Determination of HIV-1 infectivity by lymphocytic cell lines with integrated luciferase gene

We have established lymphocytic cell lines H9 and M8166 that contain integrated copy of luciferase gene under the control of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). While H9 is known to be non-permissive for or insensitive to some particular mutant strains of HIV/simian immunodeficiency virus (SIV), M8166 is one of the most susceptible lines to various HIV/SIVs. The luciferase gene driven by HIV-1 LTR was transfected into H9 and M8166 cells with the neo gene, and cell lines were selected by G418. The indicator cell lines thus obtained were designated H9/H1luc and M8166/H1luc, and monitored for their susceptibility to various HIV clones including in vitro-constructed mutants. Both cell lines, particularly M8166/H1luc, were found to be exquisitely sensitive to HIV-1 and HIV-2. Furthermore, they exhibited the response to infections by various viral clones exactly as expected from the characteristics of the original cell lines. These results indicated that our new indicator cell lines H9/H1luc and M8166/H1luc are eminently useful for a variety of molecular virological studies on HIV/SIV.     

Analysis of the function of viral protein X (VPX) of HIV-2

To investigate the function of vpx, a gene in HIV-2 and SIV, but not in HIV-1, three site-directed mutants (pMX) were constructed from a functional proviral HIV-2 plasmid clone (pSE). Transfection of COS-1 cells with all three mutants as well as pSE gave rise to equivalent amounts of virus. Each virus could be passaged in H9 and CEM lymphoid cell lines, peripheral blood lymphocytes, and monocytes with equal efficiency and demonstrated similar cytopathic effects. Hybridization data with DAN from the infected cells demonstrated the presence of similar levels of viral sequences and the mutations in each of the MX-infected cell lines. Immunoprecipitation analysis demonstrated a 16-kDa VPX protein in cells infected with SE virus, as well as in the virus particles, but not in cells infected with MX viruses or the particles themselves. However, equivalent levels of gag and env proteins were demonstrated in all infected cells and virion preparations. These data suggest that VPX is dispensable for virus replication and cytopathicity.     

人免疫缺陷病毒对不同黏膜上皮细胞系的感染能力的研究

目的 研究人免疫缺陷病毒(HIV-1)对不同黏膜上皮细胞系的感染能力.方法 用实验室株HIV-1 SF33和2株原代HIV-1(02010561,02010141)分别感染Caco-2、T斟和HeLa 3株黏膜上皮细胞和MT-4细胞.接种病毒后间隔3～4 d采集培养上清检测Ir24并用实时定量RT-PCR检测病毒载量;采集细胞提取DNA并用PCR法检测感染细胞中病毒DNA和整合入细胞基因组内的捕綝NA.结果 所用3株病毒都可以产毒性地感染阳性对照细胞MT-4,对整合病毒DNA的PCR检测发现它们均能够整合到MT-4细胞基因组内;实验室适应株HIV-1 SF33 虽然能够感染所有3株上皮细胞,但它不能整合入Cacoo2细胞的基因组中;虽然2株原代分离病毒均能感染T-84细胞,但只有HIV-1 02020141能够整合入T-84细胞的基因组中,原代分离病毒HIV-1 02010561能够感染HeLa细胞,但不能整合到其基因组中.结论 虽然HIV-1的实验室毒株和原代分离毒株都可能感染黏膜上皮细胞,但它们在黏膜上皮细胞中建立稳定产毒性感染(感染并产生病毒)的能力因细胞和毒株不同而异.     

CD8+T-cell-mediated control of HIV-1 and SIV infection

A detailed understanding of the cellular response to human immunodeficiency virus (HIV-1) infection is needed to inform prevention and therapeutic strategies that aim to contain the AIDS pandemic. The cellular immune response plays a critical role in reducing viral load in HIV-1 infection and in the nonhuman primate model of SIV infection. Much of this virus suppressive activity has been ascribed to CD8(+)T-cell-directed cytolysis of infected CD4(+)T cells. However, emerging evidence suggests that CD8(+)T cells can maintain a lowered viral burden through multiple mechanisms. A thorough understanding of the CD8(+)T-cell functions in HIV-1 infection that correlate with viral control, the populations responsible for these functions, and the elicitation and maintenance of these responses can provide guidance for vaccine design and potentially the development of new classes of antiretroviral therapies. In this review, we discuss the CD8(+)T-cell correlates of protection in HIV-1 and SIV infection and recent advances in this field.     

