Stable expression of human immunodeficiency virus type 1 Nef confers resistance against Fas-mediated apoptosis

To investigate the effect of Nef on Fas-mediated apoptosis, we compared T cells, both population and subclones stably expressing Nef from HIV-1(NL432), with Nef(-) control cells. Fas-mediated apoptosis was significantly delayed in Nef(+) cells as determined by annexin V staining and the percentage of apoptotic cells was lower in all Nef-expressing cells than in the control cells by a maximum of 10-fold. Next we measured cell surface levels of Fas to test whether the delayed apoptosis in Nef(+) cells was due to reduced cell surface expression of Fas. We found that there was no significant difference in the surface level of Fas between the Nef(+) and Nef(-) cells. To further define the steps affected by Nef in the Fas signaling pathway, the activation of caspase-3 and caspase-8 was investigated. A reasonable correlation was found between the magnitude of apoptosis measured by annexin V staining and the enzymatic activity of caspase-3. The overall level of caspase-8 activity in Nef(+) cells was also lower than in Nef(-) cells, although the extent of inhibition was not as significant as seen for caspase-3. Overall, our results indicate that long-term stable expression of Nef, which mimicks persistent or latent infection in vivo, confers resistance against anti-Fas Ab-induced apoptosis through inhibition of caspase-3 and caspase-8 activation.     

Mutational analysis of the human immunodeficiency virus type 1 Eli Nef function.

The studies presented here define an internally consistent experimental system that permits systematic analysis of the effect of nef on the rate of the human immunodeficiency virus type 1 (HIV-1) replication in a CD4+ tumor T-cell line and in primary peripheral blood mononuclear cells. The parental full-length Nef protein, derived from the Eli strain of HIV-1, accelerates virus replication in both cell types. Mutations that destabilize or alter the intracellular location of the protein affect the ability of the Nef protein to accelerate virus replication. A set of mutants was made in amino acids proposed to be required for Nef function, including threonine and serine residues proposed to be targets for phosphorylation, and in sequences thought to resemble the G-1, G-3, and G-4 sites of the family of G proteins. In most cases alterations of the critical amino acids yield stable Nef proteins of parental phenotype. These results challenge the existing theories for the mechanism of Nef function. The results also identify two residues in the carboxyl half of the protein that are important for Nef function.     

Functional domains of the human immunodeficiency virus type 1 Nef protein are conserved among different clades in Cameroon

The Nef protein of human immunodeficiency virus type 1 (HIV-1) has multiple functional domains, is immunogenic, and contains several cytotoxic T lymphocyte (CTL)-targeted epitopes. Several defined subfunctions of Nef are important for the pathogenesis of HIV-1 infection. In this study, we present the genetic diversity of the nef gene of 55 newly derived HIV-1 sequences obtained from Cameroonian patients. Four genetic subtypes and three circulating recombinant forms (CRFs) were identified: subtypes A (11%), G (7.3%), D (5.4%), F1 (1.8%), F2 (5.4%), CRF01_AE (5.4%), CRF02_AG (58.2%), and CRF11_cpx (1.8%). Two isolates clustered distinctly from the known HIV-1 genetic subtypes in nef and were designated as unclassified. Interestingly, the majority of all functional domains including the myristoylation signal, CD4 binding motif, beta turn motif, and the phosphorylation sites were well conserved in our cohort. Putative CTL-epitopic domains of the central portion of Nef were also well conserved, whereas those at the C-term were not. Our study demonstrated that despite high genetic diversity observed in the nef gene, most described functional domains and CTL epitopes were well conserved among Cameroonian HIV-1 subtypes. These findings could be used for the development of antiretroviral-acting therapeutics and anti-HIV-1 vaccines.     

Monitoring of antibodies against human immunodeficiency virus type 1 p25 core protein as prognostic marker.

Anti-p25 antibodies were evaluated by cross-sectional analysis of sera from 130 human immunodeficiency virus type 1-infected patients and in a longitudinal study of 56 patients by retrospective analysis of sequentially collected sera. High and stable antibody levels were found in Centers for Disease Control stage II or III patients, 78% of whom had levels greater than 10 arbitrary units/mL. Patients with AIDS-related complex displayed heterogeneous levels. Patients with AIDS had the lowest values: less than or equal to 10 units/mL in 96% of cases. In patients whose CD4+ cell counts eventually fell below 200/mm3 or who developed AIDS (or both), antibodies were initially less than 40 units/mL and/or they declined with a rate greater than 1 log unit/5 years, beginning at least 4 years before the index symptom. Because the only point at which CD4+ cell counts significantly differed between progressors and nonprogressors was 1 year before the disease, both initial anti-p25 values and antibody decline seemed to be better long-term prognostic markers than CD4+ cell counts.     

Functional role of human immunodeficiency virus type 1 vpu.

To investigate the role of vpu in the replication and cytopathicity of human immunodeficiency virus type 1 (HIV-1), infectious proviruses were constructed that were isogenic except for the ability to produce the protein product of vpu. The vpu-encoded protein is shown to decrease the rate of syncytium formation and cell killing in infected CD4+ human T cells, to increase greatly the export of virus particles from infected cells, and to reduce the rate of accumulation of cell-associated viral proteins. The vpu protein complements in trans the defect in a vpu- HIV-1 provirus but does not affect the simian immunodeficiency virus, which lacks vpu. These observations suggest that vpu may contribute to the AIDS epidemic by increasing the transmission efficiency of the virus.     

Angiogenic properties of human immunodeficiency virus type 1 Tat protein.

