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1
Akahata, Wataru, Zhi-yong Yang, and Gary J. Nabel. "Comparative Immunogenicity of Human Immunodeficiency Virus Particles and Corresponding Polypeptides in a DNA Vaccine." Journal of Virology 79, no. 1 (January 1, 2005): 626–31. http://dx.doi.org/10.1128/jvi.79.1.626-631.2005.
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ABSTRACT The immunogenicity of a plasmid DNA expression vector encoding both Gag and envelope (Env), which produced human immunodeficiency virus (HIV) type 1 virus-like particles (VLP), was compared to vectors expressing Gag and Env individually, which presented the same gene products as polypeptides. Vaccination with plasmids that generated VLP showed cellular immunity comparable to that of Gag and cell-mediated or humoral responses similar to those of Env as immunization with separate vectors. These data suggest that DNA vaccines encoding separated HIV polypeptides generate immune responses similar to those generated by viral particles.
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Kong, Wing-Pui, Yue Huang, Zhi-Yong Yang, Bimal K. Chakrabarti, Zoe Moodie, and Gary J. Nabel. "Immunogenicity of Multiple Gene and Clade Human Immunodeficiency Virus Type 1 DNA Vaccines." Journal of Virology 77, no. 23 (December 1, 2003): 12764–72. http://dx.doi.org/10.1128/jvi.77.23.12764-12772.2003.
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ABSTRACT The ability to elicit an immune response to a spectrum of human immunodeficiency virus type 1 (HIV-1) gene products from divergent strains is a desirable feature of an AIDS vaccine. In this study, we examined combinations of plasmids expressing multiple HIV-1 genes from different clades for their ability to elicit humoral and cellular immune responses in mice. Immunization with a modified Env, gp145ΔCFI, in combination with a Gag-Pol-Nef fusion protein plasmid elicited similar CD4+ and CD8+ cellular responses to immunization with either vector alone. Further, when mice were immunized with a mixture of Env from three clades, A, B, and C, together with Gag-Pol-Nef, the overall potency and balance of CD4+- and CD8+-T-cell responses to all viral antigens were similar, with only minor differences noted. In addition, plasmid mixtures elicited antibody responses comparable to those from individual inoculations. These findings suggest that a multigene and multiclade vaccine, including components from A, B, and C Env and Gag-Pol-Nef, can broaden antiviral immune responses without immune interference. Such combinations of immunogens may help to address concerns about viral genetic diversity for a prospective HIV-1 vaccine.
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Kong, Wei, Chunjuan Tian, Bindong Liu, and Xiao-Fang Yu. "Stable Expression of Primary Human Immunodeficiency Virus Type 1 Structural Gene Products by Use of a Noncytopathic Sindbis Virus Vector." Journal of Virology 76, no. 22 (November 15, 2002): 11434–39. http://dx.doi.org/10.1128/jvi.76.22.11434-11439.2002.
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ABSTRACT Efficient expression of the human immunodeficiency virus type 1 (HIV-1) structural gene products Gag, Pol, and Env involves the regulation by viral Rev and Rev-responsive elements (RRE). Removal of multiple inhibitory sequences (INS) in the coding regions of these structural genes or modification of the codon usage patterns of HIV-1 genes to those used by highly expressed human genes has been found to significantly increase HIV-1 structural protein expression in the absence of Rev and RRE. In this study, we show that efficient and stable expression of the HIV-1 structural gene products Gag and Env could be achieved by transfection with a noncytopathic Sindbis virus expression vector by using HIV-1 sequences from primary isolates without any sequence modification. Stable expression of these Gag and Env proteins was observed for more than 12 months. The fact that the Sindbis virus expression vector replicates its RNA only in the cytoplasm of the transfected cells and the fact that the lack of expression of HIV-1 Gag by the DNA vector containing unmodified HIV-1 gag sequences was associated with a lack of detectable cytoplasmic gag RNA suggest that a major blockage in the expression of HIV-1 structural proteins in the absence of Rev/RRE is caused by inefficient accumulation of mRNA in the cytoplasm. Efficient long-term expression of structural proteins of diverse HIV-1 strains by the noncytopathic Sindbis virus expression system may be a useful tool for functional study of HIV-1 gene products and vaccine research.
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Chen, Steve S. L., Sheau-Fen Lee, Huey-Jong Hao, and Chin-Kai Chuang. "Mutations in the Leucine Zipper-Like Heptad Repeat Sequence of Human Immunodeficiency Virus Type 1 gp41 Dominantly Interfere with Wild-Type Virus Infectivity." Journal of Virology 72, no. 6 (June 1, 1998): 4765–74. http://dx.doi.org/10.1128/jvi.72.6.4765-4774.1998.
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ABSTRACT It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615–3619, 1993; S. S.-L. Chen, J. Virol. 68:2002–2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation β-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associatedgag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.
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Moore, Anne C., Wing-pui Kong, Bimal K. Chakrabarti, and Gary J. Nabel. "Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice." Journal of Virology 76, no. 1 (January 1, 2002): 243–50. http://dx.doi.org/10.1128/jvi.76.1.243-250.2002.
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ABSTRACT The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-γ) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-γ and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-γ and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.
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zur Megede, Jan, Min-Chao Chen, Barbara Doe, Mary Schaefer, Catherine E. Greer, Mark Selby, Gillis R. Otten, and Susan W. Barnett. "Increased Expression and Immunogenicity of Sequence-Modified Human Immunodeficiency Virus Type 1 gag Gene." Journal of Virology 74, no. 6 (March 15, 2000): 2628–35. http://dx.doi.org/10.1128/jvi.74.6.2628-2635.2000.
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ABSTRACT A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1SF2 p55Gag were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55Gag protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modifiedprotease coding sequences were made, and these showed high-level Rev-independent expression of p55Gag and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag andgagprotease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gagshowed Gag-specific antibody and CD8+ cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modifiedgag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modifiedgag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.
7
Kong, Wing-Pui, Lan Wu, Timothy C. Wallstrom, Will Fischer, Zhi-Yong Yang, Sung-Youl Ko, Norman L. Letvin, et al. "Expanded Breadth of the T-Cell Response to Mosaic Human Immunodeficiency Virus Type 1 Envelope DNA Vaccination." Journal of Virology 83, no. 5 (December 24, 2008): 2201–15. http://dx.doi.org/10.1128/jvi.02256-08.
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ABSTRACT An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.
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Lee, Chun-Nan, Wei-Kung Wang, Wen-Sheng Fan, Shing-Jer Twu, Shou-Chien Chen, Ming-Ching Sheng, and Mao-Yuan Chen. "Determination of Human Immunodeficiency Virus Type 1 Subtypes in Taiwan by vpu Gene Analysis." Journal of Clinical Microbiology 38, no. 7 (2000): 2468–74. http://dx.doi.org/10.1128/jcm.38.7.2468-2474.2000.
