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M Esteban

Publications and source records attributed to M Esteban.

At least 145 records · Page 8Linked to original sources
PubMed1986

Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses.

Adenosine Triphosphate↗
PubMed1986

Mode of sensitivity and resistance of vaccinia virus replication to interferon.

In this study we show that vaccinia virus replication can be sensitive or resistant to interferon (IFN) in the same strain of mouse L cells. When IFN-treated L cells were maintained in suspension culture, infection led to a rapid inhibition of both viral and cellular protein synthesis together with breakdown of viral RNA and of rRNA. When IFN-treated L cells were maintained in monolayer culture, infection did not lead to significant inhibition of viral protein or RNA synthesis and breakdown of viral or of rRNA was not observed. The resistance of vaccinia virus replication to IFN was not dependent on the input multiplicity or state of growth of the cells (actively dividing or resting). Qualitative and quantitative differences in viral transcription and translation were observed between the two virus-cell systems. Our findings are consistent with the hypothesis that the sensitivity or resistance of vaccinia virus to IFN is mediated by specific viral products that act as activators or selective inhibitors of, at least, the dsRNA-dependent ppp(A2'p)nA synthetase/RNase system.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Biochemical and electron microscopic studies of the transcription of vaccinia DNA by RNA polymerase from Escherichia coli: localization and characterization of transcriptional complexes.

We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation. Electron microscopic studies of the interaction of E. coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with 'pre-early', 'early', and 'late' in vivo RNA; 'self-annealing' of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected. These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors. With extended in vitro incubations of the E. coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish. This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring. The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA. The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.

Animals↗
PubMed1985

Generation of a dominant 8-MDa deletion at the left terminus of vaccinia virus DNA.

Vaccinia virus mutants were obtained in high frequency from mouse Friend erythroleukemia (FEL) cells persistently infected with this virus, which contains a large (122-MDa) DNA. During long-term cell passages viral particles with deletions of the DNA are generated in FEL cells. These mutants have a major 8-MDa deletion starting between 2.2 and 3.2 MDa from the left terminus of the viral genome. More than half of the left end terminal repetition is deleted. These mutants have reduced infectivity compared to wild-type virus. The ease with which vaccinia virus mutants are obtained in FEL cells should provide a suitable system for generating mutants with other poxvirus and permit study of the genetic basis of virulence for this group of viruses.

Animals↗
PubMed1985

Interferon inhibits marker rescue of vaccinia virus.

In this investigation we have examined the effect of human interferon (IFN) type alpha on the ability of vaccinia virus to recombine within infected African green monkey kidney cells (BSC-40). We measured by marker rescue, the extent of insertion of cloned 5-kb Hind III-J restriction fragment of wild-type vaccinia DNA into the genome of temperature-sensitive mutants. We showed that IFN at doses of 100-1000 U/ml inhibited the replication of vesicular stomatitis virus (VSV) and polio viruses but not of vaccinia virus. Vaccinia virus adsorption, penetration, uncoating, protein synthesis, and yields were not inhibited. However, marker rescue of vaccinia virus was inhibited by IFN. This inhibition was not related to IFN-mediated changes in uptake of exogenous DNA or enhanced degradation of the transfected DNA. These results suggest that IFN affects homologous vaccinia DNA recombination.

Animals↗
PubMed1985

Inhibitory effect of interferon on the genetic and oncogenic transformation by viral and cellular genes.

The rodent established cell lines LTk- and NIH 3T3 have been used as recipients in gene transfer experiments to study the effect of interferon treatment on the genetic and oncogenic transformation by several genes of viral and cellular origin. Our results show that interferon severely inhibits, to a similar extent, the stable transformation of Ltk- and NIH 3T3 cells by the chicken thymidine kinase (tk) gene, Ecogpt gene, simian virus 40, v-Ha-ras, and human c-Ha-ras and c-Ki-ras oncogenes. These results are consistent with an inhibition by interferon at the level of stabilization or integration, or both, of exogenous DNA sequences in the recipient cells, with an apparent effect on gene expression.

Animals↗
PubMed1985

Interferon prevents the generation of spontaneous deletions at the left terminus of vaccinia virus DNA.

