Biomedical subjects

M Esteban

Publications and source records attributed to M Esteban.

At least 109 records · Page 6Linked to original sources

PubMed1993

The interferon-induced double-stranded RNA-activated human p68 protein kinase inhibits the replication of vaccinia virus.

The interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (p68 kinase) has long been implicated as one of the antiviral agents responsible for overcoming virus infections. To investigate the antiviral potential of p68 kinase, we have generated a recombinant vaccinia virus that expresses human p68 kinase under the control of lac operator/repressor element. Upon induction of p68 kinase gene with the inducer isopropyl-beta-D-thiogalactoside (IPTG), we observed in cultured cells a severe (> 90%) inhibition of virus protein synthesis; this inhibition correlated with autophosphorylation of p68 kinase. As a result of inhibition in the synthesis of virus polypeptides, there was a 100-fold decrease in virus yields. When cells were infected with the recombinant virus expressing lys296-->arg296 mutant p68 kinase there was no reduction in virus yields. Our findings demonstrate that human p68 kinase once activated severely inhibits vaccinia virus replication as a result of inhibition of protein synthesis.

Animals↗

PubMed1993

A mutation of the nucleoside triphosphate phosphohydrolase I (NPH-I) gene confers sensitivity of vaccinia virus to interferon.

The role of vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) in determining the resistance of this virus to alpha-interferon (IFN) was analyzed by using a well-defined temperature-sensitive mutant (ts36) in the NPH-I gene. Detailed analysis of viral proteins and mRNAs produced in cultured cells treated with or without IFN-alpha showed a strong inhibition by IFN of the synthesis of late virus polypeptides at the nonpermissive temperature, e.g., 83.8% inhibition for the luciferase reporter gene, which is associated with a decrease in steady-state mRNA levels. Sensitivity of ts36 to IFN was only the consequence of the point mutation in the NPH-1 gene, as shown by characterization of the rescued virus, R36. Our findings demonstrate that functional NPH-1 is required, at least in part, for resistance of vaccinia virus to IFN.

Acid Anhydride Hydrolases↗

PubMed1993

Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription.

We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with a 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 receptor 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.

Amino Acid Sequence↗

PubMed1993

The vaccinia virus 14-kilodalton fusion protein forms a stable complex with the processed protein encoded by the vaccinia virus A17L gene.

The mechanism by which the 14-kDa fusion protein of vaccinia virus (VV) is anchored in the envelope of intracellular naked virions (INV) is not understood. In this investigation, we demonstrate that the 14-kDa protein interacts with another virus protein with an apparent molecular mass of 21 kDa. Microsequence analysis of the N terminus of the 21-kDa protein revealed that this protein is encoded by the VV A17L gene. The 21-kDa protein is processed from a 23-kDa precursor, by cleavage at amino acid position 16, at the consensus motif Ala-Gly-Ala, previously identified as a cleavage site for several VV structural proteins. The 21-kDa protein contains two large internal hydrophobic domains characteristic of membrane proteins. Pulse-chase analysis showed that within 1 h after synthesis, the 14-kDa protein forms a stable complex with the 21-kDa protein. Formation of the complex was not inhibited by rifampin, indicating that the interaction between these two proteins occurs prior to virion morphogenesis. Immunoprecipitation analysis of disrupted virions showed the presence of the 21-kDa protein in the viral particle. Release of the 14-kDa-21-kDa protein complex from INV required treatment with the nonionic detergent Nonidet P-40 and a reducing agent. The protein complex consisted of 14-kDa trimers and of 21-kDa dimers. Since the 14-kDa fusion protein lacks a signal sequence and a large hydrophobic domain characteristic of membrane proteins, our findings suggest that the 21-kDa protein serves to anchor the 14-kDa protein to the envelope of INV.

Amino Acid Sequence↗

PubMed1993

Amplification of polyadenylated nontranslated small RNA sequences (POLADS) during superinfection correlates with the inhibition of viral and cellular protein synthesis.