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The genetic diversity of human immunodeficiency virus (HIV) type 1 (HIV-1) has been characterized mainly by analysis of theenv and gag genes. Information on thevpu genes in the HIV sequence database is very limited. In the present study, the nucleotide sequences of the vpugenes were analyzed, and the genetic subtypes determined by analysis of the vpu gene were compared with those previously determined by analysis of the gag and env genes. Thevpu genes were amplified by nested PCR of proviral DNA extracted from 363 HIV-1-infected individuals and were sequenced directly by use of the PCR products. HIV-1 subtypes were determined by sequence alignment and phylogenetic analysis with reference strains. The strains in all except one of the samples analyzed could be classified as subtype A, B, C, E, or G. The vpu subtype of one strain could not be determined. Of the strains analyzed, genetic subtypes of 247 (68.0%) were also determined by analysis of theenv or gag gene. The genetic subtypes determined by vpu gene analysis were, in general, consistent with those determined by gag and/orenv gene analysis except for those for two AG recombinant strains. All the strains that clustered with a Thailand subtype E strain in the vpu phylogenetic analyses were subtype E byenv gene analysis and subtype A by gag gene analysis. In summary, our genetic typing revealed that subtype B strains, which constituted 73.8% of all strains analyzed, were most prevalent in Taiwan. While subtype E strains constituted about one-quarter of the viruses, they were prevalent at a higher proportion in the group infected by heterosexual transmission. Genetic analysis ofvpu may provide an alternate method for determination of HIV-1 subtypes for most of the strains, excluding those in which intersubtype recombination has occurred.
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Thomas, JA, F. Cotter, AM Hanby, LQ Long, PR Morgan, B. Bramble, and BM Bailey. "Epstein-Barr virus-related oral T-cell lymphoma associated with human immunodeficiency virus immunosuppression." Blood 81, no. 12 (June 15, 1993): 3350–56. http://dx.doi.org/10.1182/blood.v81.12.3350.3350.
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Abstract Epstein-Barr virus (EBV) is generally held to infect B cells and epithelial cells, although there are now reports of EBV infection in normal T cells and neoplastic T-cell diseases. In patients with human immunodeficiency virus (HIV) infection, EBV is associated with the benign epithelial lesion, hairy leukoplakia, and has been reported in up to 80% of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma. This study shows the presence of EBV in malignant oral T-cell lymphoma in three AIDS patients, two of whom had concurrent manifestation of hairy leukoplakia. The T-cell lineage of the tumor cells was determined by positive immunophenotyping for T-cell markers and lack of B-cell or nonhematopoietic (cytokeratin) determinants. All tumors contained monoclonal T-cell populations shown by polymerase chain reaction, which showed amplification of T-cell receptor gamma chain DNA without evidence of Ig heavy chain gene rearrangement. Furthermore, these lesions showed the presence of EBV DNA and expression of EBV latent gene products in the tumor cells. EBV involvement in AIDS-related T-cell lymphoma has not been widely reported and may represent a further manifestation of opportunistic EBV infection arising in the HIV-immunocompromised host.
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Thomas, JA, F. Cotter, AM Hanby, LQ Long, PR Morgan, B. Bramble, and BM Bailey. "Epstein-Barr virus-related oral T-cell lymphoma associated with human immunodeficiency virus immunosuppression." Blood 81, no. 12 (June 15, 1993): 3350–56. http://dx.doi.org/10.1182/blood.v81.12.3350.bloodjournal81123350.
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Epstein-Barr virus (EBV) is generally held to infect B cells and epithelial cells, although there are now reports of EBV infection in normal T cells and neoplastic T-cell diseases. In patients with human immunodeficiency virus (HIV) infection, EBV is associated with the benign epithelial lesion, hairy leukoplakia, and has been reported in up to 80% of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma. This study shows the presence of EBV in malignant oral T-cell lymphoma in three AIDS patients, two of whom had concurrent manifestation of hairy leukoplakia. The T-cell lineage of the tumor cells was determined by positive immunophenotyping for T-cell markers and lack of B-cell or nonhematopoietic (cytokeratin) determinants. All tumors contained monoclonal T-cell populations shown by polymerase chain reaction, which showed amplification of T-cell receptor gamma chain DNA without evidence of Ig heavy chain gene rearrangement. Furthermore, these lesions showed the presence of EBV DNA and expression of EBV latent gene products in the tumor cells. EBV involvement in AIDS-related T-cell lymphoma has not been widely reported and may represent a further manifestation of opportunistic EBV infection arising in the HIV-immunocompromised host.
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Wei, Qing, and Patricia N. Fultz. "Differential Selection of Specific Human Immunodeficiency Virus Type 1/JC499 Variants after Mucosal and Parenteral Inoculation of Chimpanzees." Journal of Virology 76, no. 2 (January 15, 2002): 851–64. http://dx.doi.org/10.1128/jvi.76.2.851-864.2002.
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ABSTRACT Regardless of the route of transmission, it is generally accepted that the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted from an infected individual to an uninfected individual is genetically homogeneous. This finding and the observation that HIV-1 genotypes in recipients are minor variants in the donors suggest strongly that selection for specific variants occurs. However, most analyses have been limited to the V3 region of env. In addition, the exact time at which most new infections occurred was not known, making it almost impossible to analyze virus populations present in donor-recipient pairs at the time of HIV-1 transmission. To circumvent this problem, three chimpanzees were inoculated with a genetically defined stock of cell-free HIV-1/JC499 by one of three routes: intravenously or via the cervical or penile mucosa. PCR products of the C2-to-V5 region of env were amplified from both proviral DNA and virion RNA in blood samples collected soon after infection and were screened by heteroduplex analysis (HDA). Those PCR products with distinct HDA banding patterns were cloned and sequenced. In all three animals, transmitted variants encoded one of two V3-loop populations identified in the inoculum, indicating relative homogeneity in this region. However, different virus populations, defined by combinations of specific V4 and V5 sequences, were found when variants in the animal inoculated intravenously (at least 13 V4-plus-V5 combinations) were compared with those in the two animals inoculated by the mucosal routes (limited to only four V4-plus-V5 combinations). The only V4-plus-V5 population in variants found in all three chimpanzees was the major population in the inoculum, which contained viruses with more than 30 different V4-plus-V5 combinations. That the majority of the V4-plus-V5 genotypes in variants transmitted to all three animals were minor populations in the inoculum indicated that selective transmission defined by the V4-plus-V5 regions had occurred but that distinct populations were transmitted by parenteral versus mucosal routes. These results indicate that the putative homogeneity of HIV-1 variants in a newly infected individual might be an artifact of the region of the env gene evaluated and that regions other than V3 might play a major role in selective transmission.
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Ruggiero, Eliana, Roberta Bona, Claudia Muratori, and Maurizio Federico. "Virological Consequences of Early Events following Cell-Cell Contact between Human Immunodeficiency Virus Type 1-Infected and Uninfected CD4+ Cells." Journal of Virology 82, no. 16 (May 28, 2008): 7773–89. http://dx.doi.org/10.1128/jvi.00695-08.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4+ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4+ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4+ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4+ cell can occur through different mechanisms, leading to distinguishable virological outcomes.
