Biomedical subjects

M Esteban

Publications and source records attributed to M Esteban.

At least 127 records · Page 7Linked to original sources

PubMed1989

Humoral immune response elicited by highly attenuated variants of vaccinia virus and by an attenuated recombinant expressing HIV-1 envelope protein.

Attenuated variants of vaccinia virus have excellent potential for the construction of safe recombinant live vaccines. In this investigation, highly attenuated variants of vaccinia virus with several genetic markers and a variant recombinant were tested in Balb/c mice for their ability to induce humoral immune response. Mice primed with variants that had an 8-MDa deletion at the left end of the viral genome induced similar levels of circulating anti-vaccinia antibodies as the wild-type virus. However, mice primed with variants that had several genetic lesions (deletions and point mutations) induced lower levels of circulating anti-vaccinia antibodies. Mice primed and boosted with a recombinant variant with several genetic lesions, and containing the complete envelope gene of the human immunodeficiency virus (HIV) and the bacterial beta-galactosidase (beta-gal) gene, induced significant antibody response to gp 160 and beta-gal. The antibody response to gp 160 was markedly increased by successive inoculations with the recombinant variant. Our findings provide evidence that the extent of activation of the immune system by vaccinia variants can be modulated by the nature of the virus genetic lesion. In addition, when these variants are used as recombinant vaccines, it is possible to induce low levels of circulating anti-vaccinia antibodies after priming and yet achieve significant antibody response to virus-expressed foreign antigens, even after repeated boosters. Such variants could be useful in the design of live recombinant viruses as safe vaccines.

Animals↗

PubMed1989

Highly attenuated vaccinia virus mutants for the generation of safe recombinant viruses.

An attenuated vaccinia virus mutant with specific genetic lesions has been used to develop a vehicle for safer live recombinant virus vaccines. The mutant virus 48-7 has an 8-MDa deletion starting 2.2 MDa from the left end of the viral genome and point mutations in the gene encoding the 14-kDa fusion protein that determines the plaque-size phenotype of the virus. Using the highly sensitive reporter gene luciferase, we have shown that this mutant can generate recombinant viruses that infect cultured cells and animals with normal vaccinia virus tropism. Insertion of the envelope and gag genes of human immunodeficiency virus type 1 into the attenuated vaccinia mutant resulted in their efficient expression and precursor processing in infected cultured cells. Infection of mice with human immunodeficiency virus-vaccinia recombinant viruses elicited human immunodeficiency virus-specific antibodies. Using mice pretreated with cyclophosphamide as a model for immunosuppression, the reduced virulence of the mutant recombinant virus was clearly evident. These findings demonstrate that the highly attenuated vaccinia virus mutant 48-7 can be used to generate effective and safer vaccines.

Cloning, Molecular↗

PubMed1989

A single point mutation of Ala-25 to Asp in the 14,000-Mr envelope protein of vaccinia virus induces a size change that leads to the small plaque size phenotype of the virus.

The molecular defect responsible for a structural and functional abnormality of the 14,000-molecular-weight (14K) envelope protein of vaccinia virus has been identified. Through DNA sequence analysis of the entire 14K gene from wild-type vaccinia virus and three vaccinia virus mutants, a single base change of C to A was found that resulted in the substitution of Asp for Ala-25. This mutation is responsible for protein size abnormality, as documented by cell-free translation in a rabbit reticulocyte lysate of in vitro mRNA transcripts. In addition, through marker rescue experiments we show that this mutation is responsible for the small plaque size phenotype of vaccinia virus mutants. The structural consequence of the point mutation is a possible turn in an alpha-helix domain with destabilization of a hydrophobic interaction at the N terminus, resulting in monomers and trimers of vaccinia virus 14K protein with decreased electrophoretic mobilities. The functional consequence of the point mutation is a reduction in virulence of the virus.

Alanine↗

PubMed1989

Plaque size phenotype as a selectable marker to generate vaccinia virus recombinants.

In this report, we provide a new method for selection of vaccinia virus recombinants expressing foreign genes. The method is based on the use of the gene encoding the viral 14,000-molecular-weight envelope protein that rescues the small-plaque-size phenotype of a vaccinia virus variant to large-plaque-size virus. Selection of recombinants is easily obtained after visual inspection of large viral plaques.