The production and the role of POLADS in cells infected with vaccinia virus (VV) was studied in doubly infected HeLa and L cells. In cells first infected with VV followed by superinfection with UV-irradiated virus (VVuv), referred to as VV + VVuv, the course of viral polypeptide synthesis was not significantly altered. However, if cells were first infected with VVuv, followed by superinfection with VV, referred to as VVuv + VV, both host and viral polypeptide synthesis was compromised. Labeling of such infected cells with 3H-adenosine revealed that infection with VVuv or VVuv + VV, caused an amplification of incorporation of label both in the large and small size class RNAs compared to RNAs obtained from a normal infection. On the other hand, labeling of cells with 3H-adenosine which were infected with VV + VVuv, caused no significant change in the labeling pattern of large and small size class RNAs compared to labeled RNAs from normal infection. The large size class RNAs isolated from infection with VVuv or VVuv + VV, were translated in the reticulocyte lysate cell-free system much more productively compared to the same size RNAs obtained from normal infection. When these large size class RNAs were added together with HeLa cell mRNAs to the cell-free translational system, competition between the two types of mRNAs ensued. The small size class RNAs (POLADS) isolated from infection with VVuv or VVuv + VV, had little translational activity, but when added together with HeLa cell mRNAs, caused a striking inhibition of HeLa cell mRNA translation which was more pronounced than the inhibition caused by the small size class RNAs obtained from normal infections. POLADS obtained from infection with VV or VV + VVuv inhibited HeLa cell protein synthesis to the same extent and were about two times less active than the POLADS obtained from infection with VVuv or VVuv + VV. These results demonstrate that if cells are first infected with normal virus, the superinfecting UV-irradiated virus has little or no effect on the course of VV replication, suggesting that the production of POLADS under these conditions is regulated. However, when cells are first infected with VVuv, POLADS production is amplified to an "abnormal" level, thus, both viral polypeptide synthesis and replication of the superinfecting VV is compromised resulting in lower yields of virus.

Animals↗

PubMed1992

Insertional inactivation of the vaccinia virus 32-kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells.

The mechanism of poxvirus attachment to cells is poorly understood. We have identified a 32-kDa envelope protein of vaccinia virus which binds to the surface of cultured cells. This binding is specific and selective (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:22174-22180, 1990; C. Lai, S. Gong, and M. Esteban, J. Virol. 65:499-504, 1991). In this investigation, we studied the effect of inactivating the 32-kDa gene (32K gene) on the biology of vaccinia virus. We show that inactivation of the 32K gene decreases by 80% the mortality of mice infected with 32K- vaccinia virus. This reduction in mortality correlates with diminished viral gene expression in target tissues. In highly polarized epithelial cells, viral gene expression of 32K- virus was reduced (50 to 60%) at both the apical and basolateral surfaces in comparison with a 32K+ virus. Restriction of virus gene expression in polarized cell surfaces occurs for both intracellular and extracellular forms of infectious 32K- vaccinia virus. The two infectious forms of vaccinia virus 32K+ infect polarized cells preferentially by the basolateral surface. Our findings provide evidence of the importance of the 32-kDa protein in viral pathogenesis.

Animals↗

PubMed1992

Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans.

Vaccinia virus (VV) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gt11 expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of VV, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins.

Adult↗

PubMed1991

Interferon treatment inhibits early events in vaccinia virus gene expression in infected mice.

We have analyzed the role of exogenous administration of mouse interferon (IFN alpha + beta) on the replication of vaccinia virus in peritoneal cells and in the spleen of Balb/c mice. Mice were pretreated for 16 hr with IFN and then infected with a vaccinia virus recombinant expressing luciferase under an early or late virus promoter, and the enzyme activity was measured in the course of virus infection. A dose of IFN as low as 10(3) units/mouse abolished the appearance of luciferase activity in cells of the peritoneal cavity and in spleen cells. The IFN-mediated inhibition of luciferase activity was observed even when mice were infected 4 days after the administration of IFN. The IFN-treated animals were considered free of virus since neither luciferase nor viral proteins were detected in target cells several days after virus infection. Despite a severe IFN-mediated inhibition of luciferase activity, the appearance of luciferase on mRNA levels was not inhibited 6 hr after virus infection. Our finding revealed that replication of vaccinia virus in Balb/c mice is exquisitively sensitive to inhibition by IFN and that this effect occurs at early times postinfection, most likely as a result of a translational block.

Animals↗

PubMed1991

Structural properties of HIV-1 Env fused with the 14-kDa vaccinia virus envelope protein.

To gain insights into the structure-function relationship of the envelope (env) glycoprotein of the human immunodeficiency virus type 1 (HIV-1) we have generated a vaccinia virus (VV) recombinant (VV-14kENV) that expresses a fusion protein (14k-env) consisting of the VV 14-kDa envelope protein (110 amino acids) fused at the C-terminus with HIV-1 env protein (816 amino acids). The 14k-env protein displayed unique structural properties in virus-infected cells. This protein was recognized by 14 kDa-specific antisera as well as HIV-1 env antisera. It was not cleaved during virus infection of cultured cells of various origins, it was stable, it was not released to the medium, and it was not incorporated into virions. Instead of a predicted 174-kDa protein, two proteins of about 110 and 100 kDa were observed. The size reduction of the fusion protein was due to limited glycosylation (110 kDa) and formation of unglycosylated protein (100 kDa). The 14k-env protein formed oligomeric structures and was exposed on the cell surface after virus infection. When mice were inoculated with the recombinant virus that expresses the 14K-env fusion protein, humoral immune response against gp160 was observed. Our findings suggest that 14k-env protein might display novel immunogenic properties.

