Human immunodeficiency virus type 1 T-cell tropism is determined by events prior to provirus formation.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary in the ability to replicate in cells that bear the HIV-1 receptor, CD4. The mechanism responsible for these cell tropism differences is unknown. We examined different isolates of HIV-1 with regard to replication in specific tumor-derived CD4-positive T-cell lines and normal peripheral blood lymphocytes. To investigate early events in the virus life cycle at low multiplicities of infection, we used a modification of the polymerase chain reaction method. Use of a molecularly cloned primary HIV-1 isolate, HIV-1 JR-CSF, restricted for replication in T-cell lines, demonstrated that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. However, transfection of proviral DNA resulted in efficient transient virus production from these cells. Therefore, we conclude that at least one block to infection for HIV-1 strains in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.     

Differential replication of human immunodeficiency virus type 1 in CD8- and CD8+ subsets of natural killer cells: relationship to cytokine production pattern.

CD8+ and CD8- subsets of peripheral blood natural killer (NK) cells were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1) and for the ability to produce various types of interferon (IFN) and tumor necrosis factor (TNF). HIV-1 was preferentially grown in CD8+ NK cells. The ability of CD8- NK cells to suppress HIV-1 replication was related to their ability to produce alpha IFN (IFN-alpha) upon viral induction. Induction with interleukin-2 resulted in IFN-gamma production in both subsets of NK cells. In the CD8+ subset, IFN-gamma and HIV-1 mutually enhanced the production of TNF alpha, leading to hyperactivation of viral replication, whereas in CD8- NK cells IFN-gamma primed HIV-induced IFN-alpha production. The dichotomous effects of IFN-gamma on HIV-1 replication were dependent on the IFN-alpha-producing ability of the cellular targets. These findings can explain the selective depletion of the CD16+ CD8+ subset that begins early in the in vivo HIV-1 infection.     

Effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages.

Macrophages derived from human peripheral blood and cultured for 1 week were permissive for the replication of herpes simplex virus (HSV) types 1 and 2. Low titers of interferon (IFN) were produced after virus infection. The yield of infectious virions was reduced by pretreatment of cells with natural and recombinant IFN-alpha and natural IFN-beta. Recombinant and natural IFN-gamma exhibited very low antiviral activity. Treatment of cells with IFN-gamma mixed with IFN-alpha or with IFN-beta did not result in a synergistic inhibition of virus yield. We studied the synthesis of HSV type 1- and HSV type 2-coded proteins in macrophages treated with IFN-beta. Induction of the HSV beta-protein DNA polymerase was strongly inhibited in IFN-treated cells in a dose-dependent manner. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, other beta- and gamma-proteins of HSV were inhibited as well. Immunofluorescence studies revealed a strong inhibition of the expression of immediate early alpha-protein ICP4. The results indicate that IFN acts early during the viral replication cycle to inhibit the synthesis of HSV alpha- and beta-proteins.     

The inability of human immunodeficiency virus to infect chimpanzee monocytes can be overcome by serial viral passage in vivo.

Studies of lentivirus infection in ruminants, nonhuman primates, and humans suggest that virus infection of macrophages plays a central role in the disease process. To investigate whether human immunodeficiency virus type 1 (HIV-1) can infect chimpanzee macrophages, we recovered monocytes from peripheral blood mononuclear cells of HIV-1-negative animals and inoculated these and control human monocytes with a panel of four human-passaged monocytotropic virus strains and one chimpanzee-passaged isolate. HIV-1 infected human monocytes synthesized proviral DNA, viral mRNA, p24 antigen, and progeny virions. In contrast, except for the chimpanzee-passaged HIV-1 isolate, chimpanzee monocytes failed to support HIV-1 replication when cultured under both identical and a variety of other conditions. Proviral DNA was demonstrated only at background levels in these cell cultures by polymerase chain reaction for gag- and env-related sequences. Interestingly, the chimpanzee-passaged HIV-1 isolate did not replicate in human monocytes; viral p24 antigens and progeny virions were not detected. The same monocytotropic panel of HIV-1 strains replicated in both human and chimpanzee CD4+ T lymphoblasts treated with phytohemagglutinin and interleukin-2. The failure of HIV-1 to infect chimpanzee monocytes, which can be overcome by serial in vivo viral passage, occurs through a block early in the viral life cycle.     

Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.     

Endogenous IFN-alpha production by plasmacytoid dendritic cells exerts an antiviral effect on thymic HIV-1 infection

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-alpha in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-alpha production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-alpha upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-alpha in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-alpha upon engagement of TLR-9. Importantly, CpG considerably increased IFN-alpha production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-alpha. The degree of inhibition depends on the level of IFN-alpha produced by the thymic pDC.     

Direct evidence of lower viral replication rates in vivo in human immunodeficiency virus type 2 (HIV-2) infection than in HIV-1 infection

Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-na?ve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4(+) T-cell counts of >200/microl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.     

TRAF6 and IRF7 control HIV replication in macrophages

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.     

Cytofluorometry of lymphocytes infected with Epstein-Barr virus: effect of phosphonoacetic acid on nucleic acid.

DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.