Synthesis and antiretroviral activity of phospholipid analogs of azidothymidine and other antiviral nucleosides

Treatment of acquired immunodeficiency syndrome with azidothymidine (AZT, zidovudine) reduces p24 antigenemia, increases CD4 lymphocyte counts, reduces the frequency and severity of opportunistic infections and prolongs life. However, AZT and other dideoxynucleosides do not diminish the ability to isolate human immunodeficiency virus (HIV) from peripheral blood mononuclear cells. Failure to clear infectious virus may be due to inadequate inhibition of virus production by macrophages, a major reservoir of HIV infection. Cells of the macrophage lineage take up large amounts of parenterally administered liposomal material. To direct larger proportions of antiretroviral nucleosides to this important HIV reservoir, we synthesized phosphatidylAZT, AZT diphosphate dipalmitin, phosphatidylddC and phosphatidylddT, novel phospholipid prodrugs which are readily incorporated into phospholipid bilayers. These liposomal liponucleotides were shown to have antiretroviral activity in HIV-infected U937 and CEM cells. In vivo, it is anticipated that liposomes containing the antiretroviral liponucleotides will be taken up in large proportion by macrophages. This property would appear to make phosphatidylAZT and the related compounds promising candidate agents with a special potential to target drug to the macrophage reservoir of HIV infection, thereby reducing the toxicity of the antiviral nucleosides to other cells.     

Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study.

Recombinant human colony-stimulating factor-1-treated human peripheral blood-derived monocytes-macrophages are efficient host cells for recovery of the human immunodeficiency virus (HIV) from blood leukocytes of patients with acquired immunodeficiency syndrome. These cells can be maintained as viable monolayers for intervals exceeding 3 months. Infection with HIV resulted in virus-induced cytopathic effects, accompanied by relatively high levels of released progeny virus, followed by a prolonged low-level release of virus from morphologically normal cells. In both acutely and chronically infected monocytes, viral particles were seen budding into and accumulating within cytoplasmic vacuoles. The number of intravacuolar virions far exceeded those associated with the plasma membrane, especially in the chronic phase, and were concentrated in the perinuclear Golgi zone. In many instances, the vacuoles were identified as Golgi elements. Fusion of virus-laden vacuoles with primary lysosomes were rare. The pattern of cytoplasmic assembly of virus was observed with both HIV types 1 and 2 and in brain macrophages of an individual with acquired immunodeficiency syndrome encephalopathy. Immunoglobulin-coated gold beads added to acutely infected cultures were segregated from the vacuoles containing virus; relatively few beads and viral particles colocalized. The assembly of HIV virions within vacuoles of macrophages is in contrast to the exclusive surface assembly of HIV by T lymphocytes. Intracytoplasmic virus hidden from immune surveillance in monocytes-macrophages may explain, in part, the persistence of HIV in the infected human host.     

Transactivation of human immunodeficiency virus promoter by human herpesvirus 6.

Patients with acquired immunodeficiency syndrome (AIDS) are often infected with a number of other heterologous viruses in addition to the initial human immunodeficiency virus (HIV) infection, and these agents could act as potential reactivating agents of latent HIV. A new antigenically distinct herpesvirus, designated human herpesvirus 6 (HHV-6), has recently been isolated from patients with AIDS and has been shown to infect a number of different human cells, specifically human T cells, B cells, and glial cells. Since these are some of the same cells that harbor the AIDS virus, it is quite important to determine any interaction between this new herpesvirus and HIV. In this report, we demonstrate that HHV-6 can trans-activate the HIV promoter in human T-cell lines as measured by the expression of the bacterial gene chloramphenicol acetyltransferase. This indicates that stimulation of HIV gene expression by HHV-6 could play a role in HIV pathogenesis.     

Virus evolution within patients increases pathogenicity

Viruses like the human immunodeficiency virus (HIV), the hepatitis B virus (HBV), the hepatitis C virus (HCV) and many others undergo numerous rounds of inaccurate reproduction within an infected host. The resulting viral quasispecies is heterogeneous and sensitive to any selection pressure. Here we extend earlier work by showing that for a wide class of models describing the interaction between the virus population and the immune system, virus evolution has a well-defined direction toward increased pathogenicity. In particular, we study virus-induced impairment of the immune response and certain cross-reactive stimulation of specific immune responses. For eight different mathematical models, we show that virus evolution reduces the equilibrium abundance of uninfected cells and increases the rate at which uninfected cells are infected. Thus, in general, virus evolution makes things worse. An idea for combating HIV infection, however, is constructing a virus mutant that could outcompete the existing infection without being pathogenic itself.     

