinese hamster ovary (CHO) cells readily underwent apoptosis upon infection with vaccinia virus (VV). Apoptosis was observed as early as 3 h postinfection with an electrophoretic assay of DNA fragmentation and by 8 h using anin situ(TUNEL) assay. The CHOhrgene from cowpox virus, which overcomes host range restriction of VV in CHO cells, merely delayed the onset of apoptosis by approximately 3 h. Intermediate and late viral protein synthesis were not necessary for apoptosis since these events do not proceed under nonpermissive conditions. Apoptosis also occurred in the presence of cytosine arabinoside or cycloheximide, which inhibits DNA or protein synthesis, respectively, and after infection with a mutant virus that is blocked in early transcription. We also demonstrated that viral early transcription was not required for induction of apoptosis by infecting CHO cells with psoralen/UV-inactivated virus. On the other hand, apoptosis was inhibited by a neutralizing antibody to the virion L1R protein added either before or after virus attachment to cells. These results indicate that a postbinding step associated with cell entry is sufficient and required for induction of apoptosis in CHO cells. Recent progress on apoptotic signaling pathways raises the possibility that a cellular receptor for VV may be involved in apoptosis.

Repression of the A8L gene, encoding the early transcription factor 82-kilodalton subunit, inhibits morphogenesis of vaccinia virions.

The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and A8L genes, respectively. A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particles. The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle. To address these and other questions, we constructed and characterized a conditionally lethal recombinant vaccinia virus in which the A8L gene is stringently repressed by the Escherichia coli lac operator system. The amount of 82-kDa protein synthesized could be regulated by the amount of inducer: from undetectable to higher than normal levels. Virus replication, as determined by plaque formation or virus yield upon synchronous infection, was dependent on inducer. Nevertheless, de novo synthesis of the 82-kDa subunit was not required for viral early, intermediate, and late gene expression or DNA replication. Overexpression of the A8L gene alone, produced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect. Laser confocal fluorescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated in the absence of inducer, indicating that the A8L protein has a role in morphogenesis of the core and subsequent events.

Novobiocin Inhibits Vaccinia Virus Replication by Blocking Virus Assembly

Novobiocin inhibits the replication of vaccinia virus in cultured BSC40 cells. All classes of viral proteins were synthesized during synchronous infection in the presence of drug. The onset of DNA replication was delayed slightly, yet the extent of DNA replication in the presence of novobiocin was comparable to that of a control infection. A delay in the temporal transition to late viral protein synthesis was in keeping with the effects on DNA replication. Although the precursor forms of the major viral structural proteins were synthesized normally at late times, the proteolytic processing of these polypeptides was inhibited, which suggested an impediment to virus assembly. Electron microscopy revealed that novobiocin blocked virus morphogenesis at an early stage. Conversion of the concatameric DNA replication intermediates into hairpin telomeres occurred in the presence of novobiocin, confirming that telomere resolution was not coupled to virus assembly. Novobiocin is the latest addition to a class of antipoxviral agents, which includes rifampin and IMCBH, that arrest morphogenesis.

Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA.

The adenovirus L1 unit represents an example of an alternatively spliced precursor messenger (pre-mRNA) where on 5' splice can be jointed to one of two alternative 3' splice sites, producing the 52,55K or the IIIa mRNAs (Fig. 1a). Efficient usage of the distal IIIa 3' splice site requires late viral protein synthesis and is therefore confined to the late phase of virus infection. Here we show that, in extracts from uninfected cells, the classical SR proteins, which are essential splicing factors, inhibit IIIa pre-mRNA splicing by binding to an intronic repressor element and preventing recruitment of the U2 small nuclear ribonucleoprotein particle to the spliceosome. We further show that the viral repressor element has splicing-enhancer activity when appropriately placed in the pre-mRNA. Together, our results demonstrate that SR proteins function as activators or repressors of splicing depending on where on the pre-mRNA they bind.

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.

Phenotypic characterization of mutants in vaccinia virus gene G2R, a putative transcription elongation factor.

