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L Prevec

Publications and source records attributed to L Prevec.

At least 37 records · Page 2Linked to original sources
PubMed1993

Packaging capacity and stability of human adenovirus type 5 vectors.

Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.

Adenoviruses, Human↗
PubMed1992

Oral rabies vaccination of skunks and foxes with a recombinant human adenovirus vaccine.

A new recombinant rabies vaccine (human adenovirus 5 containing the rabies glycoprotein gene) was given to striped skunks (Mephitis mephitis) and red foxes (Vulpes vulpes). Groups of skunks received the vaccine in baits, by direct instillation into the mouth, or intramuscularly. Foxes were given vaccine by direct instillation into the oral cavity (DIOC). Selected groups of vaccinated skunks and foxes were challenged with street rabies virus. There were high rates of seroconversion (generally with high antibody titers) in both foxes and skunks, with survival of all challenged vaccinated animals (all challenge controls developed rabies). In skunks, vaccine given DIOC was effective over a broad range of doses (10(8.7), 10(7.6) and 10(6.4) median tissue culture infective doses). There was no evidence of pathogenic effects. The results indicate that this adenovirus recombinant has considerable potential as a wildlife oral rabies vaccine.

Adenoviruses, Human↗
PubMed1992

Function and immunogenicity of human parainfluenza virus 3 glycoproteins expressed by recombinant adenoviruses.

Human parainfluenza virus type 3 fusion (F) and hemagglutinin-neuraminidase (HN) cDNA sequences were inserted into the E3 region of the adenovirus type 5 genome. Cells infected with recombinant adenoviruses containing HPIV3 F (AdF) and HN (AdHN) sequences were shown to express HPIV3 F and HN proteins that were functional and immunogenic. The HN protein produced following AdHN infection was glycosylated, expressed on the surface of infected cells and exhibited both hemagglutinin and neuraminidase activities. AdF infection led to the synthesis of both the HPIV3 F0 precursor and its proteolytic cleavage product, F1. F proteins produced by AdF were glycosylated and expressed on the infected cell surface. Syncytium formation was observed in HeLa T4 cell monolayers upon coinfection with AdF and AdHN. The F and HN proteins expressed by recombinant adenoviruses were recognized by HPIV3 F- and HN-specific monoclonal antibodies. Mice injected intraperitoneally with AdF or AdHN produced antibodies that immunoprecipitated the appropriate HPIV3 glycoproteins and sera from immunized mice effectively neutralized HPIV3 virions. These results support future work using recombinant adenoviruses to study the immune response to individual HPIV3 glycoproteins as well as in protection studies using animal models.

Adenoviridae↗
PubMed1992

Synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the E3 region of adenovirus.

The haemagglutinin-esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.

Adenoviridae↗
PubMed1992

Sequence analysis of bovine adenovirus type 3 early region 3 and fibre protein genes.

The DNA sequences of the early region 3 (E3) and fibre protein genes of bovine adenovirus type 3 (BAd3) have been determined and the amino acid sequences predicted to be encoded by their open reading frames (ORFs) compared to those of the fibre and E3 proteins from other Ads. One of the BAd3-E3 proteins contains a region homologous to the 14.7K E3 protein of human Ad5 (HAd5). The putative BAd3 fibre protein contains a number of regions homologous to the HAd2 fibre protein sequence, but is predicted to be 244 amino acids longer owing to an increase in the number of repeating structural motifs of hydrophobic amino acid residues in the shaft region. Sequences to the left of the BAd3-E3 gene region contained the 3' end of another ORF with extensive identity with the hexon-associated protein precursor (pVIII) of HAd2. Like mouse Ad1 and canine Ad1, the BAd3 E3 gene is approximately 1.5 kb, about half the size of the E3 region of HAd2 and HAd5.

Adenoviridae↗
PubMed1991

Assembly and transcription of synthetic vesicular stomatitis virus nucleocapsids.

The functional template for transcription of vesicular stomatitis virus (VSV) RNA is a ribonucleoprotein particle (nucleocapsid) consisting of the negative-strand sense genomic RNA completely encapsidated by the viral nucleocapsid (N) protein. As an approach to create nucleocapsids in vitro, we demonstrate here the specific encapsidation by purified N protein of in vitro-synthesized RNA sequences representing the 5' end of both the negative- and positive-strand VSV genome-length RNAs. As few as 19 nucleotides from the 5'-end of positive-strand RNA allowed maximal encapsidation, although the 5' terminal 10 nucleotides would allow partial (50%) encapsidation. Sequences downstream of the binding site can be of any origin. Specific encapsidation of VSV sequences was dependent on the presence of uninfected cell cytoplasmic extracts or poly(A). The synthetic nucleocapsids have the properties of RNase resistance and a buoyant density typical of wild-type VSV nucleocapsids. We have encapsidated a synthetic virionlike RNA species which contained just the terminal sequences of the virion RNA: the N encapsidation signal from the 5' end and the leader gene from the 3' end. This assembled nucleocapsid could function in vitro as a transcription template for the VSV RNA polymerase.