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Chiang, C. S., T. Grove, M. Cooper, J. Cuan, A. Kowalski, K. Parcells, M. Tsunokawa, M. Rosenberg, E. Arcuri, and S. Franklin. "Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies." Clinical Chemistry 35, no. 6 (June 1, 1989): 946–52. http://dx.doi.org/10.1093/clinchem/35.6.946.
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Abstract We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.
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Carmichael, A., X. Jin, P. Sissons, and L. Borysiewicz. "Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection: differential CTL responses to HIV-1 and Epstein-Barr virus in late disease." Journal of Experimental Medicine 177, no. 2 (February 1, 1993): 249–56. http://dx.doi.org/10.1084/jem.177.2.249.
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Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to human persistent virus infections. Measurements of the frequency and specificity of human immunodeficiency virus type 1 (HIV-1)-specific CTL and their variation with time may indicate their relative importance in modulating the progression of HIV-1 infection. We have used limiting dilution analysis (LDA) to derive quantitative estimates of the frequency of HIV-1-specific CTL precursors in a cross-sectional study of 23 patients at different clinical stages of HIV-1 infection and to compare these with the frequency of CTL precursors specific for another persistent virus (Epstein-Barr virus [EBV]) in the same patients. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with autologous HIV-1-infected lymphoblasts and assayed for cytotoxicity in 51Cr release assays against autologous and MHC-mismatched lymphoblastoid B cells infected with recombinant vaccinia viruses expressing the three HIV-1 structural gene products. The frequency of MHC-restricted precursors was high in asymptomatic HIV-1-infected patients (env-specific CTL precursors up to 73/10(6) PBMC; gag-specific CTL precursors up to 488/10(6) PBMC), although the relative frequency against the different structural gene products varied from patient to patient. The HIV-1-specific CTL precursor frequency was reduced in patients who had more severe (< 400/microliters) CD4+ lymphocyte depletion, while in the majority of such patients the frequency of CTL precursors against EBV was maintained at levels observed in healthy controls. Direct CTL activity in unstimulated PBMC was observed in three of nine patients but no correlation was found between the presence of an activated CTL response and the magnitude of the CTL response detected after stimulation in LDA. Thus, CTL precursors were detected against all three HIV-1 structural gene products in patients with CD4+ lymphocyte counts > 400/microliters, at frequencies that are high compared with those reported for other persistent viruses. A CTL response directed against multiple protein antigens of HIV-1 may protect the patient against epitope variation. The fact that the EBV-specific CTL precursor frequencies were maintained in advanced HIV-1 infection suggests that there may be selective impairment of the HIV-1-specific CTL response associated with disease progression.
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Zheng, Natalie N., M. Juliana McElrath, Papa-Salif Sow, Stephen E. Hawes, Habibatou Diallo-Agne, Joshua E. Stern, Fusheng Li, et al. "Role of Human Immunodeficiency Virus (HIV)-Specific T-Cell Immunity in Control of Dual HIV-1 and HIV-2 Infection." Journal of Virology 81, no. 17 (June 20, 2007): 9061–71. http://dx.doi.org/10.1128/jvi.00117-07.
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ABSTRACT Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4+ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4+ and CD8+ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log10 IFN-γ spot-forming cells/106 cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4+ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4+ and HIV-2 Gag-specific IFN-γ-secreting CD8+ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.
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Arruda, Liã Bárbara, Laura I. Weber, Marisa dos Santos, Edson M. Kawakubo, and Ana Maria B. Martínez. "TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL." Revista do Instituto de Medicina Tropical de São Paulo 55, no. 2 (April 2013): 91–99. http://dx.doi.org/10.1590/s0036-46652013000200005.
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The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.
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Inghirami, G., L. Macri, E. Cesarman, A. Chadburn, J. Zhong, and DM Knowles. "Molecular characterization of CD30+ anaplastic large-cell lymphoma: high frequency of c-myc proto-oncogene activation." Blood 83, no. 12 (June 15, 1994): 3581–90. http://dx.doi.org/10.1182/blood.v83.12.3581.3581.
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Abstract Anaplastic large-cell lymphoma (ALCL) represents a morphologically distinct type of non-Hodgkin's lymphoma (NHL) characterized phenotypically by the expression of the CD30 antigen, a new member of the nerve growth factor gene family. The lymphoid origin of ALCL has been documented using immunohistochemical and molecular genetic analyses. However, very little is known so far regarding the precise pathogenetic mechanisms involved in its development and progression. Therefore, we investigated bcl-2, p53, and retinoblastoma gene (Rb) expression immunohistochemically; the occurrence of bcl-2, c-myc, and Rb gene rearrangements using Southern blotting; and the presence of ras and p53 gene somatic mutations by single-strand conformation polymorphism assay in a panel of 18 well-characterized ALCLs. In addition, the presence of Epstein-Barr (EBV) and human T-cell lymphotropic virus type I (HTLV-I) genomes were investigated using polymerase chain reaction. We identified abnormal c-myc gene products in 6 of 18 cases (33%) of ALCL. On the other hand, the bcl-2 and Rb genes were not rearranged and K-, N-, and H-ras gene somatic mutations were not found. Significant levels of p53 protein expression were found in more than 60% of ALCLs, but only a single ALCL carried a p53 gene mutation (exon 5). Only 3 ALCL cases, all occurring in human immunodeficiency virus-infected patients, were positive for EBV genomes. On the other hand, contrary to previous findings, no HTLV-I products could be identified. Despite the fact that the c-myc proto- oncogene appears to be frequently altered in ALCL, no pathognomonic abnormality could be identified and therefore additional studies and new strategies should be designed to identify the pathogenetic mechanisms involved in the development of ALCL.
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Inghirami, G., L. Macri, E. Cesarman, A. Chadburn, J. Zhong, and DM Knowles. "Molecular characterization of CD30+ anaplastic large-cell lymphoma: high frequency of c-myc proto-oncogene activation." Blood 83, no. 12 (June 15, 1994): 3581–90. http://dx.doi.org/10.1182/blood.v83.12.3581.bloodjournal83123581.
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Anaplastic large-cell lymphoma (ALCL) represents a morphologically distinct type of non-Hodgkin's lymphoma (NHL) characterized phenotypically by the expression of the CD30 antigen, a new member of the nerve growth factor gene family. The lymphoid origin of ALCL has been documented using immunohistochemical and molecular genetic analyses. However, very little is known so far regarding the precise pathogenetic mechanisms involved in its development and progression. Therefore, we investigated bcl-2, p53, and retinoblastoma gene (Rb) expression immunohistochemically; the occurrence of bcl-2, c-myc, and Rb gene rearrangements using Southern blotting; and the presence of ras and p53 gene somatic mutations by single-strand conformation polymorphism assay in a panel of 18 well-characterized ALCLs. In addition, the presence of Epstein-Barr (EBV) and human T-cell lymphotropic virus type I (HTLV-I) genomes were investigated using polymerase chain reaction. We identified abnormal c-myc gene products in 6 of 18 cases (33%) of ALCL. On the other hand, the bcl-2 and Rb genes were not rearranged and K-, N-, and H-ras gene somatic mutations were not found. Significant levels of p53 protein expression were found in more than 60% of ALCLs, but only a single ALCL carried a p53 gene mutation (exon 5). Only 3 ALCL cases, all occurring in human immunodeficiency virus-infected patients, were positive for EBV genomes. On the other hand, contrary to previous findings, no HTLV-I products could be identified. Despite the fact that the c-myc proto- oncogene appears to be frequently altered in ALCL, no pathognomonic abnormality could be identified and therefore additional studies and new strategies should be designed to identify the pathogenetic mechanisms involved in the development of ALCL.