The phenotypic defects of two mutants of vaccinia virus, the lesions of which map to gene G2R, were characterized in vivo, and the results suggest a role for the G2R protein in viral transcription elongation. Both a temperature-sensitive mutant, Cts56, and an isatin-beta-thiosemicarbazone-dependent deletion mutant, G2A, in gene G2R have a characteristic and unique defect in late viral gene expression. The G2R mutants synthesize early viral RNA, early viral proteins, and viral DNA normally under nonpermissive conditions. In G2R mutants, late viral protein synthesis begins at the normal time, low-molecular-weight viral proteins are synthesized in normal quantities, but synthesis of high-molecular-weight viral proteins is reduced in amount. Intermediate and late promoter utilization is normal in G2R mutants, but intermediate and late RNAs are reduced in size. The reduction in length of the intermediate and late mRNAs represents a truncation of mRNA 3' ends. Thus, intermediate and late RNAs are too short to encode large proteins but long enough to encode small proteins, therefore accounting for the protein synthesis phenotype. These results suggest that the G2R protein acts to regulate the elongation potential of the viral RNA polymerase late during a vaccinia virus infection.

Adenosine N1-oxide inhibits vaccinia virus replication by blocking translation of viral early mRNAs.

Adenosine N1-oxide (ANO) is a potent and highly selective inhibitor of vaccinia virus replication. We examined the impact of ANO on vaccinia virus macromolecular synthesis during synchronous infection of BSC40 cells. Viral DNA replication and viral late protein synthesis were blocked completely by ANO, effects that were attributable to a defect in the expression of viral early genes. Vaccinia virus early proteins were not synthesized in the presence of ANO, even though vaccinia virus early mRNAs were produced. Cellular protein synthesis was unaffected by ANO, and virus infection in the presence of the drug did not elicit the normal shutoff of host protein synthesis. Adenosine N1-oxide triphosphate (ANO-TP), the predominant metabolite of the drug in vivo, could substitute for ATP in RNA synthesis by purified vaccinia virus RNA polymerase. ANO-TP could support early transcription by purified virions if dATP was provided as an energy source. ANO-TP did not inhibit early transcription in the presence of ATP. These findings suggest a novel antiviral mechanism whereby incorporation of a modified nucleotide into viral mRNAs might selectively block viral gene expression at the level of translation. We believe that ANO merits consideration as an antipoxvirus drug for topical treatment of molluscum contagiosum in humans.

The Effect of Inhibitors on the Growth of the Entomopoxvirus from Amsacta moorei in Lymantria dispar (Gypsy Moth) Cells

Within the family of Poxviridae, the entomopoxviruses are the most distant relatives of the more well-known and intensively studied orthopoxviruses (vaccinia and variola). The growth of the entomopoxvirus from Amsacta moorei (AmEPV) has been characterized in cell culture and compared to that of vaccinia virus (VV), the prototypic orthopoxvirus. The overall characteristics of infected cell cultures were generally similar between the two viruses. One striking difference noted was the apparent absence of proteolytic processing of late AmEPV viral proteins, a hallmark of vertebrate poxvirus infections associated with viral morphogenesis. AmEPV, like VV, was found to be sensitive to all the inhibitors of viral infection tested including phosphonoacetic acid, 1-β-d-arabinofuranosylcytosine, and isatin-β-thiosemicarbazone (IBT), a compound associated with the rather specific inhibition of vertebrate poxviruses. While both VV and AmEPV are inhibited by IBT, the inhibition of AmEPV, unlike that of VV, is not accompanied by either a breakdown of ribosomal RNA or a global inhibition of late viral protein synthesis. Instead, in the presence of IBT, AmEPV enveloped, immature virions form devoid of a well-differentiated core, which unlike mature virions fail to insert into occlusion bodies.

Inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites.

IFN-gamma-induced nitric oxide (NO) in the murine macrophage-derived cell line RAW 264.7 was previously shown to inhibit replication of the poxviruses ectromelia and vaccinia (VV) and HSV-1. In the current study we demonstrate that murine macrophages activated as a consequence of VV infection express inducible nitric oxide synthase. These activated macrophages were resistant to infection with VV and efficiently blocked the replication of VV and HSV-1 in infected bystander cells of epithelial and fibroblast origin. This inhibition was arginine dependent, correlated with nitrite production in cultures, and reversible by the NOS inhibitor N omega-monomethyl-L-arginine. NO-mediated inhibition of VV replication was studied by treatment of virus-infected human 293 cells with the NO donor S-nitroso-N-acetyl-penicillamine. Using a VV-specific DNA probe, antibodies specific for temporally expressed viral proteins, and transmission electron microscopy, we have shown that NO inhibited viral late gene protein synthesis, DNA replication, and virus particle formation, but not expression of the early proteins that were analyzed. Putative enzymatic targets of NO were identified by reversing the NO-mediated inhibition of VV replication in the 293 cells with exogenous ferrous sulfate and L-cysteine. Reversal of inhibition may derive from the capacity of these reagents to protect or regenerate nonheme iron or thiol groups, respectively, which are essential for the catalytic activities of enzymes susceptible to inactivation by NO.