Animals↗
PubMed1991

Immune response to HIV-1 gag antigens induced by recombinant adenovirus vectors in mice and rhesus macaque monkeys.

Recombinant adenovirus vectors expressing the entire gag (p55) or CA (p24) region of human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the appropriate HIV DNA sequences into the E3 region of human adenovirus type 5 (Ad5) with and without an exogenous SV40 early promoter. The infectious recombinant adenoviruses Adgag1, AdSVgag1, and AdSVCA1 were shown to express the appropriate HIV-1 antigens in human cells in vitro, as measured by immunoprecipitation and p24 antigen capture assays. Using the p24 antigen capture assay, HIV antigen expressed by AdSVCA1 was detected earlier in infection and in greater amounts than that produced by either Adgag1 or AdSVgag1. In studies concerning the immunogenicity of these vectors, Balb/c (H-2d) mice given a single intraperitoneal injection of 10(7) or 10(8) plaque-forming units of purified vector developed serum antibodies to p24, detected by Western blotting, by 2 weeks postinjection. In the preliminary test of the immunogenicity of the recombinant adenovirus vectors in primates, two of four rhesus macaque monkeys generated antibodies to HIV-1 p24 following two injections of AdSVCA1. As expected, monkeys injected with control adenovirus failed to show any anti-HIV response, and none of the monkeys showed any adverse reactions following infection with either recombinant or control adenoviruses. These results suggest that adenovirus vectors have considerable potential in the study of possible immune therapies for HIV infection.

Adenoviruses, Human↗
PubMed1990

A recombinant human adenovirus vaccine against rabies.

Rabies continues to be a serious problem in both developed and developing nations due to the reservoir of rabies virus in wildlife vectors. The control and worldwide eradication of rabies depends on the development of safe, effective, and economical vaccines that might be used in preexposure vaccination programs for humans and animals. To this end an infectious human adenovirus type 5 recombinant virus that contains the rabies glycoprotein gene, and which may serve as the prototype for a new class of vaccines against rabies, was constructed and tested. This recombinant, when administered by either the parenteral or oronasal route, was highly effective in eliciting good levels of rabies-neutralizing antibodies in the sera of dogs and mice. Mice immunized by the recombinant virus were protected from lethal intracerebral challenge with rabies virus.

Adenoviruses, Human↗
PubMed1990

The sequence of the nucleocapsid protein (N) gene of Piry virus: possible domains in the N protein of vesiculoviruses.

A number of independent cDNA clones of the Piry virus N gene message were identified and sequenced. From the resulting sequences and previously published data, we derived the sequence of the mRNA for this protein. Sequence similarities of the translated region of Piry virus with that of other viruses suggest that Piry virus is as distantly related to Chandipura virus as it is to the vesicular stomatitis viruses of Indiana and New Jersey serotypes. Based on the relative conservation of the amino acid sequence of the nucleocapsid protein of these vesiculoviruses, the N protein can be subdivided into at least three regions, possibly indicative of functional domains.

Amino Acid Sequence↗
PubMed1989

Expression of the glycoprotein of vesicular stomatitis virus by infectious adenovirus vectors.

We have constructed recombinant human adenovirus (Ad) vectors containing the glycoprotein gene of vesicular stomatitis virus (VSV). The structural gene of the VSV glycoprotein was modified by the addition of promoter and poly(A) addition sequences from the herpes simplex virus type 1 thymidine kinase (TK) gene and inserted, in either orientation, into early region 3 (E3) of human Ad type 5. The recombinant vectors were fully infectious and replicated in HeLa cells in culture. The TK promoter was functional in both insert orientations and responsive to trans-activation by herpes virus infection; however production of VSV glycoprotein in readily detectable amounts was only obtained with the vector having an insert in the E3 parallel orientation (AdG12), and depended principally on transcripts initiating within upstream Ad sequences. The onset of expression of the glycoprotein in AdG12-infected cells was detectable at about the same time as the Ad 72K DNA-binding protein encoded by E2, and its synthesis was not prevented by blocking viral DNA synthesis. The VSV glycoprotein produced by AdG12 was fully processed and could function to direct low pH-induced fusion of infected cells. These Ad vectors have considerable potential utility for the expression of antigens in cell culture and for the immunization of animals in studies of immunity and protection.

Adenoviruses, Human↗
PubMed1989

Use of human adenovirus-based vectors for antigen expression in animals.

An infectious recombinant human adenovirus type 5 (Ad5) vector, AdG12, which carries the glycoprotein gene of vesicular stomatitis virus (VSV) and expresses that gene in cultured HeLa cells was used to examine the host range of insert expression by human Ad vectors. The VSV glycoprotein was expressed in bovine, canine and murine cells when infected with AdG12 in culture. These cell lines are respectively permissive, non-permissive and semi-permissive for human Ad5 replication. Administration of the AdG12 vector to calves, piglets or dogs by either the subcutaneous or oral route resulted in the production of high titres of neutralizing antibodies to VSV. Mice injected intraperitoneally with the vector produced neutralizing antibodies and were protected against subsequent intravenous challenge with normally lethal doses of VSV. This work demonstrates the utility of human adenoviral vectors for antigen expression in a number of non-human cell lines and for the induction of an immune response to the delivered antigen in a number of species.