Specific cell surface requirements for the infection of CD4-positive cells by human immunodeficiency virus types 1 and 2 and by Simian immunodeficiency virus.

Human CD4 was expressed on a range of mammalian cell lines. CD4+ non-primate cells, derived from rat, hamster, mink, cat, and rabbit, bind recombinant gp120 of human immunodeficiency virus type 1 (HIV-1) but are resistant to HIV-1 infection. CD4 expression on various human, rhesus, and African green monkey cell lines confers differential susceptibilities for HIV-1, HIV-2, and simian immunodeficiency (SIV) strains. For example, CD4+ TE671 rhabdomyosarcoma cells are sensitive to HIV-1 and HIV-2 but resistant to SIV, whereas CD4+ U87 glioma cells are resistant to HIV-1 infection but sensitive to HIV-2 and SIV. HIV-1 infection was not dependent on human major histocompatibility class I expression. Studies of cell fusion and of infection by vesicular stomatitis virus pseudotypes bearing HIV-1 and HIV-2 envelopes showed that the differential cell tropisms of HIV-1, HIV-2, and SIV are determined at the cell surface.     

Evidence of productively infected CD8+ T cells in patients with AIDS: implications for HIV-1 pathogenesis

CD8+ T lymphocytes play an important protective role against HIV infection. The onset of AIDS is associated with a decline in both the number of CD8+ T lymphocytes and anti-HIV cytotoxic activity in CD8+ T cells. The reason for this progressive failure of CD8+ T cells in HIV-1 infection remains unknown. Earlier reports have shown presence of viral DNA in CD8+ cells of HIV-1-infected patients; under some conditions, CD8+ T cells have been shown to express CD4 in vitro and can be susceptible to infection with HIV-1. However, whether CD8+ lymphocytes in vivo can be productively infected with HIV-1 remains unclear. In this study, we generated multiple CD8+ T-cell clones from two patients with AIDS. These clones were CD8+/CD3+ but did not express CD4. Several of these CD8+ clones from both patients were found to be endogenously infected with HIV-1 and spontaneously produced these viruses. CD8+ cell-produced HIV-1 was biologically competent because viruses produced by most of these clones could efficiently infect and replicate in peripheral blood lymphocytes from HIV-negative donors. In addition, some of these viruses were able to form syncytia in MT-2 cells indicating syncytium-inducing phenotype. Comparison of the sequences in V3 loop areas among different viruses showed changes in some of the clones from both patients. For the first time, this report provides direct evidence that mature CD8+ T cells can be productively infected with HIV-1 in patients with AIDS. Direct infection of CD8+ T lymphocytes may play a role in the eventual failure of these cells in HIV-1 infection.     

CD4-Dependent and CD4-independent utilization of coreceptors by human immunodeficiency viruses type 2 and simian immunodeficiency viruses

More than 10 G protein-coupled receptors (GPCRs) have been reported to act as coreceptors for entry of human and simian immunodeficiency viruses (HIV and SIV). We investigated the utilization of six GPCRs as coreceptors by T-cell-line-adapted HIV-2 strains (CBL-20, CBL-21, CBL-23, GH-1, ROD, and SBL6669) and SIV strains (SIVagmTYO-1, SIVmac251, and SIVmndGB-1). NP-2/CD4 cells were transduced with CCR3, CCR5, CCR8, CXCR4, GPR1, or APJ, and examined for susceptibilities to cell-free HIV/SIV. HIV-2 strains were grouped into two types by their coreceptor usage. The first group, CBL-20 and CBL-21, used CXCR4 exclusively; the other four strains used a few or all of the six coreceptors. These strains could further infect CD4-negative NP-2/CXCR4 or NP-2/CCR5 cells in the presence (all strains) or absence (SBL6669 and ROD strains) of soluble CD4. SIVagm and SIVmnd infected NP-2/CD4/GPR1 cells. The coreceptors CCR3, CCR8, GPR1, and APJ did not mediate the CD4-independent infection. Although HIV-2ROD and SIVmnd infected both NP-2/CD4/CXCR4 and NP-2/CD4/CCR5 cells, only CXCR4 and CCR5, respectively, were used in CD4-independent infection. Binding of virions to CD4-negative cells occurred at 4 degrees C. These findings suggest that there may be a correlation between the promiscuous use of coreceptors by HIV-2/SIV strains and their ability to infect CD4-negative cells.     