Animals↗

PubMed1991

Vaccinia virus preferentially enters polarized epithelial cells through the basolateral surface.

The uptake of vaccinia virus in polarized epithelial cells was studied to determine whether the site of entry was confined to either the apical or the basolateral membrane. Virus infection was monitored with a recombinant vaccinia virus carrying the luciferase reporter gene. Using cell lines MDCK and MDCK-D11, a clonal line with high transepithelial electrical resistance, we determined that vaccinia virus preferentially enters through the basolateral membrane. The possibility that there is a polarized cell surface distribution of vaccinia virus receptors which may be involved in systemic poxvirus infections is discussed.

Animals↗

PubMed1991

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.

Animals↗

PubMed1991

The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia coli induces virus immunity in animals.

Vaccinia virus (VV) was successfully used as a live vaccine to eradicate smallpox, but the nature of viral proteins involved in eliciting viral immunity has not yet been identified. A potential candidate is a 14-kDa VV envelope protein that is involved in virus penetration at the level of virus-cell fusion, in cell-cell fusion late in infection, and in virus dissemination. The 14-kDa envelope protein has been produced in Escherichia coli, with properties similar to those of the native protein found in the virus particle and in infected cells (C. Lai, S. Gong, and M. Esteban, J. Biol. Chem. 256:22174-22180, 1990). In this investigation, we showed that mice immunized with purified VV 14-kDa protein synthesized in E. coli in the form of a monomer or a trimer develop high-titer neutralizing antibodies and are protected when challenged with lethal doses of wild-type VV. Our findings demonstrate that it is possible to confer protection against VV through immunization with the 14-kDa envelope protein.

Ammonium Sulfate↗

PubMed1991

Mechanism of selective translation of vaccinia virus mRNAs: differential role of poly(A) and initiation factors in the translation of viral and cellular mRNAs.

We have recently demonstrated that the poly(A) moieties of short RNAs obtained from both in vitro transcription and from vaccinia virus (VV)-infected cells exhibit dissimilar effects on the in vitro translation of cellular and VV mRNAs (R. Bablanian, G. Coppola, P. Masters, and A. K. Banerjee, Virology 148:375-380, 1986; M. J. Su and R. Bablanian, Virology 179:679-693, 1990). In the present study, we have investigated the roles of poly(A), m7GTP, and initiation factors in the mechanism of selective translation of VV mRNAs. The effects of unfractionated poly(A) [termed poly(A)un, with various chain lengths up to 3,000 nucleotides] and a 150- to 300-nucleotide fraction of synthetic poly(A) [termed poly(A)150-300] on the translation of HeLa cell mRNAs and early and late VV mRNAs were studied. Both the poly(A)un and the poly(A)150-300 completely inhibited the translation of HeLa cell mRNAs obtained from total cytoplasmic RNA in the nuclease-treated reticulocyte lysates. Viral mRNAs from total cytoplasmic RNA also were slightly inhibited (15 to 38%) by the poly(A)un, whereas the poly(A)150-300 had no significant effect on their translation. The translation of oligo(dT)-cellulose-selected HeLa mRNAs was as sensitive to inhibition by poly(A)150-300 as the mRNAs found in total cytoplasmic RNA. However, the translations of oligo(dT)-cellulose-selected viral mRNAs become more sensitive to the inhibitory effect of poly(A)150-300 than the translations of viral mRNAs found in the total cytoplasmic RNA. Both HeLa and VV mRNAs became more resistant to the poly(A)-mediated inhibition when these mRNAs were deadenylated, but the relative resistance to inhibition by poly(A)150-300 of deadenylated VV mRNAs was much greater than that of HeLa cell mRNAs. The translation of VV mRNAs was significantly less inhibited than the translation of HeLa mRNAs when the cap analog, m7GTP, was added to the cell-free system. The inhibition of HeLa cell mRNA translation by both poly(A)un and poly(A)150-300 was completely restored when poly(A)-binding protein (PAB) was added to the cell-free translational system. The addition of eukaryotic initiation factor 4A (eIF-4A) did not restore translation when poly(A)un was used to inhibit translation; however, inhibition by poly(A)150-300 was significantly reversed by this initiation factor. The reversal of poly (A)-mediated inhibition of HeLa cell mRNA translation was additive when PAB was used together with eIF-4A. Early VV mRNA translation was only slightly inhibited by poly(A)un (15%), and this inhibition was completely reversed by either PAB or eIF-4A.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrier Proteins↗

PubMed1990

Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli.

Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene. We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus. 1) 14 kDa is a trimer of identical subunits. 2) A monomer binds to itself more strongly than to a dimer or a trimer. 3) Oligomerization does not require cellular factors. 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity. 5) 14-kDa binds with specificity to the cell surface of cultured cells.

