Mario Stevenson

SH3-mediated Hck Tyrosine Kinase Activation and Fibroblast Transformation by the Nef Protein of HIV-1

Tyrosine kinases of the Src family are regulated via their Src homology 2 (SH2) and SH3 domains. The Nef protein of human immunodeficiency virus-1 (HIV-1) has previously been shown to bind with high affinity and specificity in vitro to the SH3 domain of Hck, a Src family member expressed primarily in myeloid cells. However, the effect of Nef on Hck activity in living cells is unknown. Here we show that Rat-2 fibroblasts co-expressing Hck and Nef rapidly developed transformed foci, whereas control cells expressing either protein alone did not. Nef formed a stable complex with Hck and stimulated its tyrosine kinase activity in vivo. Mutagenesis of the Nef proline-rich motif essential for SH3 binding completely blocked complex formation, kinase activation, and transformation, indicating that the Nef SH3-binding function is required for its effects on Hck. These results provide direct evidence that SH3 engagement is sufficient to activate a Src family kinasein vivo and suggest that Hck may be activated by Nef in HIV-infected macrophages.

Inhibition of human immunodeficiency virus type 1-mediated cytopathic effects by poly(L-lysine)-conjugated synthetic antisense oligodeoxyribonucleotides.

The ability of poly(L-lysine)-conjugated and methylphosphonate-modified synthetic human immunodeficiency virus type 1 (HIV-1) antisense oligodeoxyribonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection was studied. The abundance of viral antigens in oligomer-treated cultures indicated that the oligomers did not significantly affect viral infectivity. Similarly, no significant effects on relative viral RNA accumulation were apparent. The presence of poly(L-lysine)-modified oligomer complementary to the HIV-1 splice donor site resulted in a significant reduction in the production of viral structural proteins and virus titre in infected cultures. In addition, these cells were protected from HIV-1-mediated cytopathic effects while the other cultures rapidly succumbed to the cytotoxic effects of HIV-1 infection. The presence of poly(L-lysine)-conjugated oligomer resulted in the establishment of a persistent HIV-1 infection characterized by a highly productive virus infection in the absence of cell death while treatment of persistently infected cells with phorbol ester resulted in renewed cytopathicity. These results demonstrate the ability of synthetic antisense oligonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection.

Susceptibility of human glial cells to infection with human immunodeficiency virus (HIV)

Three human brain‐derived cell lines (including two of astrocytic origin) were exposed in vitro to the human immunodeficiency virus (HIV), the etiologic agent of immunodeficiency in AIDS. In all three lines, HIV transcripts were detected by in situ hybridisation in 20–30% of cells 48 h afterinfection. Synthesis of virus gag gene products p24 and p55 was demonstrated by immunoblotting. No cytopathic effects typical of HIV‐infected human T lymphocytes were observed. Our data indicate that HIV is neurotropic, and support the hypothesis that this virus may infect astrocytes in the brain.

A human T-cell line resistant to cytopathic effects of the human immunodeficiency virus (HIV)

Infection of human helper T lymphocytes with the human immunodeficiency virus (HIV) results in a rapid induction of cytopathic effects and cell lysis. We isolated a variant of the human T-lymphoblastoid cell line, CEM, that is fully susceptible to HIV infection but resistant to virally induced cytopathic effects. Exposure of the cells, designated CR-10, to HIV resulted in the expression of viral antigens in 100% of cells within 6-9 days. Virus-infected cells remained fully viable and could be cultivated under standard culture conditions for a desired period of time. Parental CEM cells died within 9-12 days after HIV infection. Proviral DNA could be detected in the HIV-infected CR-10 cells by Southern blot and molecular hybridization 4-5 days after infection; the relative amount of proviral DNA reached maximum at Days 6-10 and remained stable during an 8-month follow-up period. Virus production by HIV-infected CR-10 cells was documented by electron microscopy and detection of reverse transcriptase activity in cell culture supernatants. HIV-infected CR-10 cells exhibited a down modulation of the OKT-3, OKT-4, OKT-4A, OKT-8, and OKT-11 T-cell surface markers, but not of the OKT-9 (transferrin receptor). One of the HIV persistently infected CR-10 cell clones has been kept in continuous culture for over 8 months. During this period, the cells remained fully viable, 100% positive for HIV antigens, and negative for most of the T-cell surface markers tested and continued to produce biologically active HIV. The CR-10 and HIV-infected CR-10 cell lines will be useful in studies on the biology of HIV and in the isolation and large-scale propagation of this virus.

Efficient synthesis of viral nucleic acids following monocyte infection by HIV-1

The replication of human immunodeficiency virus type 1 in mononuclear phagocytes (blood monocytes, tissue macrophages, and dendritic cells) is an important feature of HIV-1 pathogenesis. Although most primary HIV-1 isolates are able to productively infect monocytes, some reports suggest that rates of viral DNA synthesis and virus replication are reduced in HIV-1-infected monocytes as compared to infected T cells. In this study we compare kinetics of viral DNA synthesis in CD4+ T cells and monocytes following HIV-1 infection. Our results indicate that reverse transcription of viral nucleic acids following infection of monocytes occurs at rates equal to or greater than that observed following infection of T cells. These studies reveal no postentry restrictions to HIV-1 replication following infection in monocytes. Moreover, the results support the notion that both monocytes and CD4+ T cells are equally permissive for virus replication in infected individuals.