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Ikuta, Kazufumi, Shamala K. Srinivas, Tim Schacker, Jun-ichi Miyagi, Rona S. Scott, and John W. Sixbey. "Points of Recombination in Epstein-Barr Virus (EBV) Strain P3HR-1-Derived Heterogeneous DNA as Indexes to EBV DNA Recombinogenic Events In Vivo." Journal of Virology 82, no. 23 (September 25, 2008): 11516–25. http://dx.doi.org/10.1128/jvi.01036-08.
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ABSTRACT Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation.
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Przybylski, GK, J. Goldman, VL Ng, MS McGrath, BG Herndier, DP Schenkein, JG Monroe, and LE Silberstein. "Evidence for early B-cell activation preceding the development of Epstein-Barr virus-negative acquired immunodeficiency syndrome-related lymphoma." Blood 88, no. 12 (December 15, 1996): 4620–29. http://dx.doi.org/10.1182/blood.v88.12.4620.bloodjournal88124620.
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To investigate the origin and pathogenesis of acquired immunodeficiency syndrome (AIDS)-related lymphoma (ARL), we studied 14 cases in which Epstein-Barr virus (EBV) infection was not an etiologic factor. By histology, 8 of the specimens were of the small noncleaved cell type and 6 consisted of the large diffuse cell type. Southern analysis using a J(H) probe was consistent with a monoclonal B-cell tumor in 13 cases. To characterize the expressed Ig genes, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing of PCR products. Eight cases expressed IgM and 1 case expressed IgG. V(H)3 genes were found in 5 cases, V(H)4 genes in 3 cases, V(H)1 genes in 2 cases, and a V(H)7 gene in 1 case. The nucleotide homology to known germline V(H) genes ranged from 80% to 97%, suggesting significant somatic diversification of expressed V(H) genes. The large proportion of V(H)3-expressing lymphomas in this series corresponds to the frequency of V(H)3-expressing B cells in the peripheral blood from healthy and (recent) human immunodeficiency virus (HIV)-seropositve individuals and contrasts with the V(H)3 clonal deficit observed in late stages of HIV infection. Similar to the Ig heavy chain genes, the corresponding Ig light chain genes showed significant deviation from known germline gene sequences. The large proportion of V(H)3-expressing lymphomas as well as the high degree of somatic deviation from germline suggest that these EBV-negative lymphomas might arise from antigen-selected expanded B-cell clones before transformation. Further support for this hypothesis is provided by sequential Ig sequence analysis in 1 patient with large-cell lymphoma. It was shown that 3 years before the diagnosis of axillary lymphoma, there existed several B-cell clones in this patient's bone marrow. One of these clones present in the bone marrow expressed the same rearranged V(H) gene as the axillary lymphoma. Taken together, the current findings from Ig gene analyses suggest that activation of B cells in the early phase of HIV infection may be a predisposing factor for subsequent B-cell transformation.
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Veronese, F. D., B. Joseph, T. D. Copeland, S. Oroszlan, R. C. Gallo, and M. G. Sarngadharan. "Identification of simian immunodeficiency virus SIVMAC env gene products." Journal of Virology 63, no. 3 (1989): 1416–19. http://dx.doi.org/10.1128/jvi.63.3.1416-1419.1989.
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Learn, Gerald H., David Muthui, Scott J. Brodie, Tuofu Zhu, Kurt Diem, James I. Mullins, and Lawrence Corey. "Virus Population Homogenization following Acute Human Immunodeficiency Virus Type 1 Infection." Journal of Virology 76, no. 23 (December 1, 2002): 11953–59. http://dx.doi.org/10.1128/jvi.76.23.11953-11959.2002.
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ABSTRACT Understanding the properties of human immunodeficiency virus type 1 (HIV-1) variants capable of establishing infection is critical to the development of a vaccine against AIDS. Previous studies of men have shown that the HIV-1 env gene is homogeneous early in infection, leading to the suggestion that infection is established by a single transmitted variant. However, we report here that all of eight homosexual men evaluated beginning 3.7 to 9 weeks following onset of symptoms of acute infection harbored diverse virus populations in their blood, with median genetic distances averaging 1.08% in the env C2V5 region and 0.81% in the gag p17 gene. Within another 4.7 to 11 weeks, the variant lineage in env became more homogeneous, while gag sequences continued to diversify. Thus, the homogenization that has been reported to characterize acute infection is actually preceded by the replication of multiple virus variants. This early selective process focuses on viral properties within Env but not Gag p17. Hence, the viral homogeneity observed early in HIV-1 infection results from a selective process that occurs during the establishment of infection.
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Riboldi, P., G. Gaidano, EW Schettino, TG Steger, DM Knowles, R. Dalla-Favera, and P. Casali. "Two acquired immunodeficiency syndrome-associated Burkitt's lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments." Blood 83, no. 10 (May 15, 1994): 2952–61. http://dx.doi.org/10.1182/blood.v83.10.2952.2952.
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Abstract We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4–21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4–21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4–21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4–21 gene in the human population, the nature of the nucleotide differences in the expressed VH4–21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4–21 gene. Thus, the expression of VH4–21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.
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Pang, Shen, Duan Yu, Dong Sung An, Gayle C. Baldwin, Yiming Xie, Betty Poon, Yen-Hung Chow, No-Hee Park, and Irvin S. Y. Chen. "Human Immunodeficiency Virus Env-Independent Infection of Human CD4− Cells." Journal of Virology 74, no. 23 (December 1, 2000): 10994–1000. http://dx.doi.org/10.1128/jvi.74.23.10994-11000.2000.
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ABSTRACT CD4− epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4− human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4− cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.
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Scott, J. M., and M. J. Imperiale. "Promoter-proximal poly(A) sites are processed efficiently, but the RNA products are unstable in the nucleus." Molecular and Cellular Biology 17, no. 4 (April 1997): 2127–35. http://dx.doi.org/10.1128/mcb.17.4.2127.
