Biomedical subjects

J Overbaugh

Publications and source records attributed to J Overbaugh.

At least 73 records · Page 4Linked to original sources

PubMed1995

In vivo selection of long terminal repeat alterations in feline leukemia virus-induced thymic lymphomas.

To determine what genetic changes are selected in the enhancer sequences of the feline leukemia virus (FeLV) long terminal repeat in cats that develop T cell tumors, we cloned proviral U3 sequences in cats that died with thymic lymphoma following infection with molecularly cloned FeLV. Analysis of the U3 enhancer region revealed single base changes, including point mutations in the core and FLV-1 sequences. Additionally, in clones from two of four cat tumors, portions of the enhancer including Lvb and core were duplicated with respect to the single enhancer unit of the inoculating virus. In contrast, a PCR survey of necropsy DNA samples derived from five cats that did not develop tumors revealed that all retained the single enhancer unit of the infecting virus. These results demonstrate that viruses with duplicated enhancers can be generated and selected after only a single passage in cats, and furthermore, that such viruses may be particularly selected in tumors.

Animals↗

PubMed1995

Human immunodeficiency virus type 1-infected cells in breast milk: association with immunosuppression and vitamin A deficiency.

Breast milk samples from human immunodeficiency virus type 1 (HIV-1)-seropositive women were analyzed by polymerase chain reaction to determine the prevalence and determinants of HIV-1-infected cells in breast milk. Breast milk samples (212) were collected from 107 women, and 58% of the samples had detectable HIV-1 DNA. The proportion of HIV-1-infected cells in the milk samples ranged from 1 to 3255/10(4) cells. Breast milk samples with detectable HIV-1 DNA were more likely to be from women with absolute CD4 cell counts of < 400 (odds ratio, 3.1; 95% confidence interval [CI], 1.5-7.0). Severe vitamin A deficiency (< 20 micrograms/dL) was associated with a 20-fold increased risk of having HIV-1 DNA in breast milk among women with < 400 CD4 cells/mm3 (95% CI, 2.1-188.5). Women with CD4 cell depletion, especially those with vitamin A deficiency, may be at increased risk of transmitting HIV-1 to their infants through breast milk.

Adolescent↗

PubMed1995

Human immunodeficiency virus DNA in urethral secretions in men: association with gonococcal urethritis and CD4 cell depletion.

To evaluate the prevalence and correlates of human immunodeficiency virus (HIV)-infected cells in urethral secretions, samples were collected from 106 HIV-seropositive men with and without urethritis. HIV DNA was detected by polymerase chain reaction in 27% of 184 urethral specimens and was associated with CD4 cell depletion (P for trend, .03) and with urethritis (odds ratio [OR], 2.4; 95% confidence interval [CI], 1.2-4.6) or gonorrhea (OR, 2.9; 95% CI, 1.5-5.8). Two multivariate models were constructed that included age, CD4 cell count < 200/mm3, and either urethritis or gonococcal infection. Detection of HIV-infected cells in urethral secretions was independently associated with < 200 CD4 cells/mm3 (OR, 2.2; 95% CI, 0.9-5.2; P = .05) and urethritis (OR, 2.7; 95% CI, 1.3-5.3; P = .003) in the first model and with gonococcal infection (OR, 3.2; 95% CI, 1.6-6.4; P < .001) in the second model. Successful treatment of gonococcal urethritis was associated with a 2-fold reduction in urethral HIV DNA (44% vs. 21%; P = .02). Thus, treatment of gonococcal urethritis may be an effective strategy for reducing HIV transmission.

Adult↗

PubMed1995

Diversity in virus populations from genital secretions and peripheral blood from women recently infected with human immunodeficiency virus type 1.

In order to develop a human immunodeficiency virus type 1 vaccine with global efficacy, it is important to evaluate the virus populations that are transmitted to individuals living in high-incidence areas. To determine the nature of the human immunodeficiency virus type 1 population transmitted to women during heterosexual contact, we examined the diversity of the proviral envelope gene in infected cells in both genital secretions and peripheral blood from six recently seroconverted Kenyan women. Heterogeneous virus populations were present in cervical secretions and/or peripheral blood shortly after seroconversion for five of six infected individuals, and tissue-specific variants were identified in several cases.