Extracellular human immunodeficiency virus type 1 (HIV-1) Tat protein promotes growth of spindle cells derived from AIDS-associated Kaposi sarcoma (AIDS-KS), an angioproliferative disease very frequent in HIV-1-infected individuals. Normal vascular cells, progenitors of AIDS-KS cells, proliferate in response to Tat after exposure to inflammatory cytokines, whose levels are augmented in HIV-1-infected individuals and in KS lesions. Here we show that Tat also promotes AIDS-KS and normal vascular cells to migrate and to degrade the basement membrane and stimulates endothelial cell morphogenesis on a matrix substrate. These effects are obtained at picomolar concentrations of exogenous Tat and are promoted by the treatment of the cells with the same inflammatory cytokines stimulating expression of the receptors for Tat, the integrins alpha 5 beta 1 and alpha v beta 3. Thus, under specific circumstances, Tat has angiogenic properties. As Tat and its receptors are present in AIDS-KS lesions, these data may explain some of the mechanisms by which Tat can induce angiogenesis and cooperate in the development of AIDS-KS.     

Vpu: a multifunctional protein that enhances the pathogenesis of human immunodeficiency virus type 1

The Vpu protein is the smallest of the proteins encoded by human immunodeficiency virus type 1 (HIV-1). This transmembrane protein interacts with the CD4 molecule in the rough endoplasmic reticulum (RER), resulting in its degradation via the proteasome pathway. Vpu also has been shown to enhance virion release from infected cells. While much has been learned about the function of Vpu in cell culture systems, its exact role in HIV-1 pathogenesis is still unknown. This has been primarily due to the lack of a suitable primate model system since vpu is found only in HIV-1 and simian immunodeficiency viruses isolated from chimpanzees (SIVcpz), and three species of old world monkeys within the genus Cercopithecus. Several laboratories have developed pathogenic molecular clones of simian-human immunodeficiency virus (SHIV) in which the tat, rev, vpu and env genes of HIV-1 are expressed in the genetic background of SIV. The availability of such clones has allowed investigators to assess the role of Vpu in pathogenesis using a relevant animal model. This review will focus on the current understanding of the structure-function relationships of Vpu protein and recent advances using the SHIV model to assess the role of Vpu in HIV-1 pathogenesis.     

Acquired immunodeficiency syndrome-associated T-cell lymphoma: evidence for human immunodeficiency virus type 1-associated T-cell transformation.

The majority of lymphomas in the setting of acquired, iatrogenic, or congenital immunodeficiencies are B-cell lymphoproliferations. We describe a rare T-cell lymphoma in a fulminantly ill patient infected with human immunodeficiency virus type 1 (HIV-1). The T-cell nature of the process was defined genotypically (monoclonal T-cell receptor beta-chain [CT beta] rearrangement) and phenotypically (CD45RO+, CD4+, CD5+, CD25+, CD8-, CD3- and negative for a variety of B-cell and monocyte markers). The CD4+, CD25+ (interleukin-2 receptor [IL-2R]) phenotype with production of IL-2 and IL-2R RNA is analogous to human T-lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATLL); however, no HTLV-1 could be detected. Southern blot analysis did demonstrate monoclonally integrated HIV-1 within the tumor genome. Furthermore, the tumor cells were producing HIV p24 antigen as shown by immunohistochemistry. This is the first case of acquired immunodeficiency syndrome (AIDS)-associated non-Hodgkin's lymphoma in which HIV-1 infection may have played a central role in the lymphocyte transformation process.     

Gene inoculation generates immune responses against human immunodeficiency virus type 1.

Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.     

Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.

Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.     

Attenuated Mengo virus as a vector for immunogenic human immunodeficiency virus type 1 glycoprotein 120.

Introduction of a sequence encoding 147 amino acids from human immunodeficiency virus type I (HIV-1) strain MN glycoprotein gp120 into the RNA genome of the stably attenuated Mengo virus strain vM16 yielded an infectious recombinant virus, vMLN450, which expressed the heterologous HIV-1 sequence along with the normal Mengo virus proteins. The HIV-1 gp120 sequence, fused to the amino terminus of the short, nonstructural Mengo virus leader polypeptide was recognized by a gp120 V3 loop-specific monoclonal antibody. When inoculated into mice, recombinant virus vMLN450 elicited a high-titer anti-HIV-1 antibody response as well as an HIV-1MN-specific cytotoxic cellular immune response. An anti-HIV-1 antibody response could also be detected in cynomolgus monkeys after a single immunization. We propose that attenuated Mengo virus can serve as an effective expression vector in cell systems and various animal species and offers another approach to the development of new, live recombinant vaccines.     

Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.

The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.     

Herpes simplex virus type 1 and human immunodeficiency virus type 1 antigens in platelets from a hemophilia B patient with human immunodeficiency virus type 1-related thrombocytopenia.

We analyzed platelet-associated antigens from a hemophilia B patient with human immunodeficiency virus type 1 (HIV-1)-related thrombocytopenia. Two bands appeared at 31,000 and 37,000 daltons in the platelet lysate after reaction with autologous serum in SDS-PAGE and Western blots. The band at 37,000 daltons was obtained using anti-herpes simplex type 1 (HSV-1) rabbit antiserum. Doublet bands at 36,000 and 37,000 daltons also appeared after reaction with HSV-1 seropositive human serum. The band at 31,000 daltons appeared after reaction with anti-HIV-1 rabbit serum. These results suggest that the platelet-associated antigens in this patient are components of both HSV-1 and HIV-1 antigens. In addition, acyclovir decreased his PAIgG level and increased his platelet count, and zidovudine increased his platelet count. Thus, we concluded that each of the platelet-associated antigens is partially responsible for the thrombocytopenia by causing deposition of immune complexes in this patient.