CD4 and CD8 T cells from SIV-infected macaques have defective signaling responses after perturbation of either CD3 or CD2 receptors

Single-cell clones, designated E11S, C11R, and A1S, were obtained from the HuT-78 T cell line persistently infected with an isolate of Simian immunodeficiency virus (SIV), SIV/Mne. The infected clones, unlike uncloned uninfected HuT-78 cells, no longer expressed the CD4 marker and, after their CD3 receptors were cross-linked, had dramatically reduced intracellular free calcium ([Ca2+]i) responses. In one clone, E11S, the unresponsiveness was not limited to the inositol phospholipid pathway of signaling since a reduction in CD3-mediated activation of protein tyrosine kinase-dependent phosphorylation also was evident in this SIV-infected clone. These results led us to test whether T lymphocytes from animals infected with SIV had defective [Ca2+]i responses prior to detectable changes in CD4 levels or lymphadenopathy. The [Ca2+]i responses to both CD3 mAb and CD2 mAb were 10-50% less in T cells from Walter Reed stage 2 animals than in healthy controls. This anergy was more pronounced in chronically infected animals progressing to Walter Reed stage 3/4. The responses of these animals could not be augmented even when combinations of CD3 and CD4 mAb were used. Both CD4+CD44lo T cells, which are not infected with SIV, and the CD4+CD44hi T cell subset, previously shown to be the reservoir of SIV infection in blood, had pronounced defective responses to CD3 mAb. Similarly, both CD4+ and CD8+ T cells were consistently unresponsive in chronically infected animals, again implying that an indirect mechanism, rather than SIV infection per se, may be responsible for this immune dysfunction.     

A comparison of 2 T-lymphoid cell lines persistently infected with the human immunodeficiency virus (HIV-1) with different productive capacities]

T-lymphoid cell lines (H9/CBL-4 and CEM/CBL-4) persistently infected with HIV-1 were observed simultaneously for 6.5 months. The virus activity was characterized by such parameters as the number of infected cells determined by fluorescent antibody technique, the total level of virus--specific protein synthesis determined by immune blotting method, and the capacity to infect H9 and CEM cells. A comparative analysis of the two cell lines helped define the evaluation criteria for high and low productivity cultures. It was shown that a short-term virus persistence could exist in high-productivity cultures and long-term persistence in low-productivity cultures. The cytopathic activity of virus in cultures could be judged by accumulation of virus protein p24 in cell-free supernatants, this being one of the factors defining the efficacy of infection of H9 and CEM T-lymphoid cells.     

The Vif accessory protein alters the cell cycle of human immunodeficiency virus type 1 infected cells

The viral infectivity factor gene (vif) of HIV-1 increases the infectivity of viral particles by inactivation of cellular anti-viral factors, and supports productive viral replication in primary human CD4 T cells and in certain non-permissive T cell lines. Here, we demonstrate that Vif also contributes to the arrest of HIV-1 infected cells in the G(2) phase of the cell cycle. Viruses deleted in Vif or Vpr induce less cell cycle arrest than wild-type virus, while cells infected with HIV-1 deleted in both Vif and Vpr have a cell cycle profile equivalent to that of uninfected cells. Furthermore, expression of Vif alone induces accumulation of cells in the G(2) phase of the cell cycle. These data demonstrate a novel role for Vif in cell cycle regulation and suggest that Vif and Vpr independently drive G(2) arrest in HIV-1 infected cells. Our results may have implications for the actions and interactions of key HIV-1 accessory proteins in AIDS pathogenesis.