Feline immunodeficiency virus tropism

Feline immunodeficiency virus (FIV) induces a disease similar to acquired immunodeficiency syndrome (AIDS) in cats, yet in contrast to human immunodeficiency virus (HIV), CD4 is not the viral receptor. We identified a primary receptor for FIV as CD134 (OX40), a T cell activation antigen and costimulatory molecule. CD134 expression promotes viral binding and renders cells permissive for viral entry, productive infection, and syncytium formation. Infection is CXCR4-dependent, analogous to infection with X4 strains of HIV. Thus, despite the evolutionary divergence of the feline and human lentiviruses, both viruses use receptors that target the virus to a subset of cells that are pivotal to the acquired immune response. Further, we applied the new method for FIV receptor to Ebola virus entry factors with some modifications, and identified receptor-type tyrosine kinases, Axl and Dtk (members of Tyro3 family). Distribution of the molecules matches well with the Ebola virus tropism.     

The value of primate models for studying human immunodeficiency virus pathogenesis

Abstract: Research on human immunodeficiency virus (HIV) infection is compromised by the obvious limitation in having for study only virus-infected individuals or those exposed to the virus. Steps involved in transmission or pathogenesis require planned experimentation. The identification of animal models of acquired immunodeficiency syndrome (AIDS) has therefore been helpful for evaluating phases of HIV pathogenesis. Of the seven subgenera of lentiviruses now recognized, two share the characteristics with HIV of a T cell tropism and the associated loss of CD4+ cells in the host associated with disease: the feline immunodeficiency virus (FIV) and the simian immunodeficiency virus (SIV) (Table 1). The other animal lentiviruses grow best in macrophages and their infection generally reflects clinical sequellae of infection of this cell type. This review addresses those features of SIV, HIV, and SHIV infections of non-human primates that illustrate the importance of the animal models of AIDS.     

Differentiation of human embryonal carcinoma cells induces human immunodeficiency virus permissiveness which is stimulated by human cytomegalovirus coinfection.

Human immunodeficiency virus (HIV) replicates in differentiated but not undifferentiated NTERA-2 human embryonal carcinoma cells; neither cell type expresses CD4. Susceptibility of the differentiated cells is enhanced by coinfection with cytomegalovirus. HIV infection induces lactoseries glycolipids, suggesting a mechanism whereby HIV might interfere with normal embryogenesis.     

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.     

The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species transmission.

The primate immunodeficiency virus Vif proteins are essential for replication in appropriate cultured cell systems and, presumably, for the establishment of productive infections in vivo. We describe experiments that define patterns of complementation between human and simian immunodeficiency virus (HIV and SIV) Vif proteins and address the determinants that underlie functional specificity. Using human cells as virus producers, it was found that the HIV-1 Vif protein could modulate the infectivity of HIV-1 itself, HIV-2 and SIV isolated from African green monkeys (SIVAGM). In contrast, the Vif proteins of SIVAGM and SIV isolated from Sykes' monkeys (SIVSYK) were inactive for all HIV and SIV substrates in human cells even though, at least for the SIVAGM protein, robust activity could be demonstrated in cognate African green monkey cells. These observations suggest that species-specific interactions between Vif and virus-producing cells, as opposed to between Vif and virus components, may govern the functional consequences of Vif expression in terms of inducing virion infectivity. The finding that the replication of murine leukemia virus could also be stimulated by HIV-1 Vif expression in human cells further supported this notion. We speculate that species restrictions to Vif function may have contributed to primate immunodeficiency virus zoonosis.     

DC-SIGN interactions with human immunodeficiency virus: virus binding and transfer are dissociable functions

The C-type lectins DC-SIGN and DC-SIGNR capture and transfer human immunodeficiency virus (HIV) to susceptible cells, although the underlying mechanism is unclear. Here we show that DC-SIGN/DC-SIGNR-mediated HIV transmission involves dissociable binding and transfer steps, indicating that efficient virus transmission is not simply due to tethering of virus to the cell surface.     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

Quantitative expression and virus transmission analysis of DC-SIGN on monocyte-derived dendritic cells

The C-type lectins DC-SIGN and DC-SIGNR efficiently bind human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains and can transmit bound virus to adjacent CD4-positive cells. DC-SIGN also binds efficiently to the Ebola virus glycoprotein, enhancing Ebola virus infection. DC-SIGN is thought to be responsible for the ability of dendritic cells (DCs) to capture HIV and transmit it to T cells, thus promoting HIV dissemination in vitro and perhaps in vivo as well. To investigate DC-SIGN function and expression levels on DCs, we characterized a panel of monoclonal antibodies (MAbs) directed against the carbohydrate recognition domain of DC-SIGN. Using quantitative fluorescence-activated cell sorter technology, we found that DC-SIGN is highly expressed on immature monocyte-derived DCs, with at least 100,000 copies and often in excess of 250,000 copies per DC. There was modest variation (three- to fourfold) in DC-SIGN expression levels between individuals and between DCs isolated from the same individual at different times. Several MAbs efficiently blocked virus binding to cell lines expressing human or rhesus DC-SIGN, preventing HIV and SIV transmission. Interactions with Ebola virus pseudotypes were also blocked efficiently. Despite their ability to block virus-DC-SIGN interactions on cell lines, these antibodies only inhibited transmission of virus from DCs by approximately 50% or less. These results indicate that factors other than DC-SIGN may play important roles in the ability of DCs to capture and transmit HIV.     