AIDS Vaccines↗

PubMed1995

Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity.

Amino Acid Sequence↗

PubMed1994

Isolation of a novel subgroup B feline leukemia virus from a cat infected with FeLV-A.

Proviruses were cloned directly from a cat that developed neurological disorders approximately 28 months after inoculation with a molecularly cloned, minimally pathogenic subgroup A feline leukemia virus (FeLV-A). In addition to FeLV-A proviruses that were nearly identical to the inoculated virus, we detected a subgroup B-like variant in brain, bone marrow, and lymph node that apparently had acquired the major portion of its extracellular envelope gene (gp70) from endogenous FeLV-related sequences. A similar recombinant was also detected, by PCR, at low levels in bone marrow from an early time postinfection (2.5 months). A full-length proviral variant with this recombinant structure cloned from brain tissue encoded a replication-defective virus. A chimera encoding the 5' gag-pol portion of FeLV-A and the 3' env-LTR portion of the defective brain-derived clone was replication-competent and had the extended host range properties of FeLV-B.

Amino Acid Sequence↗

PubMed1994

Intrarectal inoculation of macaques by the simian immunodeficiency virus, SIVmne E11S: CD4+ depletion and AIDS.

Macaca nemestrina and Macaca fascicularis were inoculated with various doses of a single-cell clone of SIVmne-infected HuT 78 cells (E11S) by both the intravenous and intrarectal routes. Animals inoculated intravenously at each dose seroconverted and virus was isolated from peripheral blood mononuclear cells, but only the high-dose intrarectally exposed macaques became viremic and seroconverted. However, some seronegative, virus isolation negative intrarectally inoculated macaques showed evidence of infection and disease.

Animals↗

PubMed1994

Evolution of feline leukemia virus variant genomes with insertions, deletions, and defective envelope genes in infected cats with tumors.

In order to study retroviral variation, selection, and viral correlates of in vivo pathogenicity, we documented the evolution of feline leukemia virus (FeLV) variants in cats that died with thymic lymphoma after infection with molecularly cloned subgroup A FeLV. Using genomic DNA from cat necropsy samples, we employed PCR to amplify and clone the envelope gene, which is a major determinant of the specific pathogenicity of different FeLV variants. In the envelope gene, mutations encoded scattered amino acid changes that did not cluster into clearly definable variable regions; however, characterization of these terminal variant sequences revealed a predominance of G-to-A and A-to-G nucleotide substitutions. Additionally, some cats harbored variants with recombinant subgroup B-like envelope genes, while the major variant from one cat had a 12-bp insertion in a region previously characterized as an immunodeficiency-inducing determinant. Finally, proviruses from tumor DNA frequently possessed envelope genes predicted to encode a protein truncated in the N-terminal half because of either premature termination codons or deletions ranging from 29 to 1,666 bp. In contrast, all envelope genes cloned from the bone marrow of one cat were predicted to encode full-length envelope product, and only a minority of proviral clones from a cat that did not develop a tumor had defective envelope genes. Thus, in the cat, viruses evolved from subgroup A FeLV that had point mutations, insertions, deletions, or recombinant envelope genes. Furthermore, defective variants were particularly prominent in T-cell tumors.

Amino Acid Sequence↗

PubMed1994

Persistence of simian immunodeficiency virus Mne variants upon transmission.