Genetic Markers↗

PubMed1988

Antiviral activity of a synthetic analog of prostaglandin A in mice infected with influenza A virus.

We have previously shown that prostaglandins of the A series potently inhibit virus replication in several virus-host systems in vitro. In the present report we have studied the effect of a long-acting synthetic analog of PGA, 16,16-dimethyl-PGA2(Di-M-PGA2), on virus infection in vivo, using as a model Balb/c mice infected with influenza A (PR8) virus. Depending upon the dose of viral inoculum, PR8 virus caused the death of 50 to 100% of the animals in a period of 8-20 days. Di-M-PGA2-treatment significantly increased mouse survival by an average of 40%, independently of the dose of inoculum and the age of the animals. The fact that Di-M-PGA2-treatment decreased virus titers in the lungs and did not alter the host immune response, suggested that PGA's therapeutic action was due to suppression of virus replication. Finally, two anti-inflammatory compounds, which inhibit prostaglandin synthesis, aspirin and indomethacin, were shown not to significantly alter mouse survival in this system.

Animals↗

PubMed1988

Stability of vaccinia virus DNA during persistent infections: accumulation of left-end deletions and of tandem repeats at both ends of the viral genome and prevention by interferon.

The stability of the large vaccinia virus genome (122 MDa) has been studied in long-term cultures of persistently infected Friend erythroleukemia (FEL) cells. Deletions of about 8 MDa at the left terminus of vaccinia DNA are generated at early passages, and are maintained even after 2 years of continuous cell passages. The generation of deletions is followed by a stable accumulation of tandem repeats up to 6 MDa at the left end and up to 2 MDa at the right end of vaccinia DNA. Neither translocations nor rearrangements of DNA are observed during persistent infection. A recombinational mechanism within the tandem repeats or a mechanism similar to that described for adenovirus might explain the reiteration of tandem repeats at both ends. Significantly, in persistently infected cultures continuously treated with interferon (IFN) both the deletion and the large accumulation of tandem repeats were completely blocked. We suggest that reiteration of tandem repeats at the termini of vaccinia DNA might provide signals for more efficient virus multiplication in FEL cells.

Animals↗

PubMed1988

Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state.

Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals.

We have introduced the firefly luciferase gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infection. Luminescence produced by the action of luciferase [Photinus-luciferin:oxygen 4-oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of beta-galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals.

Animals↗

PubMed1987

Studies on the mechanism of entry of vaccinia virus in animal cells.

In order to study the mechanism of entry of vaccinia virus into cells the fate of virion associated polypeptides was investigated during infection of african green monkey kidney (BSC-40) cells with 35 S-methionine labelled virus. Approximately 12-15 percent of the virion polypeptides were degraded to acid-soluble products by 3 hours post-infection. Proteolysis was inhibited (50 percent) by methylamine, suggesting a lysosomal site of degradation. Neither methylamine or chloroquine inhibited virus infectivity or uncoating indicating a non-acid endocytic mechanism of entry. Subcellular fractionation studies on density gradients indicated that the bulk of the input virion polypeptides were associated with the plasma membrane fraction. In addition, input virion DNA was partially resolved from the membrane fraction. The results are most consistent with a mechanism of entry involving fusion of the virus with the plasma membrane.

Animals↗

PubMed1987

Selective inhibition of protein synthesis by synthetic and vaccinia virus-core synthesized poly(riboadenylic acids).

Studies were undertaken to compare the effect of poly(A)s from various sources on selective inhibition of protein synthesis in the reticulocyte lysate system programmed with viral and cellular mRNAs. RNA synthesized in vitro by vaccinia virus cores in the presence of only ATP inhibited overall HeLa cell polypeptide synthesis by over 80% with a minimal effect on translation of vaccinia virus mRNAs. Hybridization of the [alpha-32P]AMP-labeled RNA made in vitro by vaccinia virus cores in the presence of only ATP, showed no complementary to HindIII restriction fragments of vaccinia virus DNA indicating that the in vitro product was poly(A). Fractionation of synthetic and core-synthesized poly(A) into three size classes showed that the larger the size of poly(A), the greater its inhibitory activity of protein synthesis in the cell-free system. Inhibition of translation of mRNAs from vaccinia virus-infected HeLa cells was also observed in the presence of poly(A). However, virus-induced polypeptide synthesis was more resistant to the effect of poly(A) than were cellular polypeptides. Oligo(dT) when added to the reticulocyte lysate system was capable of reversing the inhibition of protein synthesis caused by both core-synthesized poly(A) and core-transcribed RNAs. These results indicate that poly(A) synthesized by the virion-associated enzyme has inhibitory properties similar to those of synthetic poly(A).