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine.

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.     

Short-lived infected cells support virus replication in sooty mangabeys naturally infected with simian immunodeficiency virus: implications for AIDS pathogenesis

Sooty mangabeys (SMs) naturally infected with simian immunodeficiency virus (SIV) do not develop AIDS despite high levels of virus replication. At present, the mechanisms underlying this disease resistance are poorly understood. Here we tested the hypothesis that SIV-infected SMs avoid immunodeficiency as a result of virus replication occurring in infected cells that live significantly longer than human immunodeficiency virus (HIV)-infected human cells. To this end, we treated six SIV-infected SMs with potent antiretroviral therapy (ART) and longitudinally measured the decline in plasma viremia. We applied the same mathematical models used in HIV-infected individuals and observed that SMs naturally infected with SIV also present a two-phase decay of viremia following ART, with the bulk (92 to 99%) of virus replication sustained by short-lived cells (average life span, 1.06 days), and only 1 to 8% occurring in longer-lived cells. In addition, we observed that ART had a limited impact on CD4(+) T cells and the prevailing level of T-cell activation and proliferation in SIV-infected SMs. Collectively, these results suggest that in SIV-infected SMs, similar to HIV type 1-infected humans, short-lived activated CD4(+) T cells, rather than macrophages, are the main source of virus production. These findings indicate that a short in vivo life span of infected cells is a common feature of both pathogenic and nonpathogenic primate lentivirus infections and support a model for AIDS pathogenesis whereby the direct killing of infected cells by HIV is not the main determinant of disease progression.     

A bispecific antibody composed of a nonneutralizing antibody to the gp41 immunodominant region and an anti-CD89 antibody directs broad human immunodeficiency virus destruction by neutrophils

In addition to the direct neutralization of virus, there is a broader potential for antibody-mediated inhibition of human immunodeficiency virus (HIV) by targeting HIV to effector cells. We demonstrate here that a bispecific antibody incorporating a broadly reactive anti-gp41 antibody, F240, and an anti-IgA receptor (CD89) antibody is effective at directing neutrophils to destroy HIV. Not only are neutrophils the predominant type of white blood cells and very efficient at mediating cell cytotoxicity, they are relatively resistant to infection with HIV. Therefore, they represent a significant weapon against infection if they can be directed and armed to destroy HIV and infected cells.     

Immune complexes containing human immunodeficiency virus type 1 primary isolates bind to lymphoid tissue B lymphocytes and are infectious for T lymphocytes

This study investigated the interaction of tonsil B lymphocytes with immune complexes containing human immunodeficiency virus (HIV IC) primary isolates and the infectivity of the B cell-bound HIV IC. Treatment of virus with a source of antibody and complement increased HIV IC binding to B cells by 5.6-fold. Most of the HIV IC that bound to B cells were not internalized but remained on the cell surface and were gradually released over 72 h. Cell-bound HIV IC were highly infectious for T cells while virus released by cultured B cells was only slightly infectious. Removal of HIV IC from the B-cell surface by protease treatment reduced the infection of T cells to near-background levels, indicating that infectious virus remained on the B-cell surface. These studies show that B lymphocytes can carry and transfer infectious HIV IC to T cells and thus suggest a novel mode of infection of T cells in lymphoid tissue that could be important for pathogenesis during HIV infection.     

基于调控趋化性细胞因子受体CCR5抗HIV-1的研究进展

获得性免疫缺陷综合征(AIDS),又称"艾滋病",是威胁着人类生命健康的重大传染性疾病。趋化性细胞因子受体5(Chemotactic cytokine receptor 5,CCR5)作为嗜巨噬细胞性1型人类免疫缺陷病毒(Human immunodeficiency virus type 1,HIV-1)进入宿主细胞的必需的辅助受体自发现以来被广泛关注。CCR5在HIV靶细胞表面的表达水平与靶细胞的病毒载量呈正相关且和艾滋病的进程强相关。因此,通过干预CCR5在靶细胞表面的表达能在一定程度上阻止HIV进入靶细胞。因此,本文从分子水平阐述干预CCR5的主要生物学机制,并对近几年的研究进行综述。     

A biological response modifier, PSK, inhibits human immunodeficiency virus infection in vitro

PSK, a biological response modifier (BRM), was studied to determine its anti-viral activity on human immunodeficiency virus (HIV) in vitro. Either a novel infection system using human T-cell lymphotropic virus type I (HTLV-I)-carrying MT-4 cells or a coculture system using MOLT-4 cells and its virus-producing cells MOLT-4/HIVHTLV-IIIB which induces multinucleated giant cells very efficiently was used. PSK almost completely blocked the cytopathic effect such as giant cell formation and HIV-specific antigen expression both in MT-4 cells and MOLT-4 cells at a concentration of 0.4 and 0.8 mg/ml, respectively. Pretreatment of the virus with PSK may specifically interfere with early stages of HIV infection by modifying the viral receptor.