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The presence of two polyadenylation signals in the primary transcript of the human immunodeficiency virus type 1 (HIV-1) provirus leads to a requirement for regulation of 3'-end processing. To ensure that viral genome replication and gene expression occur, polyadenylation must occur at the poly(A) site transcribed from the 3' long terminal repeat (LTR) but not the 5' LTR. Models that have been proposed to explain this regulation include (i) inhibition of the 5' site as a result of proximity to the promoter and (ii) enhancement of the 3' site by U3 sequences that are transcribed upstream of only the 3' poly(A) site. In previous studies designed to investigate these models, a reduction in the levels of steady-state RNA was observed when the HIV-1 poly(A) site was placed within 500 nucleotides of the cap site. Although these findings were interpreted to be the result of promoter proximity effects on 3'-end processing, in vitro studies demonstrated that the HIV-1 poly(A) site was equally functional in promoter-proximal and promoter-distal positions. These results led to the hypothesis that, in vivo, the poly(A) site is fully active at this close distance but the short transcripts produced are highly unstable in the nucleus and undergo rapid degradation, precluding their appearance as abundant mRNAs in the steady-state pool. To investigate the biogenesis of these short RNAs in vivo, experiments were performed to examine directly the nuclear processing rates of the HIV-1 poly(A) site in intact cells. By using recombinant adenoviruses as expression vectors, it is now demonstrated conclusively that the HIV-1 poly(A) site is indeed processed at equivalent levels independent of its distance from the promoter. Although transcripts containing the promoter-proximal poly(A) site are processed efficiently, most undergo degradation in the nucleus instead of nucleocytoplasmic transport.
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Rangel, Hector R., Jan Weber, Bikram Chakraborty, Arantxa Gutierrez, Michael L. Marotta, Muneer Mirza, Patti Kiser, Miguel A. Martinez, Jose A. Este, and Miguel E. Quiñones-Mateu. "Role of the Human Immunodeficiency Virus Type 1 Envelope Gene in Viral Fitness." Journal of Virology 77, no. 16 (August 15, 2003): 9069–73. http://dx.doi.org/10.1128/jvi.77.16.9069-9073.2003.
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ABSTRACT A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.
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Wang, Bao-Zhong, Weimin Liu, Sang-Moo Kang, Munir Alam, Chunzi Huang, Ling Ye, Yuliang Sun, et al. "Incorporation of High Levels of Chimeric Human Immunodeficiency Virus Envelope Glycoproteins into Virus-Like Particles." Journal of Virology 81, no. 20 (August 1, 2007): 10869–78. http://dx.doi.org/10.1128/jvi.00542-07.
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ABSTRACT The human immunodeficiency virus (HIV) envelope (Env) protein is incorporated into HIV virions or virus-like particles (VLPs) at very low levels compared to the glycoproteins of most other enveloped viruses. To test factors that influence HIV Env particle incorporation, we generated a series of chimeric gene constructs in which the coding sequences for the signal peptide (SP), transmembrane (TM), and cytoplasmic tail (CT) domains of HIV-1 Env were replaced with those of other viral or cellular proteins individually or in combination. All constructs tested were derived from HIV type 1 (HIV-1) Con-S ΔCFI gp145, which itself was found to be incorporated into VLPs much more efficiently than full-length Con-S Env. Substitution of the SP from the honeybee protein mellitin resulted in threefold-higher chimeric HIV-1 Env expression levels on insect cell surfaces and an increase of Env incorporation into VLPs. Substitution of the HIV TM-CT with sequences derived from the mouse mammary tumor virus (MMTV) envelope glycoprotein, influenza virus hemagglutinin, or baculovirus (BV) gp64, but not from Lassa fever virus glycoprotein, was found to enhance Env incorporation into VLPs. The highest level of Env incorporation into VLPs was observed in chimeric constructs containing the MMTV and BV gp64 TM-CT domains in which the Gag/Env molar ratios were estimated to be 4:1 and 5:1, respectively, compared to a 56:1 ratio for full-length Con-S gp160. Electron microscopy revealed that VLPs with chimeric HIV Env were similar to HIV-1 virions in morphology and size and contained a prominent layer of Env spikes on their surfaces. HIV Env specific monoclonal antibody binding results showed that chimeric Env-containing VLPs retained conserved epitopes and underwent conformational changes upon CD4 binding.
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Fikkert, Valery, Peter Cherepanov, Kristel Van Laethem, Anke Hantson, Barbara Van Remoortel, Christophe Pannecouque, Erik De Clercq, Zeger Debyser, Anne-Mieke Vandamme, and Myriam Witvrouw. "env Chimeric Virus Technology for Evaluating Human Immunodeficiency Virus Susceptibility to Entry Inhibitors." Antimicrobial Agents and Chemotherapy 46, no. 12 (December 2002): 3954–62. http://dx.doi.org/10.1128/aac.46.12.3954-3962.2002.
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ABSTRACT We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.
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Himathongkham, Sunee, Nancy S. Halpin, Jinling Li, Michael W. Stout, Christopher J. Miller, and Paul A. Luciw. "Simian-Human Immunodeficiency Virus Containing a Human Immunodeficiency Virus Type 1 Subtype-E Envelope Gene: Persistent Infection, CD4+ T-Cell Depletion, and Mucosal Membrane Transmission in Macaques." Journal of Virology 74, no. 17 (September 1, 2000): 7851–60. http://dx.doi.org/10.1128/jvi.74.17.7851-7860.2000.
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ABSTRACT The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with theenv ectodomain from the subtype-E strain HIV-1CAR402, which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by the intravenous or intravaginal route; virus replicated in these animals but did not produce hematological abnormalities. In an attempt to elicit the pathogenic potential of the recombinant clone, we serially passaged this viral clone via transfusion of blood and bone marrow through juvenile macaques to produce SHIV-E-P4 (fourth-passage virus). The serially passaged virus established productive infection and CD4+ T-cell depletion in juvenile macaques inoculated by either the intravenous or the intravaginal route. Determination of the coreceptor usage of SHIV-E-CAR and serially passaged SHIV-E-P4 indicated that both of these viruses utilized CXCR4 as a coreceptor. In summary, the serially passaged SHIV subtype-E chimeric virus will be important for studies aimed at developing a nonhuman primate model for analyzing the functions of subtype-E env genes in viral transmission and pathogenesis and for vaccine challenge experiments with macaques immunized with HIV-1 env antigens.
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Liu, Shan-Lu, Christine L. Halbert, and A. Dusty Miller. "Jaagsiekte Sheep Retrovirus Envelope Efficiently Pseudotypes Human Immunodeficiency Virus Type 1-Based Lentiviral Vectors." Journal of Virology 78, no. 5 (March 1, 2004): 2642–47. http://dx.doi.org/10.1128/jvi.78.5.2642-2647.2003.
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ABSTRACT Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the parental Env, do not transform rodent fibroblasts but are still capable of pseudotyping lentiviral and oncoretroviral vectors.
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Lederman, Michael. "Haemophilia, human immunodeficiency virus and human immunodeficiency virus pathogenesis." Thrombosis and Haemostasis 104, no. 11 (2010): 911–14. http://dx.doi.org/10.1160/th10-02-0096.