Cell-Free System↗

PubMed1987

Isolation and characterization of attenuated mutants of vaccinia virus.

Attenuated variants of vaccinia virus with two specific and stable genetic markers were obtained after long-term passage of persistently infected Friend erythroleukemia (FEL) cells. The genetic markers were an 8-MDa deletion on the left HindIII-C terminus of the viral genome and sequence alterations localized in the middle of the HindIII-A DNA fragment. This latter genetic marker led to small plaque size phenotype of these variants. The mode of replication of these variants in tissue culture cells and their virulence in mice were analyzed. In cultured cells, these variants have greatly reduced virus yields in cell lines of different origins. These variants penetrate into cells, synthesize early and late viral proteins, and replicate their DNA with about the same efficiency as wild-type virus. The defect of these variants appears at some step during virus maturation. When groups of BALB/c mice were inoculated intraperitoneally (ip) with these variants, 50% of the mice survived with greater than or equal to 1 X 10(9) plaque-forming units (PFU) as opposed to about 50% survival for mice inoculated with 1 X 10(6) PFU of wild-type virus. Mice inoculated with these variants were fully protected when challenged ip with lethal doses of wild-type virus. The reduced virulence of these variants correlated with the 8-MDa deletion; in addition, the plaque size phenotype marker contributes to a further decrease of the virulence of vaccinia virus. Due to their limited virus production and protective immune response, these variants may be potentially useful as vaccines.

Animals↗

PubMed1987

A 14K envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus.

The phenomenon of genetic variability and attenuation or virulence of poxviruses is poorly understood. We have identified mutants of vaccinia virus from untreated and interferon (IFN)-treated persistently infected Friend erythroleukemia (FEL) cells that have major alterations in the size of a virus structural protein. This protein is part of the virus envelope as documented with specific monoclonal antibody (mAbC3). This protein, under reducing conditions, has a molecular weight of about 14,000 (14K) Da in wild-type virus but gained 1.5 K in mutants from untreated, persistently infected cells and about 0.5 K in mutants from IFN-treated persistently infected cells. Under nonreducing conditions, this protein forms covalently linked oligomers which also differ in size between wild-type and mutant viruses. The 14K protein elicits humoral immune response as assessed by immunoblots of two-dimensional SDS-PAGE analysis using rabbit anti-vaccinia serum. Two molecular forms of the 14K protein with different isoelectric points were found only in mutants from untreated, persistently infected cells. Protein modifications were the result of DNA sequence alterations in the virus population since the 15.5 K protein could be reverted to 14K after marker-rescue with the cloned 14K encoding gene. We provide direct evidence that changes in size of the 14K envelope protein are responsible for the small plaque size phenotype of these variants. From our previous studies (J. F. Rodriguez, R. Janeczko, and M. Esteban, 1985, J. Virol. 56, 352-356; J. F. Rodriguez, E. Paez, and M. Esteban, 1987, J. Virol. 61, 395-404) and those described here, we established that the 14K protein plays an important role in virus-host cell interactions. This is because this protein is part of the virus envelope, is highly antigenic, elicits neutralizing antibodies, has a role in virus penetration acting in cell fusion, is involved in the virus plaque size phenotype and is highly conserved among Orthopoxviruses. In addition, because mutants with altered 14K protein that have gained 1.5K in size are attenuated (S. Dallo and M. Esteban, 1987, Virology 159, 408-422), it suggests that a modified form of this protein contributes to the decrease of the virulence of vaccinia virus.