In macaques infected with a clone of simian immunodeficiency virus (SIV) Mne, viral variants consistently evolve multiple new potential glycosylation sites in the first variable region (V1) prior to the development of AIDS. In the present study, we asked whether viruses with these glycosylation sites persist when they are transmitted to a naive macaque. Variants that evolved after transmission to a recipient macaque were compared with virus that evolved in the donor, which had been infected by cloned SIV Mne. Upon transmission, the specific serine/threonine-rich motifs potentially encoding novel O-linked glycosylation site(s) in V1 were conserved in virus isolated from lymph node, spleen, and liver tissue from the recipient. There was some accumulation of changes in V3 of envelope in virus from the recipient, whereas changes in this region were not observed in virus from the donor macaque. Some variants detected in the tissue of the recipient at necropsy were most closely related to viruses present in the donor inoculum even though these particular variants were not detected early after infection in the recipient's peripheral blood mononuclear cells. Overall, virus with the predominant V1 sequences associated with progression to disease are transmitted to and persist in the recipient animal.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence of the splice junction of feline leukemia virus envelope mRNA.

The splice donor and acceptor sequences for the subgenomic envelope messenger RNA of feline leukemia virus (FeLV) were determined. The splice junction was characterized for viral mRNA from a prototype subgroup A FelV and an immunodeficiency-inducing FeLV variant. Thirty-seven of 38 partial envelope cDNAs, cloned after PCR amplification from T cells infected with either virus, utilized the same splice donor and acceptor sites to generate a mRNA that would be predicted to encode envelope protein. One novel cDNA, which could also code for envelope protein, was generated from a cryptic splice acceptor 109 nucleotides downstream of the normal junction. No additional subgenomic FeLV gene products were detected using reverse transcription and polymerase chain reaction amplification of FeLV sequences from chronically infected cells.

Base Sequence↗

PubMed1993

Coincident involvement of flvi-2, c-myc, and novel env genes in natural and experimental lymphosarcomas induced by feline leukemia virus.

The flvi-2 locus is a target of insertional mutagenesis in thymic lymphosarcomas induced by feline leukemia virus (FeLV). flvi-2 encodes the gene bmi-1, whose product is implicated as a myc-collaborator in the induction of B- and T-cell lymphoma. We have examined the involvement of flvi-2 and myc in natural and experimentally induced FeLV-positive feline lymphosarcomas which are heterogeneous in anatomical origin, geographic origin, and strain of FeLV involved. We further compared these findings with previous reports of novel FeLV env genes in the same tumors. The results show that proviral insertion at flvi-2 occurs commonly in natural and experimental feline thymic lymphosarcomas of diverse origins [52% overall], and that alterations in c-myc commonly accompany insertional mutagenesis of flvi-2 [54% overall]. However, 46% of tumors with flvi-2 insertions apparently lack involvement of c-myc. These observations support the hypothesis that interruption of flvi-2 may be an early event in a multistep cascade, one possibility for completion of which is activation of c-myc. Interruption of flvi-2 was not observed in nonthymic lymphosarcomas of alimentary or multicentric origin, although c-myc may be involved. A proportion of both thymic and nonthymic tumors have been shown previously to contain FeLV proviruses with recombinant or mutant env genes. Our findings strongly implicate the insertional mutagenesis of flvi-2, the activation of c-myc, and the emergence of novel env genes in FeLV-mediated lymphomagenesis, particularly in the induction of thymic lymphosarcoma. The data show that these events may overlap, but do not necessarily occur concurrently.

Animals↗

PubMed1993

Delayed cytopathicity of a feline leukemia virus variant is due to four mutations in the transmembrane protein gene.

Two molecularly cloned, replication-defective variants of feline leukemia virus, called 61B and 61C, have both been shown to cause fatal immunodeficiency in cats when coinfected with a replication-competent, minimally pathogenic helper virus, but 61B exhibits a longer latency period between infection and disease (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). Infection of the 3201 feline T-cell line with 61B plus helper virus also results in longer time from infection to cytopathic effect compared with 61C plus helper virus, providing an in vitro system with which to study the mechanism for this difference. We report that the primary determinant of cytopathicity of 61B maps to gp70, the extracellular envelope glycoprotein. The long latency of 61B, on the other hand, maps to the extracellular portion of the envelope transmembrane protein, in which there are only four predicted amino acid differences between 61B and 61C. These differences render 61B replication defective, and two of the predicted amino acid changes lie in a region that is highly conserved among many retroviruses. The eventual onset of 61B cytopathicity in cell culture was associated with the outgrowth of an apparent recombinant virus that encodes the pathogenic gp70 of 61B and replaces the transmembrane protein of 61B with that of the helper virus. Thus, during in vitro infection, a cytopathic virus evolved from a replication-defective virus and a nonpathogenic virus, suggesting that recombination between multiple variants in natural infection may influence progression of feline leukemia virus-associated immunodeficiency disease.