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SummaryIn July 1982, the occurrence of three cases of acquired immunodeficiency syndrome (AIDS) in men with haemophilia was an immediate signal to Oscar Ratnoff that AIDS was transmissible through blood products. Work that he led provided important and clear indication that the AIDS agent was transmissible through pooled plasma products and had rapidly infected many men who had haemophilia. Before the blood supply was protected, the risk for infection in haemophilia was related directly to the intensity of therapy with pooled anti-haemophilic factor concentrates. Studies performed among the small proportion of haemophiliacs who remained uninfected despite heavy exposure to these plasma products revealed that the rare protective genotype – homozygosity for the 32 base pair deletion in the CCR5 gene was heavily concentrated in this population. Among those who did not have this protective genotype, a state of diminished immune activation distinguished these high risk uninfected haemophiliacs from haemophiliacs who later acquired human immunodeficiency virus (HIV) infection and from healthy uninfected controls. Immune activation state may not only predict risk for HIV acquisition but also appears to be an important predictor and likely determinant of HIV disease progression. The potential drivers of immune activation in chronic HIV infection include HIV itself, other co-infecting pathogens, homeostatic responses to cytopenia as well as the recently recognised phenomenon of translocation of microbial products across a damaged gut mucosal surface. This latter process is particularly compelling as clinical studies have shown a good relationship between indices of microbial translocation and markers of both immune activation and T cell homeostasis in chronic HIV infection. More recently, we have also found evidence that these microbial products also may drive a heightened tendency to thrombus formation in HIV infection via induction of monocyte tissue factor expression. Thus systemic exposure to microbial elements that are translocated through a gut mucosa damaged in the first few weeks of HIV infection may contribute to the pathogenesis of both immune deficiency and the heightened risk for vascular events that have been noted in persons with HIV infection.
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Le Tortorec, Anna, and Stuart J. D. Neil. "Antagonism to and Intracellular Sequestration of Human Tetherin by the Human Immunodeficiency Virus Type 2 Envelope Glycoprotein." Journal of Virology 83, no. 22 (September 9, 2009): 11966–78. http://dx.doi.org/10.1128/jvi.01515-09.
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ABSTRACT Tetherin (CD317/BST-2), an interferon-induced membrane protein, restricts the release of nascent retroviral particles from infected cell surfaces. While human immunodeficiency virus type 1 (HIV-1) encodes the accessory gene vpu to overcome the action of tetherin, the lineage of primate lentiviruses that gave rise to HIV-2 does not. It has been previously reported that the HIV-2 envelope glycoprotein has a Vpu-like function in promoting virus release. Here we demonstrate that the HIV-2 Rod envelope glycoprotein (HIV-2 Rod Env) is a tetherin antagonist. Expression of HIV-2 Rod Env, but not that of HIV-1 or the closely related simian immunodeficiency virus (SIV) SIVmac1A11, counteracts tetherin-mediated restriction of Vpu-defective HIV-1 in a cell-type-specific manner. This correlates with the ability of the HIV-2 Rod Env to mediate cell surface downregulation of tetherin. Antagonism requires an endocytic motif conserved across HIV/SIV lineages in the gp41 cytoplasmic tail, but specificity for tetherin is governed by extracellular determinants in the mature Env protein. Coimmunoprecipitation studies suggest an interaction between HIV-2 Rod Env and tetherin, but unlike studies with Vpu, we found no evidence of tetherin degradation. In the presence of HIV-2 Rod Env, tetherin localization is restricted to the trans-Golgi network, suggesting Env-mediated effects on tetherin trafficking sequester it from virus assembly sites on the plasma membrane. Finally, we recapitulated these observations in HIV-2-infected CD4+ T-cell lines, demonstrating that tetherin antagonism and sequestration occur at physiological levels of Env expression during virus replication.
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Kitrinos, Kathryn M., Julie A. E. Nelson, Wolfgang Resch, and Ronald Swanstrom. "Effect of a Protease Inhibitor-Induced Genetic Bottleneck on Human Immunodeficiency Virus Type 1 env Gene Populations." Journal of Virology 79, no. 16 (August 15, 2005): 10627–37. http://dx.doi.org/10.1128/jvi.79.16.10627-10637.2005.
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ABSTRACT The initiation of drug therapy or the addition of a new drug to preexisting therapy can have a significant impact on human immunodeficiency virus type 1 (HIV-1) populations within a person. Drug therapy directed at reverse transcriptase and protease can result in dramatic decreases in virus load, causing a contraction in the virus population that represents a potential genetic bottleneck as a subset of virus with genomes carrying resistance mutations repopulate the host. While this bottleneck exerts an effect directly on the region that is being targeted by the drugs, it also affects other regions of the viral genome. We have applied heteroduplex tracking assays (HTA) specific to variable regions 1 and 2 (V1/V2) and variable region 3 (V3) of the HIV-1 env gene to analyze the effect of a genetic bottleneck created by the selection of resistance to ritonavir, a protease inhibitor. Subjects were classified into groups on the basis of the extent of the initial drop in virus load and the duration of virus load reduction. Subjects with a strong initial drop in virus load exhibited a loss of heterogeneity in the env region at virus load rebound; in contrast, subjects with a weak initial drop in virus load exhibited little to no loss of heterogeneity at virus load rebound in either region of env examined. The duration of virus load reduction also affected env populations. Subjects that had prolonged reductions exhibited slower population diversification and the appearance of new V1/V2 species after rebound. The longer reduction of virus load in these subjects may have allowed for improved immune system function, which in turn could have selected for new escape mutants. Subjects with rapid rebound quickly reequilibrated the entry env variants back into the resistant population. When the pro gene developed further resistance mutations subsequent to virus load rebound, no changes were observed in V1/V2 or V3 populations, suggesting that the high virus loads allowed the env populations to reequilibrate rapidly. The rapid equilibration of env variants during pro gene sequence transitions at high virus load suggests that recombination is active in defining the HIV-1 sequence population. Conversely, part of the success of suppressive antiviral therapy may be to limit the potential for evolution through recombination, which requires dually infected cells.
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Hammarskjöld, M. L., J. Heimer, B. Hammarskjöld, I. Sangwan, L. Albert, and D. Rekosh. "Regulation of human immunodeficiency virus env expression by the rev gene product." Journal of Virology 63, no. 5 (1989): 1959–66. http://dx.doi.org/10.1128/jvi.63.5.1959-1966.1989.
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Wu, Kunyu, Gyoung Nyoun Kim, and C. Yong Kang. "Expression and processing of human immunodeficiency virus type 1 gp160 using the vesicular stomatitis virus New Jersey serotype vector system." Journal of General Virology 90, no. 5 (May 1, 2009): 1135–40. http://dx.doi.org/10.1099/vir.0.009019-0.
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The Indiana serotype of vesicular stomatitis virus (VSVIND), but not the New Jersey serotype (VSVNJ), has been widely used as a gene expression vector. In terms of prime–boost-based vaccine strategies, it would be desirable to use two different VSV serotypes to avoid immunity against the priming viral vector. Here, we report that we have applied the VSVNJ vector system for expression of the env gene of human immunodeficiency virus type 1 (HIV-1). The HIV-1 env gene was inserted into the VSVNJ vector system at two different sites: between the P and M genes (NP-gp160-MGL) and between the G and L genes (NPMG-gp160-L). The HIV-1 env gene product, gp160, was efficiently expressed and processed in cells infected with either of these two recombinant VSV–HIV-1gp160 viruses. In this study, we have investigated the applicability of the VSVNJ vector system for foreign gene expression.