Adenoviruses, Human↗
PubMed1986

Persistence of vesicular stomatitis virus in cloned interleukin-2-dependent natural killer cell lines.

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.

Animals↗
PubMed1985

Phosphorylation sites on phosphoprotein NS of vesicular stomatitis virus.

The phosphoprotein NS of vesicular stomatitis virus which accumulates within the infected cell cytoplasm is phosphorylated at multiple serine and threonine residues (G. M. Clinton and A. S. Huang, Virology 108:510-514, 1981; Hsu et al., J. Virol. 43:104-112, 1982). Using incomplete chemical cleavage at tryptophan residues, we mapped the major phosphorylation sites to the amino-terminal half of the protein. Analysis of phosphate-labeled tryptic peptides suggests that essentially all of the label is within the large trypsin-resistant fragment predicted from the sequence of Gallione et al. (J. Virol. 39:52-529, 1981). A similar result has been obtained for NS protein isolated from the virus particle by C.-H. Hsu and D. W. Kingsbury (J. Biol. Chem., in press). Analysis of phosphodipeptides utilizing the procedures of C. E. Jones and M. O. J. Olson (Int. J. Pept. Protein Res. 16:135-142, 1980) enabled us to detect as many as six distinct phosphate-containing dipeptides. From these studies, together with the known sequence data, we conclude that the major phosphate residues on cytoplasmic NS protein are located in the amino third of the NS molecule and most probably between residues 35 and 106, inclusive. The studies also provide formal chemical proof that NS protein has a structure consistent with a monomer of the sequence of Gallione et al. as modified by J. K. Rose (personal communication). The low electrophoretic mobility of this protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not therefore due to dimerization.

Hydrolysis↗
PubMed1984

Protein kinase activity associated with immunoprecipitates of the vesicular stomatitis virus phosphoprotein NS.

Monospecific antisera prepared to denatured N and NS proteins of the Indiana serotype of VSV were used to investigate the protein associations in extracts of virus-infected cells. Complexes precipitated from the cytoplasm of infected cells with either anti-N or anti-NS serum both contained N and NS proteins but could be differentiated by the absence of any trace of M protein in the complexes precipitated with anti-NS serum. Immunoprecipitation with anti-NS serum of NS protein from the soluble cytoplasmic protein pool always resulted in coprecipitation of N protein suggesting a possible functional association of these proteins in the soluble fraction. The association of protein kinase activity with complexes containing NS protein was demonstrated by the phosphorylation of NS serine residues when immunoprecipitates containing NS protein were incubated with [32P]ATP in vitro. In protein aggregates precipitated with antibody after high salt dissociation of viral proteins it was also possible to demonstrate the presence of a c-src-like protein kinase activity as previously shown by G.M. Clinton, N.G. Guerina, H. Guo, and H.S. Huang (J. Biol. Chem. 257, 3313-3319 (1982) ).

Animals↗
PubMed1982

Linear mapping of tryptophan residues in Vesiculovirus M and N proteins by partial chemical cleavage.

Nonlimit chemical cleavage at tryptophan residues of protein labeled at the amino terminus afforded a simple procedure for generating specific fragments and for mapping tryptophan positions. A comparison of the matrix (M) and nucleocapsid (N) proteins of four members of the Vesiculovirus group by this procedure suggests considerable conservation of tryptophan number and location in the four serotypes examined.

Capsid↗
PubMed1982

Characterization of the electrophoretic mobility mutation in the N protein of the tsD1 mutant of vesicular stomatitis virus New Jersey serotype.

Some isolates of the temperature sensitive mutant tsD1 of complementation group D of vesicular stomatitis virus of New Jersey serotype have a nucleocapsid (N) protein which shows an increased electrophoretic mobility on sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) when compared with wild type. Utilizing techniques involving specific chemical cleavage at tryptophan or methionine residues, as well as enzymatic cleavage with carboxypeptidases A and B, we have determined that residues near the carboxyterminus are responsible for the electrophoretic difference of the mutant protein. We have further shown that there are no differences in the tryptic peptides of the mutant compared with the wild type or a non-ts revertant in this region of the protein. We have identified a tryptic peptide located outside the relevant carboxyterminal region which is distinct in mutant and revertant. We conclude that the mutation producing the aberrant electrophoretic mobility of N protein of the tsD1 mutant is a missense point mutation located at least 40 amino acid residues from the carboxyterminus and which interacts with a more proximal carboxyregion so as to influence electrophoretic mobility on SDS-PAGE.

Electrophoresis, Polyacrylamide Gel↗