Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.
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The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses. (+info)
Sites of simian foamy virus persistence in naturally infected African green monkeys: latent provirus is ubiquitous, whereas viral replication is restricted to the oral mucosa.
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Foamy viruses (FV), retroviruses of the genus Spumavirus, are able to infect a wide variety of animal species and replicate in nearly all types of cultured cells. To identify the cells targeted by FV in the natural host and define the sites of viral replication, multiple organs of four African green monkeys naturally infected with simian FV type 3 were investigated for the presence of FV proviral DNA and viral transcripts. All organs contained significant amounts of FV proviral DNA. In addition to proviruses containing the complete transactivator gene taf, proviral genomes carrying a specific 295-bp deletion in the taf gene were detected in all monkeys. As in the case of human foamy virus the deletion leads to the formation of the bet gene that is regarded to be instrumental in the regulation of viral persistence. FV RNA was detected by RT-PCR and in situ hybridization only in the oral mucosa of one monkey. No other samples contained detectable levels of viral transcripts. Histopathological changes were not observed in any of the tissue samples analyzed. Our results show that the natural history of FV is characterized by latent infection in all organs of the host and by minimal levels of harmless viral replication in the oral mucosa. The broad host cell range in vivo further encourages the development of FV-derived vectors for therapeutic gene delivery. (+info)
Replication and budding of simian immunodeficiency virus in polarized epithelial cells.
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Simian immunodeficiency virus (SIV) infection of primates provides an important model for infection of humans by HIV. Since mucosal epithelium is likely to be an important portal of entry, we decided to study aspects of the interaction of SIV with epithelial cells. SIV was shown to produce virus efficiently in polarized epithelial cells (Vero C1008) transfected with SIVmac239 proviral DNA. The virus titer in the epithelial cell culture fluid reached 10(3) TCID50/ml at day 3 posttransfection. Initially after transfected epithelial cells were plated on a permeable membrane, virus budded at both the apical and the basolateral domains. However, after the cells formed a tight monolayer, 95-100% of the virus particles budded basolaterally, as assessed by release of p27 antigen into the fluid above and below the monolayer. This finding was confirmed by electron microscopy, which showed that the mature virus budded basolaterally in polarized cells. After introduction of the CD4 gene into Vero cells by a retrovirus vector, polarizable cells were able to be infected by cell-free SIVmac239 virus. The virus titer reached 10(4) TCID50/ml in culture fluid and virions also budded basolaterally, the same as the virus from transfected cells. Two viruses (SIVmac1A11 and SIVmac251) that contain truncated TMgp28 instead of TMgp41 also budded basolaterally. Furthermore, we found that HIV-1 with full-length or truncated TMgp41 also budded basolaterally. (+info)
A canarypox vaccine expressing multiple human immunodeficiency virus type 1 genes given alone or with rgp120 elicits broad and durable CD8+ cytotoxic T lymphocyte responses in seronegative volunteers.
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Induction of CD8+ cytotoxic T cells is considered one of the important correlates for the protective efficacy of candidate human immunodeficiency virus type 1 (HIV-1) vaccines. To induce CD8+ cytotoxic T lymphocytes (CTLs) along with neutralizing antibody and CD4+ T cell help, a live canarypox virus construct expressing gp120, transmembrane gp41, the gag and protease genes, and sequences containing CTL epitopes in nef and pol was given simultaneously with, or followed by, rgp120 SF2. CD8+ CTLs were detected in 61% of volunteers at some time during the trial. Three to 6 months after the last immunization, the gene-specific responses were gag, 26/81; env, 17/77; nef, 12/77; and pol, 3/16. Simultaneous immunization with the canarypox vector and the subunit, beginning with the initial immunization, resulted in earlier antibody responses. In summary, a strategy of immunization with a canarypox vector expressing multiple genes of HIV-1 given with gp120 results in durable CD8+ CTL responses to a broad range of epitopes. (+info)
The cytoplasmic domain of HIV-1 gp41 interacts with the carboxyl-terminal region of alpha-catenin.
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To know the cellular protein interactions with the viral protein can give an insight into the molecular mechanisms of the virus life cycle. As the function of the cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) gp41 is not known clearly, we searched for a cellular protein that interacts with the cytoplasmic domain of the HIV-1 gp41 using the yeast two-hybrid assay system. Screening of HeLa cell cDNA library yielded alpha-catenin cDNA. The cytoplasmic domain of the HIV-1 gp41 and the simian immunodeficiency virus (SIV) gp41 were able to interact with the carboxyl-terminal region of alpha-catenin specifically. Mapping of the interaction sites revealed that the interaction between the domain containing the second helix structure from the carboxyl-terminus of HIV-1 gp41 and the carboxyl-terminal region of alpha-catenin was stronger than other domains of gp41. (+info)
Replication of a foamy virus mutant with a constitutively active U3 promoter and deleted accessory genes.
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Foamy viruses (FVs) are complex retroviruses which require for their replication the activity of a transcriptional trans-activator (Tas) as well as Tas-responsive elements in the viral promoters. A mutant of the chimpanzee FV strain, CFV/hu (previously called human FV), genome in which most of the U3 promoter of the CFV long terminal repeat was substituted by the constitutively active human cytomegalovirus immediate early gene enhancer/promoter was constructed. This plasmid (pTS12) and a derivative (pTS13), which has a deletion in the tas gene, gave rise to replication-competent virus. Compared with parental CFV, both mutants replicated only very poorly, with retarded growth kinetics and maximal cell-free virus titres reduced by approximately three orders of magnitude. Mutation of the DD35E motif of the CFV integrase to DA35E rendered the recombinant TS virus replication-deficient. This indicated that provirus integration is probably still required for this FV derivative, which had been converted from a complex regulated retrovirus into a simple one by incorporation of a constitutively active promoter from another virus which regularly does not integrate into the host cell genome. (+info)
Mitochondrial targeting of the p13II protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I).
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The X region of the HTLV-I genome contains four major open reading frames (ORFs), two of which, termed x-I and x-II, are of still undefined biological significance. By indirect immunofluorescence and dual labeling with marker proteins, we demonstrate that p13II, an 87-amino acid protein coded by the x-II ORF, is selectively targeted to mitochondria. Mutational analysis revealed that mitochondrial targeting of p13II is directed by an atypical 10-amino acid signal sequence that is not cleaved upon import and is able to target the Green Fluorescent Protein to mitochondria. Expression of p13II results in specific alterations of mitochondrial morphology and distribution from a typical string-like, dispersed network to round-shaped clusters, suggesting that p13II might interfere with processes relying on an intact mitochondrial architecture. Functional studies of mitochondria with the cationic fluorochrome tetramethylrhodamine revealed that a subpopulation of the cells with p13II-positive mitochondria show a disruption in the mitochondrial inner membrane potential (Apsi), an early event observed in cells committed to apoptosis. Taken together, these results suggest novel virus-cell interactions that might be important in HTLV-I replication and/or pathogenicity. (+info)
Monomer-trimer equilibrium of the ectodomain of SIV gp41: insight into the mechanism of peptide inhibition of HIV infection.
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The monomer-trimer equilibrium of the ectodomain of SIV gp41 (residues 27-149, e-gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e-gp41 trimer is 3.1 x 10(11) M(-2). The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C-labeled e-gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e-gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer-trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides. (+info)