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Gao, Feng, Eric A. Weaver, Zhongjing Lu, Yingying Li, Hua-Xin Liao, Benjiang Ma, S. Munir Alam, et al. "Antigenicity and Immunogenicity of a Synthetic Human Immunodeficiency Virus Type 1 Group M Consensus Envelope Glycoprotein." Journal of Virology 79, no. 2 (January 15, 2005): 1154–63. http://dx.doi.org/10.1128/jvi.79.2.1154-1163.2005.
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ABSTRACT Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6 env gp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated “consensus” env genes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.
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Giron, M. L., F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Peries, and R. Emanoil-Ravier. "Human foamy virus polypeptides: identification of env and bel gene products." Journal of Virology 67, no. 6 (1993): 3596–600. http://dx.doi.org/10.1128/jvi.67.6.3596-3600.1993.
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Dudley, Dawn M., Jennifer L. Wentzel, Matthew S. Lalonde, Ronald S. Veazey, and Eric J. Arts. "Selection of a Simian-Human Immunodeficiency Virus Strain Resistant to a Vaginal Microbicide in Macaques." Journal of Virology 83, no. 10 (March 11, 2009): 5067–76. http://dx.doi.org/10.1128/jvi.00055-09.
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ABSTRACT PSC-RANTES binds to CCR5, inhibits human immunodeficiency virus type 1 (HIV-1) entry, and has been shown as a vaginal microbicide to protect rhesus macaques from a simian-human immunodeficiency virus chimera (SHIVSF162-p3) infection in a dose-dependent manner. In this study, env gene sequences from SHIVSF162-p3-infected rhesus macaques treated with PSC-RANTES were analyzed for possible drug escape variants. Two specific mutations located in the V3 region of gp120 (K315R) and C-helical domain of gp41 (N640D) were identified in a macaque (m584) pretreated with a 100 μM dose of PSC-RANTES. These two env mutations were found throughout infection (through week 77) but were found at only low frequencies in the inoculating SHIVSF162-p3 stock and in the other SHIVSF162-p3-infected macaques. HIV-1 env genes from macaque m584 (env m584) and from inoculating SHIVSF162-p3 (env p3) were cloned into an HIV-1 backbone. Increases in 50% inhibitory concentrations to PSC-RANTES with env m584 were modest (sevenfold) and most pronounced in cells expressing rhesus macaque CCR5 as compared to human CCR5. Nonetheless, virus harboring env m584, unlike inoculating virus env p3, could replicate even at the highest tissue culture PSC-RANTES concentrations (100 nM). Dual-virus competitions revealed a dramatic increase in fitness of chimeric virus containing env m584 (K315R/N640D) over that containing env p3, but again, only in rhesus CCR5-expressing cells. This study is the first to describe the immediate selection and infection of a drug-resistant SHIV variant in the face of a protective vaginal microbicide, PSC-RANTES. This rhesus CCR5-specific/PSC- RANTES resistance selection is particularly alarming given the relative homogeneity of the SHIVSF162-p3 stock compared to the potential exposure to a heterogeneous HIV-1 population in human transmission.
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Wyatt, Linda S., Patricia L. Earl, Wei Xiao, Jeffrey L. Americo, Catherine A. Cotter, Jennifer Vogt, and Bernard Moss. "Elucidating and Minimizing the Loss by Recombinant Vaccinia Virus of Human Immunodeficiency Virus Gene Expression Resulting from Spontaneous Mutations and Positive Selection." Journal of Virology 83, no. 14 (May 6, 2009): 7176–84. http://dx.doi.org/10.1128/jvi.00687-09.
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ABSTRACT While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.
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Saunders, N. A., S. Alexander, and I. Tatt. "env Gene Typing of Human Immunodeficiency Virus Type 1 Strains on Electronic Microarrays." Journal of Clinical Microbiology 43, no. 4 (April 1, 2005): 1910–16. http://dx.doi.org/10.1128/jcm.43.4.1910-1916.2005.
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Bagnarelli, Patrizia, Francesca Mazzola, Stefano Menzo, Maria Montroni, Luca Butini, and Massimo Clementi. "Host-Specific Modulation of the Selective Constraints Driving Human Immunodeficiency Virus Type 1env Gene Evolution." Journal of Virology 73, no. 5 (May 1, 1999): 3764–77. http://dx.doi.org/10.1128/jvi.73.5.3764-3777.1999.
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ABSTRACT To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0.0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.
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Jordan-Paiz, Ana, Sandra Franco, and Miguel Angel Martinez. "Synonymous Codon Pair Recoding of the HIV-1 env Gene Affects Virus Replication Capacity." Cells 10, no. 7 (June 29, 2021): 1636. http://dx.doi.org/10.3390/cells10071636.
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Synonymous codon pair deoptimization is an efficient strategy for virus attenuation; however, the underlying mechanism remains controversial. Here, we optimized and deoptimized the codon pair bias (CPB) of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene to investigate the influence of env synonymous CPB recoding on virus replication capacity, as well as the potential mechanism. We found that env CPB deoptimization did not always generate attenuation, whereas CPB optimization attenuated virus replication in MT-4 cells. Furthermore, virus attenuation correlated with reduced Env protein production but not with decreased viral RNA synthesis. Remarkably, in our model, increasing the number of CpG dinucleotides in the 5′ end of env did not reduce the replication capacity of HIV-1. These results indicate that factors other than CPB or CpG content may have impacted the viral fitness of the synonymously recoded study variants. Our findings provide evidence that CPB recoding-associated attenuation can affect translation efficiency. Moreover, we demonstrated that an increased number of CpGs in the 5′ end of HIV-1 env is not always associated with reduced virus replication capacity.
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Takehisa, Jun, Léopold Zekeng, Eiji Ido, Yumi Yamaguchi-Kabata, Innocent Mboudjeka, Yosuke Harada, Tomoyuki Miura, Lazare Kaptué, and Masanori Hayami. "Human Immunodeficiency Virus Type 1 Intergroup (M/O) Recombination in Cameroon." Journal of Virology 73, no. 8 (August 1, 1999): 6810–20. http://dx.doi.org/10.1128/jvi.73.8.6810-6820.1999.
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ABSTRACT Here we describe, for the first time, recombinants between two highly divergent major groups of human immunodeficiency virus type 1 (HIV-1), M and O, within a Cameroonian woman infected with three different HIV-1 strains, a group O virus, a subtype D virus, and a recently reported IBNG (A/G)-like recombinant virus. Using nested extra-long PCR amplification, we sequenced from the polregion to the env region including accessory genes of the viral genome obtained from the patient’s uncultured peripheral blood mononuclear cells and examined the phylogenetic position of each gene. Compared with sequential blood samples obtained in 1995 and 1996, there were multiple segmental exchanges between three HIV-1 strains (O, D, and IBNG) and all the recombinants appeared to be derived from a common M/O ancestor. Importantly, recombination between groups M and O occurred, even though the homology between these two groups is 69, 76, 68, and 55% in the gag, pol,vif-vpr, and env regions, respectively. Recombination between strains with such distant lineages may contribute substantially to generating new HIV-1 variants.