Expression of the T4 molecule (AIDS virus receptor) by human brain-derived cells

Three human cell lines of astrocytic origin were evaluated for expression of a human T‐lymphocyte surface glycoprotein, T4, which also serves as a cellular receptor for the human immunodeficiency virus (AIDS virus, HIV). T4 antigen was detected on the cell surface of 2 of these cell lines using monoclonal OKT‐4 antibody and flow cytometry. Gene transcripts encoding the T4 molecule were detected by a ribonuclease protection assay in surface T4‐positive and ‐negative cells. Our results suggest that astrocytes may serve as targets for HIV infection in the brain.

Antibodies to HTLV-III/LAV in Venezuelan patients with acute malarial infections.

Malarial infections have been associated with an immunosuppressive state similar to that seen in acquired immunodeficiency syndrome (AIDS). Its manifestations include severe lymphopenia depletion of the T4-lymphocyte subset activation of B cells and diminished in vitro lymphocyte responses. Because of these similarities the authors tested Venezuelan patients with acute malaria for antibodies to human T-lymphotropic virus type III (HTLV-III). The sample included 12 patients with acute Plasmodium vivax infection and 12 with P. falciparum infection. None of the patients were receiving antimalarial drugs and none belonged to any of the recognized risk groups for AIDS. 3 of the patients with P. falciparum infection (25%) and 5 with P. vivax (41%) were found to be positive for HTLV-III antibodies by indirect immunofluorescence. Western blot and radioimmunoprecipitation tests. The pattern of the Western blot and radioimmunoprecipitation bands obtained with samples from malaria patients was similar to that noted in AIDS patients. Since acute malaria does not evolve into an AIDS-like syndrome it is concluded that exposure to HTLV-III and the occurrence of severe immunoregulatory disturbances are not always sufficient for the induction of AIDS.

1,2,3-Triazoles as Amide Bioisosteres: Discovery of a New Class of Potent HIV ‑ 1 Vif Antagonists

RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G) expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC50 = 6 μM) by isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, 1d (IC50 = 1.2 μM). We identified several potent HIV-1 inhibitors from a 1d based library including 5ax (IC50 = 0.01 μM), 5bx (0.2 μM), 2ey (0.4 μM), 5ey (0.6 μM), and 6bx (0.2 μM).

HIV controllers with different viral load cut-off levels have distinct virologic and immunologic profiles

Background:The mechanisms behind natural control of HIV replication are still unclear, and several studies pointed that elite controllers (ECs) are a heterogeneous group. Methods:We performed analyses of virologic, genetic, and immunologic parameters of HIV-1 controllers groups: (1) ECs (viral load, <80 copies/mL); (2) ebbing elite controllers (EECs; transient viremia/blips); and viremic controllers (VCs; detectable viremia, <5000 copies/mL). Untreated noncontrollers (NCs), patients under suppressive highly active antiretroviral therapy (HAART), and HIV-1–negative individuals were analyzed as controls. Results:Total and integrated HIV-1 DNA for EC were significantly lower than for NC and HAART groups. 2-LTR circles were detected in EEC (3/5) and VC (6/7) but not in EC. Although EC and EEC maintain normal T-cell counts over time, some VC displayed negative CD4+ T-cell slopes. VC and EEC showed a higher percentage of activated CD8+ T cells and microbial translocation than HIV-1–negative controls. EC displayed a weaker Gag/Nef IFN-&ggr; T-cell response and a significantly lower proportion of anti-HIV IgG antibodies than EEC, VC, and NC groups. Conclusion:Transient/persistent low-level viremia in HIV controllers may have an impact on immunologic and virologic profiles. Classified HIV controller patients taking into account their virologic profile may decrease the heterogeneity of HIV controllers cohorts, which may help to clarify the mechanisms associated to the elite control of HIV.

Occludin controls HIV transcription in brain pericytes via regulation of SIRT-1 activation.

HIV invades the brain early after infection; however, its interactions with the cells of the blood‐brain barrier (BBB) remain poorly understood. Our goal was to evaluate the role of occludin, one of the tight junction proteins that regulate BBB functions in HIV infection of BBB pericytes. We provide evidence that occludin levels largely control the metabolic responses of human pericytes to HIV. Occludin in BBB pericytes decreased by 10% during the first 48 h after HIV infection, correlating with increased nuclear translocation of the gene repressor C‐terminal‐binding protein (CtBP)‐1 and NFκB‐p65 activation. These changes were associated with decreased expression and activation of the class III histone deacetylase sirtuin (SIRT)‐1. Occludin levels recovered 96 h after infection, restoring SIRT‐1 and reducing HIV transcription to 20% of its highest values. We characterized occludin biochemically as a novel NADH oxidase that controls the expression and activation of SIRT‐1. The inverse correlation between occludin and HIV transcription was then replicated in human primary macrophages and differentiated monocytic U937 cells, in which occludin silencing resulted in 75 and 250% increased viral transcription, respectively. Our work shows that occludin has previously unsuspected metabolic properties and is a target of HIV infection, opening the possibility of designing novel pharmacological approaches to control HIV transcription.—Castro, V., Bertrand, L., Luethen, M., Dabrowski, S., Lombardi, J., Morgan, L., Sharova, N., Stevenson, M., Blasig, I. E., Toborek, M., Occludin controls HIV transcription in brain pericytes via regulation of SIRT‐1 activation. FASEB J. 30, 1234–1246 (2016). www.fasebj.org