In this report we have shown that Friend erythroleukemia cells persistently infected with vaccinia virus maintain the persistent infection even after 1 year of continuous interferon (IFN) treatment. The persistently infected cultures were responsive to IFN as determined by their ability to induce 2-5A synthetase, to increase the intracellular levels of 2-5A, and to cause rRNA cleavage. While large deletions at the left terminus of vaccinia DNA occurred readily in the virus population from untreated cells, IFN completely suppressed the generation of these spontaneous deletions. Removal of IFN from these cultures led to the appearance of similar deletions at the left terminus of the viral genome. The regions deleted contain more than half of the left-end inverted terminal repetition of the vaccinia genome. These findings show that IFN alters specific events associated with the generation of vaccinia DNA deletions.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.

Antibodies, Monoclonal↗
PubMed1984

Resistance of vaccinia virus to interferon is related to an interference phenomenon between the virus and the interferon system.

In this investigation the sensitivity of vaccinia virus to interferon (IFN) has been examined in cultured cells. In a variety of mouse and human cells of different origins vaccinia virus functions (RNA, protein, and virus yields) were found to be relatively resistant to IFN. In these systems, the levels of the IFN-mediated enzyme activities (2-5A synthetase and protein kinase) were severely impaired by the virus. This virus-mediated inhibitory effect developed with time after infection and was dependent on viral protein synthesis. Mixed infections between vaccinia virus and viruses (VSV or polio) which are sensitive to IFN showed that both protein synthesis and virus yields were not inhibited. These findings show that vaccinia virus can overcome the antiviral action of IFN and that viral gene functions appear to be involved in this interference phenomenon.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Nature and mode of action of vaccinia virus products that block activation of the interferon-mediated ppp(A2'p)nA-synthetase.

In this report it has been shown that inhibition of the 2-5A synthetase in IFN-treated, vaccinia virus-infected mouse L and human HeLa S3 cells is related to specific viral functions. This inhibition occurs concomitantly with degradation of ATP and with dephosphorylation of ppp(A2'p)nA. At least two viral-mediated enzyme activities are thought to be involved in this process, an ATPase and a phosphatase. The ATPase activity was established after determining the extent of hydrolysis of ATP, the nature of 2-5A, and the relative abundance of the different oligomers. Cytoplasmic cell extracts and purified vaccinia virions were bound to poly (I):(C) agarose, incubated with [3H]ATP, [alpha-32P]ATP, or [gamma-32P]ATP, and the extent of hydrolysis of ATP was determined by TLC. Authentic 2-5A and the relative abundance of the various oligomers were characterized by enzymatic and alkali treatments and identification by TLC and HPLC analysis. The phosphatase activity was measured by TLC after determining the degree of dephosphorylation of 2-5A from the extent of labeling at the 5'-OH termini with [gamma-32P]ATP and polynucleotide kinase. While free 5'-OH termini were not observed in oligomers synthesized with bound poly (I):(C) agarose enzyme fractions from IFN-treated, uninfected cells, a strong phosphorylation was found in oligomers from IFN-treated, infected cells. These findings suggest that it is the contribution of these viral enzyme activities that renders vaccinia virus resistant to interferons.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Effect of interferon on integrity of vaccinia virus and ribosomal RNA in infected cells.

The state of vaccinia and ribosomal RNAs in IFN-treated, vaccinia virus-infected mouse L cells grown in suspension is examined. In these cells a drastic inhibition (approximately 90%) of both viral and cellular protein synthesis occurs after virus infection of IFN-treated cells. The findings show that (1) primary transcription of vaccinia virus is not impaired by IFN, but is rather enhanced; (2) viral RNAs produced in IFN-treated, infected cells are predominantly of the early class; (3) these viral RNAs can be translated in a cell-free protein-synthesizing system; (4) in IFN-treated, infected cells there is extensive cleavage of 28 and 18 S rRNA at early times post infection, resulting in a characteristic cleavage pattern; (5) cleavage of rRNA is viral RNA dependent. The results indicate that in this virus-cell system the IFN-mediated inhibition of vaccinia and cellular protein synthesis is correlated with an alteration in ribosomal integrity.

Animals↗
PubMed1984

Induction of an anti-viral response and 2',5'-oligo A synthetase by interferon in several thymidine kinase-deficient cell lines.