Restriction of Vaccinia Virus Replication in CHO Cells Occurs at the Stage of Viral Intermediate Protein Synthesis

Vaccinia virus (VV) and cowpox virus (CPV) differ in their abilities to replicate in Chinese hamster ovary (CHO) cells because VV has a disrupted host range ( hr) gene. To facilitate an examination of the molecular events associated with abortive infection of CHO cells with VV, we constructed two sets of recombinant viruses that contain a viral early promoter regulating the cat gene encoding chloramphenicol acetyltransferase and viral intermediate or late promoters regulating the lacZ gene encoding β-galactosidase. The first set has the disrupted hr gene and the second set has the intact CPV homolog, allowing replication in CHO cells. Reporter chloramphenicol acetyltransferase and β-galactosidase assays demonstrated that early gene expression was unperturbed, whereas intermediate and late gene expression were severely inhibited under abortive conditions. Metabolic labeling studies confirmed the absence of viral late protein synthesis. The accumulation of viral DNA under abortive conditions was consistent with the synthesis of viral early proteins and established that inhibition of late protein synthesis was not primarily due to a replicative block. Analysis of steady state levels of viral mRNAs revealed substantial quantities of early and intermediate species but only very small amounts of late mRNAs under nonpermissive conditions. Despite the presence of viral intermediate mRNAs, the corresponding intermediate proteins, which function as late transcription factors, were not detected by immunoprecipitation of lysates from metabolically labeled infected CHO cells. Furthermore, when expression of lacZ was regulated by an intermediate promoter, no β-galactosidase was detected even though lacZ transcripts were present. Thus, the abortive phenotype in CHO cells can be explained by a block to translation of intermediate mRNAs which prevents the synthesis of late transcription factors.

Action of brefeldin A on translation in Semliki Forest virus-infected HeLa cells and cells doubly infected with poliovirus.

Brefeldin A (BFA) is a macrolide antibiotic that blocks membrane traffic through the vesicular system and affects the glycosylation of viral glycoproteins. Treatment of HeLa cells infected with Semliki Forest virus (SFV) with BFA enhances the synthesis of late viral proteins. Proteolytic cleavage of p107 is partially blocked and viral glycoproteins accumulate in BFA-treated cells. This enhanced synthesis of late SFV proteins is due, at least in part, to an increase in the formation of the subgenomic SFV 26S mRNA. Since BFA blocks the replication of poliovirus genomes without affecting the cleavage of the translation initiation factor p220, protein synthesis was analysed in doubly infected cells. HeLa cells infected with SFV and poliovirus at the same multiplicity predominantly synthesize poliovirus proteins. But if these cells are treated with BFA they synthesize significant amounts of SFV capsid protein C for several hours, despite the fact that p220 has been degraded.

Cyclosporin A inhibits vaccinia virus replication in vitro.

The effect of the immune modulator, Cyclosporin A (CsA) on vaccinia virus replication has been examined in cell cultures. In the present study we report that CsA is anti-viral towards vaccinia virus. Viral yield was inhibited by more than 97% after 24 h postinfection in the presence of 16 microM to 40 microM CsA. An analysis of the infectious cycle in greater detail revealed that CsA did not effect the total level of [35S] methionine incorporation into vaccinia infected cells. However, both early and late viral gene expression were inhibited by CsA. Late viral protein synthesis appeared to be more sensitive to the drug. At least one late viral polypeptide of approximately Mr 38,000 was virtually undetected up to 8 h postinfection in the presence of 40 microM CsA. Host protein synthesis which is normally inhibited by the virus was not turned off until very late in infection. Viral DNA replication was also inhibited by the addition of CsA at levels comparable to those observed for late protein synthesis.

Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors

Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.