Animals↗

PubMed1987

Role of prostaglandins and non-steroid anti-inflammatory drugs in the pathogenicity of vaccinia virus.

The effect of prostaglandins (PGs) of the A series (A1 and dimethyl PGA2), E1, D2, F2 alpha and PGI2 (prostacyclin) and of inhibitors of PG synthesis (aspirin and indomethacin) on the pathogenicity of vaccinia virus was studied in BALB/c mice. PGs of the A series, D2 and F2 alpha conferred little or no protection to mice against the lethal effects of vaccinia virus. Mice treated with PGE1 showed a dramatic increase in mortality after viral infection. However, when mice were treated with PGI2, their survival was greatly enhanced. Mice treated with aspirin and indomethacin showed a marked increase in mortality. Increased mortality correlated with higher virus yields in target tissues (spleen) and with inhibition of antibody response, whereas the increase in survival correlated with lower virus yields and with normal antibody responses. The significance of our findings is that PGI2 can block the outcome of the disease caused by vaccinia virus whereas other PGs and their inhibitors not only worsen the disease, but may activate and enhance viral infections through immune suppression.

Animals↗

PubMed1987

Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein.

A library of rabbit poxvirus DNA fragments contained in the expression cloning vector lambda gt11 was screened with monoclonal antibodies that react specifically against a 14-kilodalton envelope protein of vaccinia virus and rabbit poxvirus. The 14-kilodalton protein appears to play an important role in virus penetration at the level of cell fusion; it also elicits neutralizing antibodies, and it forms covalently linked trimers on the surface of virions and in infected cells (Rodriguez et al., J. Virol. 56:482-488, 1985; Rodriguez et al., J. Virol. 61:395-404, 1987). Two recombinant bacteriophages expressing beta-galactosidase fusion proteins were isolated. Restriction enzyme analysis and hybridization studies mapped the 14-kilodalton encoding sequences in the middle of vaccinia virus HindIII A DNA fragment. Nucleotide sequence analysis revealed an open reading frame (ATG) preceded by a characteristic TAA sequence of late genes. The sequence spans 330 nucleotides and codes for a protein with a molecular weight of 12,500 and an isoelectric point of 6.3. There are two small hydrophobic regions, one at the C terminus (11 amino acids) and the other at the N terminus (5 amino acids). The protein contains two cysteines for oligomer formation and one glycosylation site. Inspection of the deduced amino acid sequence of the 14-kilodalton protein revealed consensus sites with the hemagglutinin precursor of influenza A virus and with adenylate kinase and cytochrome c of various species.

Amino Acid Sequence↗

PubMed1987

Structural and functional studies of a 39,000-Mr immunodominant protein of vaccinia virus.

Little is known about the nature of poxvirus proteins involved in the host immune response. Screening a lambda gt11 expression library of genomic rabbit poxvirus DNA with hyperimmune rabbit anti-vaccinia virus serum and selection of monospecific antibodies identified a highly antigenic viral protein of about 39,000 molecular weight (39K protein). The same-size protein of vaccinia virus was also identified with a monoclonal antibody (MAb B6) obtained from hybridomas generated after fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Structural analysis revealed that the 39K protein is an acidic polypeptide, that it can exist in two molecular forms because of intramolecular disulfide linkages, and that it is part of the virus core. This protein shares antigenic determinants with a cytoplasmic component(s) from uninfected cells. Functional studies revealed that the 39K protein is synthesized at late times postinfection and appears to be required for virus assembly. This protein is highly conserved in members of the Orthopoxvirus group, but in cowpox virus, a 41K virion protein was specifically recognized by antibodies that reacted against the vaccinia virus 39K protein. Significantly, during long-term passages of Friend erythroleukemia cells persistently infected with vaccinia virus, some virus mutants were found to increase or decrease by about 2 kilodaltons the size of the 39K protein. Mapping analysis localized sequences encoding the 39K protein in a rifampin-sensitive gene cluster between the two major core-associated viral polypeptides, 4a and 4b. The fact that the 39K core protein of vaccinia virus elicits strong humoral immune response, induces antibodies that react against a host component(s), and is subjected to genetic variability suggests that this protein has important biological functions.