Amino Acid Sequence↗

PubMed1990

Structural and functional characterization of a cell surface binding protein of vaccinia virus.

The nature of the interaction between the enveloped DNA-containing poxviruses and the surfaces of host cells as a first step in virus infection is not known. In this investigation we have identified and defined structural and functional properties of a 32-kDa protein of vaccinia virus. This protein is part of the virus envelope and binds to the cell surface of various cultured cells. The gene encoding the 32-kDa viral protein was mapped and sequenced. It was found to code a 35,426-Da protein with a large N-terminal domain with sequence homology to carbonic anhydrases and a C-terminal domain with sequences similar to those of the attachment glycoprotein VP7 of rotavirus and to transmembrane proteins. A potential cell surface binding domain was within the last 50 amino acid residues of the C terminus. The 32-kDa protein is basic, predicted pI 8.67, is synthesized at late times post-infection, may form dimers held by disulfide bonds at the single cysteine 262, and is apparently non-glycosylated. The 32-kDa protein is a vaccinia virus antigen, with predicted antigenic sites located near amino acids 108-110 (carbonic anhydrase domain) and 298-299 (transmembrane domain). Several lines of evidence suggest that the 32-kDa protein is needed for efficient virus replication in cultured cells but that in addition to this protein other viral proteins are involved in the process of virus entry into cells.

Amino Acid Sequence↗

PubMed1990

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression.

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.

Base Sequence↗

PubMed1990

Vaccinia virus induces cell fusion at acid pH and this activity is mediated by the N-terminus of the 14-kDa virus envelope protein.

The mechanism by which the large-size poxviruses enter animal cells is not known. In this investigation we show that acid pH treatment of wild-type vaccinia virus-infected cells triggers strong fusion of cells in culture, with an optimum at pH 4.8. We have identified the virus-induced fusion protein as a 14-kDa envelope protein, based on the ability of a 14-kDa specific monoclonal antibody (mAbC3) to block vaccinia virus-induced fusion-from-within and fusion-from-without. We provide genetic evidence for a role of the 14-kDa protein in cell fusion, since insertion of the 14-kDa encoding gene into the genome of nonfusogenic mutant viruses generates heterozygous viruses that now acquire acid pH-dependent fusion activity. DNA sequence analyses of the 14-kDa encoding gene of the mutant viruses, 65-16 and 101-14, reveal N-terminal deletions of 46 and 10 amino acids, respectively. These deletions remove a small hydrophobic region at the N-terminus of the 14-kDa protein and prevent fusion. Our findings demonstrate that vaccinia virus can induce strong fusion of cells in culture at acid pH implying some entry of the virus by endocytosis, that the 14-kDa virus envelope protein is the fusogenic protein, and that the N-terminal proximal region is involved in fusion.

Amino Acid Sequence↗

PubMed1990

Regulated expression of nuclear genes by T3 RNA polymerase and lac repressor, using recombinant vaccinia virus vectors.

Recombinant vaccinia viruses that express the bacteriophage T3 RNA polymerase (VV-T3pol) or the Escherichia coli lac repressor (VV-lacI) under control of the early-late vaccinia promoter P7.5 were constructed. To determine whether phage polymerase and lac repressor can function in the nucleus of mammalian cells, the bacterial chloramphenicol acetyltransferase (CAT) gene was cloned downstream of a T3 promoter (PT3-CAT) or downstream of a T3 promoter-lac operator fusion element (PT3Olac-CAT), and these reporter gene cassettes were introduced stably into NIH 3T3 or Ltk- cells. Infection of 3T3/PT3-CAT or Ltk-/PT3-CAT cells by VV-T3pol led to rapid expression of CAT (greater than 20 ng of CAT protein per 10(6) cells). The presence of hydroxyurea (which blocks virus DNA replication) did not prevent CAT production. When 3T3/PT3Olac-CAT cells were infected with both VV-T3pol and VV-lacI (multiplicities of infection of 2.5 and 10, respectively), greater than 30-fold repression of CAT gene activity by lac repressor was observed. This could be reversed to unrepressed levels by the presence of 10 mM o-nitrophenyl-beta-D-galactoside (IPTG) in the medium. Regulated expression of the target gene was observed with cell lines that had been maintained for over 1 year (greater than 50 passages in culture), and Southern blot analysis revealed the presence of the CAT gene only in the nuclear fraction in these cells, demonstrating the stability of the target gene. These results indicate that vaccinia virus-encoded proteins can function in the mammalian nucleus and provide the basis for a genetic system in which essential vaccinia virus genes, placed in the chromosome of a cell, can be used to complement defective virus particles. This approach may prove useful for other virus systems.

Animals↗