Amino Acid Sequence↗

PubMed1993

Replication and persistence of simian immunodeficiency virus variants after passage in macaque lymphocytes and established human cell lines.

In lentivirus infections, there are typically few cells in the host that harbor the provirus. For this reason, molecular clones of human and simian immunodeficiency viruses (HIV and SIV) are generally derived after passage and amplification of the virus in cell culture. To determine whether SIV variants that persist in culture are similar to the variants that predominate in the host, we examined the proviral sequence of the SIV envelope (env) gene before and after cocultivation of lymphocytes from a macaque with AIDS with naive macaque lymphocytes or human cell lines. Many of the predominant variants in the monkey replicated and persisted in macaque lymphocytes and CEMx174 cells in culture, but a more limited population of variants replicated in C8166 cells. Passage of virus, harvested after 4 weeks of coculture, onto naive cells further demonstrated that the majority of proviruses detected by polymerase chain reaction were also viral variants that were expressed and packaged into infectious virions.

Amino Acid Sequence↗

PubMed1993

flvi-2, a target of retroviral insertional mutagenesis in feline thymic lymphosarcomas, encodes bmi-1.

LC-FeLV is a myc-containing strain of feline leukemia virus which induces thymic lymphosarcoma in the domestic cat with short latency. A locus in feline DNA, termed flvi-2, is commonly interrupted in naturally occurring and experimentally induced thymic lymphosarcomas containing LC-FeLV; thus, interruption of a gene encoded by flvi-2 may cooperate with the myc oncogene in the induction of T-cell tumors by LC-FeLV. Clones homologous to flvi-2 have been isolated from a normal human thymus cDNA library. Nucleotide sequence analysis of the cDNA clones demonstrates that flvi-2 encodes bmi-1, a gene previously identified as a target for MoMuLV integration and as a myc-collaborator in retrovirally-induced B-cell lymphomas in E mu-myc transgenic mice. In feline thymic lymphomas, retroviral integrations occur downstream of the gene, and result in enhanced expression of a bmi-1 transcript of normal size. These findings demonstrate the interruption of bmi-1 in natural as well as experimentally induced tumors, implicate the activation of bmi-1 in the induction of T-cell as well as B-cell lymphoma, and support the premise that bmi-1 functions as a myc collaborator.

Amino Acid Sequence↗

PubMed1992

Baboon T cell lymphomas expressing the B cell-associated surface proteins CD40 and Bgp95.

Papio hamadryas baboons in the Sukhumi colony develop enzootic outbreaks of malignant lymphomas with an incidence of about 1.5% per year among adults of the high-risk stock. We investigated the surface phenotypes of cells from normal and lymphomatous animals using antibodies against human lymphocyte antigens. We found that more than 80% of the lymphomas that developed during the last 3 years were characterized histologically to be of the peripheral T cell type. Generally, the lymphomatous cells also expressed high levels of MHC class II DR protein, CD18 (LFA-1 beta chain), and CD45RO. Surprisingly, these cells also expressed on their surface two proteins previously characterized as being relatively B cell-restricted: CD40 and Bgp95. These proteins were never found on the peripheral blood T cells from normal animals. The expression of these two gene products was confirmed by RNA blotting and immunoprecipitation. In most cases, the two B cell-associated proteins were expressed on the predominant T cell subsets; we found both B cell proteins on CD4+, CD8+ as well as on the CD4/8 double-positive cells when these subsets were expressed at high levels. About 90% of these animals are seropositive for Herpesvirus papio and human T cell leukemia virus-1 (HTLV-1) before developing outright lymphomas. In all of the lymphoma samples, HTLV-1 tax DNA sequences were detected by PCR amplification. Whether or not HTLV-1 or the Herpesvirus papio gene products influence the surface expression of CD40 and Bgp95 remains to be determined.