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Chen, Steve S. L., Sheau-Fen Lee, Chin-Kai Chuang, and V. Samuel Raj. "trans-Dominant Interference with Human Immunodeficiency Virus Type 1 Replication and Transmission in CD4+ Cells by an Envelope Double Mutant." Journal of Virology 73, no. 10 (October 1, 1999): 8290–302. http://dx.doi.org/10.1128/jvi.73.10.8290-8302.1999.
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ABSTRACT We previously reported that a human immunodeficiency virus type 1 (HIV-1) envelope (Env) mutant with the whole cytoplasmic domain deleted, denoted mutant TC, is able to dominantly interfere with wild-type (wt) virus infectivity. In the present study, the feasibility of developing a dominant negative mutant-based genetic anti-HIV strategy targeting the gp41 cytoplasmic domain was investigated. Mutants TC and 427,TC, a TC derivative with a Trp-to-Ser substitution introduced into residue 427 in the CD4-binding site, and a series of mutants with deletions in the cytoplasmic domain, effectivelytrans-dominantly interfered with wt Env-mediated viral infectivity, as demonstrated by an env trans-complementation assay. The syncytium formation-defective 427,TC double mutant not only inhibited heterologous LAV and ELI Env-mediated viral infectivity but also interfered with syncytium formation and infectivity mediated by the Env proteins of the two primary isolates 92BR and 92US. Stable HeLa-CD4-LTR-β-gal clones that harbored Tat-controlled expression cassettes encoding the control ΔKS, which had a deletion in the env gene, wt, or mutantenv gene were generated. Viral transmission mediated by laboratory-adapted T-cell-tropic HXB2 and NL4-3 viruses was greatly reduced in the TC and 427,TC transfectants compared to that observed in the control ΔKS and wt transfectants. Viral replication caused by HXB2 and NL4-3 viruses and by macrophage-tropic ConB and ADA-GG viruses was delayed or reduced in human CD4+ T cells transfected with the 427,TC env construct compared to that observed in cells transfected with the control ΔKS or TC envconstruct. The lack of significant interference by TC mutant was due neither to the lack of TC env gene integration into host DNA nor to the lack of TC Env expression upon Tat induction. These results indicate that this 427,TC Env double mutant has a role in the development of trans-dominant mutant-based genetic anti-HIV strategies.
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Mochizuki, Hideki, Joan P. Schwartz, Koichi Tanaka, Roscoe O. Brady, and Jakob Reiser. "High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells." Journal of Virology 72, no. 11 (November 1, 1998): 8873–83. http://dx.doi.org/10.1128/jvi.72.11.8873-8883.1998.
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ABSTRACT Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266–15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/β-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5′ and 3′ long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 × 107 CFU/μg of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 × 106 to 8 × 106 CFU/μg of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.
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Vzorov, Andrei N., Dabney W. Dixon, Jenna S. Trommel, Luigi G. Marzilli, and Richard W. Compans. "Inactivation of Human Immunodeficiency Virus Type 1 by Porphyrins." Antimicrobial Agents and Chemotherapy 46, no. 12 (December 2002): 3917–25. http://dx.doi.org/10.1128/aac.46.12.3917-3925.2002.
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ABSTRACT We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-β-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 μg/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.
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Machuca, Roberto, Louise B. Jørgensen, Peter Theilade, and Claus Nielsen. "Molecular Investigation of Transmission of Human Immunodeficiency Virus Type 1 in a Criminal Case." Clinical Diagnostic Laboratory Immunology 8, no. 5 (September 1, 2001): 884–90. http://dx.doi.org/10.1128/cdli.8.5.884-890.2001.
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ABSTRACT Very few criminal cases involving human immunodeficiency virus type 1 (HIV-1) transmission have been described. We report on an HIV-1 transmission case with a child being infected by an HIV-1-positive man. The objective was to determine through molecular epidemiology and phylogenetic analyses whether HIV-1 from the HIV-1-positive man could be the source of infection in the HIV-1-positive child, as claimed by the authorities. We conducted genetic analysis of three different parts of the HIV-1 genome (gag, pol, and env) by PCR, direct-sequencing, and phylogenetic analyses. We used maximum likelihood, maximum parsimony, and neighbor-joining methods for the phylogenetic analyses to investigate whether the sequences from the man and the child were related. We found that the viral sequences from the man and the child formed separate clusters in all of the phylogenetic analyses compared to the local controls. A unique amino acid deletion was identified in the C2-V3-C3 region of the env gene in the virus from the man and the child. These results were used in the criminal court to elucidate whether the virus from the man was related to the virus from the child. In summary, the results from the phylogenetic analyses, the sequence distances between the virus from the man and the virus from the child, and the identification of the unique molecular fingerprint in the env gene together indicated that the virus from the man and the virus from the child were epidemiologically linked.
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Martinez-Picado, Javier, Simon D. W. Frost, Nuria Izquierdo, K. Morales-Lopetegi, Silvia Marfil, Teresa Puig, Cecilia Cabrera, Bonaventura Clotet, and Lidia Ruiz. "Viral Evolution during Structured Treatment Interruptions in Chronically Human Immunodeficiency Virus-Infected Individuals." Journal of Virology 76, no. 23 (December 1, 2002): 12344–48. http://dx.doi.org/10.1128/jvi.76.23.12344-12348.2002.
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ABSTRACT We analyzed the evolution of the human immunodeficiency virus type 1 (HIV-1) env gene in 12 chronically infected individuals who underwent structured treatment interruptions (STIs). Analyses of length variation and of clonal sequences demonstrated highly unpredictable evolution, which may limit the strengthening of HIV-specific immune responses by STIs because of the variability in exposure to viral antigens.
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Xin, Ruolei, Xiang He, Hui Xing, Feng Sun, Mingjian Ni, Yuanzhi Zhang, Zhefeng Meng, et al. "Genetic and temporal dynamics of human immunodeficiency virus type 1 CRF07_BC in Xinjiang, China." Journal of General Virology 90, no. 7 (July 1, 2009): 1757–61. http://dx.doi.org/10.1099/vir.0.009290-0.
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To explore the temporal genetic variation of human immunodeficiency virus type 1 CRF07_BC and reconstruct its epidemic in Xinjiang, China, we studied 216 C2–V4 fragments of env genes sampled from 1996 to 2008. Phylogenetic analysis indicates that the viruses prevailing in Xinjiang form a large monophyletic cluster and may have originated from a common ancestor. The epidemic in Xinjiang was probably established around 1995 (95 % confidence interval, 1994–1996). We noted an increased diversity of CRF07_BC over time, with a rapid evolutionary rate we estimated to be 8.3×10−3 substitutions per site per year in the env gene. After 5–6 years of the epidemic (1997–2002), the transmission rate of CRF07_BC in Xinjiang slowed down, although CRF07_BC infection remained at a high prevalence.
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Kimura, T., and Jonathan Karn. "Murine retrovirus vector expressing env gene of human immunodeficiency virus type 1(HIV-1)." Uirusu 41, no. 2 (1991): 85–94. http://dx.doi.org/10.2222/jsv.41.85.
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