It has previously been shown that a strain of thymidine kinase (tk)-deficient mouse L-929 cells was unable to respond to murine beta-interferon by induction of an anti-viral state and synthesis of double-stranded, RNA-dependent enzymes. Sensitivity to interferon can be restored by introducing into the cells a segment of Herpes simplex virus DNA containing the viral tk gene. It is shown here that not all Ltk(-) cell strains are resistant to interferon, suggesting that expression of a tk gene is not a prerequisite for response to interferon. Introduction of various genes into the resistant Ltk(-) strain, either alone or together with DNA containing the Herpes virus tk gene, leads to restoration of interferon sensitivity only when tk-containing DNA is inserted, showing that the activation of interferon responsiveness is not an artifact of the gene transfer, selection, and cloning procedures. The results imply that a component of the Herpes virus DNA introduced into the cells is able to activate interferon sensitivity.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Defective vaccinia virus particles in interferon-treated infected cells.

Vaccinia virus particles formed in interferon-treated, infected cells have been isolated. These particles have been characterized with regard to polypeptide composition, and ability to adsorb, penetrate, uncoat, and synthesize proteins in infected cells. As determined by one-dimensional SDS-PAGE analysis, with interferon concentrations of 100-500 u/ml, the pattern of [35S]methionine-labeled virion proteins was not altered; higher doses of interferon resulted in decreased labeling of some proteins. However, interferon doses of 100-500 u/ml decreased phosphorylation of vaccinia virus basic core polypeptide (P-11) by 30-70%; the same doses of interferon decreased the labeling of virus glycoproteins by 40-80%. Virus purified from interferon-treated cells adsorb, penetrate, and uncoat to a lesser extent than virus control. During infection to cells, these virus particles caused shutoff and synthesized the same spectrum of viral proteins as normal virus. These findings show that there are protein alterations in vaccinia virus particles isolated from interferon-treated, infected cells. These alterations may contribute to limit the spread of virus infection.

Animals↗
PubMed1984

Analysis of replicating vaccinia DNA in interferon-treated, virus-infected cells.

The effect of interferon (IFN) on the replication of vaccinia DNA has been examined. Studies were carried out in infected mouse L cells grown in monolayer cultures. We examined discontinuous synthesis of small DNA fragments, ligation of the small fragments to form intermediate-sized DNA molecules, completion of unit-length DNA molecules, and introduction of crosslinks at each end of the newly replicated DNA molecules late in infection. We measured by pulse-label and pulse-chase experiments the extent of incorporation of 3H-thymidine into DNA and the conversion of small size DNA into mature and cross-linked viral DNA molecules. Interferon inhibited the initiation of viral DNA and this correlated with an overall inhibition of protein synthesis. Interferon inhibited elongation of viral DNA but this did not correlate with an inhibition of protein synthesis. The effect on elongation was not the result of increased degradation of newly synthesized DNA. It is suggested that the effects of IFN on initiation and elongation of vaccinia DNA may be the result of a decrease in the availability of enzymes or factors involved in the regulation of viral DNA synthesis.

Animals↗
PubMed1984

Selective inhibition of viral gene expression as the mechanism of the antiviral action of PGA1 in vaccinia virus-infected cells.

We have previously shown that prostaglandins of the A series are potent inhibitors of the replication of several animal viruses in cultured cells. In this report we have studied the mechanism of the antiviral action of PGA1 in vaccinia virus-infected mouse L cells, where there is an alteration in both the rate and extent of the synthesis of some virus proteins. When cytoplasmic RNAs from PGA-treated, vaccinia virus-infected cells were translated in cell-free systems, similar selective inhibition of the synthesis of some viral polypeptides was observed. The lack of translation of some viral RNAs was not due to an impairment of the methylation process nor to a difference in ionic requirements. PGA1, even at doses as high as 10 micrograms/ml, did not exert any direct inhibitory action on transcription in vitro as measured in two cell-free systems, and had no effect on primary transcription-translation of vaccinia virus RNAs when assayed in coupled cell-free systems. Southern blot hybridization analysis of cytoplasmic RNAs to EcoRI restriction fragments of vaccinia DNA showed that PGA1 was able to induce major changes in the pattern of RNA transcripts during the course of viral infection. We propose that changes in the transcription programme of vaccinia virus RNAs could be due either to an alteration of specific viral proteins that regulate transcription by direct binding of PGA1, or to the synthesis and/or activation of a host product that mediates the antiviral action.

Animals↗