Animals↗

PubMed1992

Structure and pathogenicity of individual variants within an immunodeficiency disease-inducing isolate of FeLV.

We previously described the molecular cloning of a replication-defective variant of feline leukemia virus (FeLV) that induced fatal immunodeficiency in cats. Eighteen proviruses have now been molecularly cloned from cats inoculated with the original isolate (FeLV-FAIDS) or its in vivo passages. Three were replication-competent and each of these was noncytopathic for the feline T-cell line, 3201. Replication of the prototype, FeLV-61E, in cats was associated with development of T cell tumors in some cats. The remaining 15 proviruses were replication-defective, but each of six of these tested was found to be cytopathic for 3201 cells when rescued with the noncytopathic helper virus, 61E. Three defective/helper virus mixtures were inoculated into cats and all induced fatal immunodeficiency, but with varied efficiency and kinetics. Each of these virus mixtures was attenuated relative to a mixture containing 61E and the intestine-targeted, FeLV-FAIDS-61C prototype defective molecular clone. Furthermore, one replication-competent virus chimera generated using the envelope and LTR of the defective pathogenic variant was incapable of inducing viremia in cats. The observed differences in the biological activity between the defective viruses could be attributed to no more than 10 scattered amino acid changes in envelope and either one or two nucleotide changes in the LTR.

Amino Acid Sequence↗

PubMed1992

Photodynamic inactivation of retrovirus by benzoporphyrin derivative: a feline leukemia virus model.

The ability of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), and either broad-spectrum (400-1200 nm) or narrow-band (600-700 nm) red light to kill feline leukemia virus (FeLV) and FeLV-infected cat T cells (cell line 3201) was investigated in culture medium containing fetal calf serum and in blood from infected cats. A molecular clone of FeLV, 61E, is minimally pathogenic and productively infects 3201 cells while causing no change in rate of cell division, viability, or size. Active virus (either free or within infected cells) was quantified by using a limiting dilution assay that involved cocultivation of test samples with naive 3201 cells, after which either the polymerase chain reaction or a reverse transcriptase assay was used to detect the presence of virus. It was shown that 61E-infected T cells in culture were slightly more sensitive to photodynamic killing than were uninfected cells. Infected cells and free virus were eliminated from whole blood taken from infected cats by using 4 micrograms per mL of BPD-MA and 40 J per cm2 of red light. These results correlate well with previous results with BPD-MA and vesicular stomatitis virus in whole human blood and suggest that this photosensitizer is a promising agent for the elimination of retroviruses that are either free or located within infected cells in blood.

Animals↗

PubMed1992

Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques.

Genetic diversity is a hallmark of the human immunodeficiency virus (HIV) genome, but the role of distinct HIV variants in the development of AIDS is unclear. Envelope (env) is the most highly variable gene in HIV as well as in other retroviruses. We have previously demonstrated that variation in simian immunodeficiency virus (SIV) env is primarily localized in two regions (V1 and V4) during progression to simian AIDS. To determine whether there is a common genotype that evolves as AIDS develops, a total of 160 SIV env genes isolated directly from the tissue DNAs of four macaques infected with cloned virus were compared. Common amino acid sequence changes were identified within V1, V4, and, in the late stages of disease, near V3. At several positions, the same amino acid change was seen frequently in the variant genomes from all four animals. As AIDS developed, the majority of viruses evolved an extended sequence in V1 that was rich in serine and threonine residues and shared similarity with proteins modified by O-linked glycosylation. Several of the predominant common sequence changes in V1 and V4 created new sites for N-linked glycosylation. Thus, common features of the SIV variants that evolve during progression to AIDS are motifs that potentially allow for structural and functional changes in the env protein as a result of carbohydrate addition.

Amino Acid Sequence↗