Animals↗

PubMed1987

A 14,000-Mr envelope protein of vaccinia virus is involved in cell fusion and forms covalently linked trimers.

A monoclonal antibody, MAbC3, that reacts with a 14,000-molecular-weight envelope protein (14K protein) of vaccinia virus completely inhibited virus-induced cell fusion during infection. Immunoblot and immunofluorescence studies revealed that the 14K protein was synthesized at about 6 to 7 h postinfection and transported from the cytoplasm to the cell surface. Synthesis and transport of the 14K protein during infection occurred in the presence of rifampin, an inhibitor of virus maturation. One- and two-dimensional gel electrophoretic analyses demonstrated that the 14K protein forms largely trimers (42K) that are covalently linked by disulfide bonds. The facts that MAbC3 prevents virus uncoating and blocks virus-induced cell fusion but does not prevent virus attachment to cells and the 14K envelope protein forms trimers all suggest that this protein plays major role in virus penetration.

Animals↗

PubMed1987

Virus attenuation and identification of structural proteins of vaccinia virus that are selectively modified during virus persistence.

To investigate the genetic stability of vaccinia virus DNA, we have tested whether alterations occurred in the polypeptide composition of this complex virus during persistent infections. We found that variants isolated at various passages in Friend erythroleukemia cells persistently infected with vaccinia virus contained, in addition to an 8-megadalton (MDa) deletion on the left terminus of the viral genome, major alterations in the sizes of three structural proteins with molecular masses of about 39, 21, and 14 kDa. Alterations in isoelectric points were also observed in proteins of 48, 27, and 14 kDa. The 14-kDa protein is part of the virus envelope, and the variants increased the size of this protein from 0.5 to 3 kDa with increasing passage number. Alteration in size of the 14-kDa protein is a dominant trait since it appeared in the whole virus population by passage 48. With more passages, some variants were found to increase or decrease the size of a 39-kDa core protein by about 2 kDa and to decrease the size of an envelope protein of 21 kDa by about 2 kDa. These three proteins were immunogenic in mice and elicited a strong host immune response. Major alterations in the sizes of these proteins were prevented by continuous treatment of the persistently infected cultures with interferon. However, after interferon was removed, protein modifications appeared with increasing passage number. Generation of the 8-MDa deletion and alterations in the size of the 14-kDa protein correlated with a marked decrease in virulence of these variants. Our findings suggest that during virus persistence, specific mutations are introduced in the vaccinia virus genome that lead to protein alterations and to highly attenuated viruses.

Animals↗

PubMed1987

Resistance of vaccinia virus to interferons: modulation of the 2-5A system in interferon-treated, vaccinia virus infected cells.

In this study we have examined if resistance of vaccinia virus to interferon (IFN) correlates with virus-induced alterations of the 2-5A system. We have shown that in various IFN-treated vaccinia virus infected cells of mouse, monkey and human origins, the intracellular levels of 2-5A are low early in infection but exhibit a sharp rise late in infection. In spite of the presence of 2-5A, activation of the 2-5A dependent RNase, as measured by the rRNA cleavage assay, does not occur or is delayed in the course of virus infection. However, when cycloheximide, an inhibitor of protein synthesis is added at the time of virus infection, extensive cleavage or rRNA is observed in IFN-treated, infected cells. If cycloheximide is added at various times after virus infection, rRNA cleavage is gradually prevented and a virus-induced inhibitor of the 2-5A system can be detected between 1-2 hr post infection. A function encoded by a ts 22 mutant of vaccinia virus blocked rRNA cleavage. Restriction of rRNA cleavage during virus infection correlated with dephosphorylation of 2-5A. Our findings suggest that modulation of the 2-5A system by vaccinia virus involves the production of an activator and simultaneous synthesis of an inhibitor(s). Viral ds-RNA is likely to be the activator while a function encoded by ts 22 mutant is involved in inhibition of the 2-5A system. Other viral functions (ATPase and phosphatase) may also be involved in modifications of the 2-5A system by regulating 2-5A levels and altering the integrity of 2-5A. Modifications of the 2-5A system, during vaccinia virus infection might contribute to the resistance of this cytoplasmic DNA virus to IFN.

2',5'-Oligoadenylate Synthetase↗