Host factors mediating HIV-1 replication

Human immunodeficiency virus type 1(HIV-1) infection is the leading cause of death worldwide in adults attributable to infectious diseases. Although the majority of infections are in sub-Saharan Africa and Southeast Asia, HIV-1 is also a major health concern in most countries throughout the globe. While current antiretroviral treatments are generally effective, particularly in combination therapy, limitations exist due to drug resistance occurring among the drug classes. Traditionally, HIV-1 drugs have targeted viral proteins, which are mutable targets. As cellular genes mutate relatively infrequently, host proteins may prove to be more durable targets than viral proteins. HIV-1 replication is dependent upon cellular proteins that perform essential roles during the viral life cycle. Maraviroc is the first FDA-approved antiretroviral drug to target a cellular factor, HIV-1 coreceptor CCR5, and serves to intercept viral–host protein–protein interactions mediating entry. Recent large-scale siRNA and shRNA screens have revealed over 1000 candidate host factors that potentially support HIV-1 replication, and have implicated new pathways in the viral life cycle. These host proteins and cellular pathways may represent important targets for future therapeutic discoveries. This review discusses critical cellular factors that facilitate the successive steps in HIV-1 replication.     

Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.     

High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3′ half of the viral genome of subtype A was replaced with the corresponding subtype C3′ half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5′ half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.     

Characterization of membrane-bound IL-22+ T cell subsets in HIV-1 patients coinfected with Mycobacterium tuberculosis

BackgroundPreviously, we have found that IL-22 could be not only secreted outside of cells, but also highly expressed on the T cells membrane in HIV-1 negative patients with tuberculosis (TB). However, the study on membrane-bound IL-22+ cells of HIV-1 infected patients is rare. Therefore, we investigated antigen-specific membrane-bound IL-22+ T cell subsets in Mycobacterium tuberculosis (M.tb) coinfection of HIV-1 infected individuals.MethodsA case–control study that enrolled 74 HIV-1 infected participants was carried out, including HIV-1 monoinfection (HIV+TB?, n = 43), HIV-1 infected patients with latent TB (HIV+LTB, n = 18) and HIV-1 coinfected patients with active TB (HIV+TB+, n = 13). We made use of an IFN-γ release assay (IGRA) to screen LTB individuals. Purified protein derivative (PPD) and phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) were used as specific-stimulators to detect the levels of peripheral blood membrane-bound IL-22+ T cell subsets via cell surface staining and flow cytometry among three groups.ResultsAn approximate rate of 24.3% (n = 18 out of 74) of latent M.tb infection among HIV-1 positive population in Eastern China. Interestingly, HMBPP-specific CD3+Vγ2+ T cells were impaired in HIV+TB+patients compared with HIV+LTB patients (P < 0.05). Furthermore, increases of PPD-specific and HMBPP-specific membrane-bound IL-22+ T cell subsets including CD3+, CD3+CD4+ and CD3+Vγ2+ T cells were observed in HIV+TB+group rather than HIV+LTB groups (all P < 0.05).ConclusionAntigen-specific membrane-bound IL-22+ T cells were highly expressed in M.tb coinfection of HIV-1 infected individuals, and may play an important role in anti-TB immune response during coinfection with HIV-1.     

Regulation of primary HIV-1 isolate replication in dendritic cells

The potential role of dendritic cells (DC) in the immunopathology of human immunodeficiency virus 1 (HIV-1) disease remains controversial. This study examines replication of a panel of HIV-1 strains (both laboratory adapted and primary) within DC, in the context of the well-established monocyte-DC and monocyte-macrophage transition. Viral replication was assessed by p24 ELISA assay. All strains of HIV-1 tested replicated in DC. Only CCR5-tropic virus replicated in macrophages. Lipopolysaccharide (LPS) induced DC maturation (as reflected in altered cell phenotype) and at the same time diminished the ability of DC to support HIV-1 replication. In contrast the presence of activated T cells, which had been fixed to prevent them acting as a site for viral replication, enhanced the ability of the DC to support viral replication, as has been reported previously for macrophages. Thus cells that are DC by phenotype, but are not activated, act as the optimum reservoir for HIV-1 replication. If this form of DC is present in peripheral tissues, this will be permissive for amplification of the in vivo viral load at sites where there are few responder cells available, and hence contribute to the persistent immunopathology.     

HIV-1 induces IL-10 production in human monocytes via a CD4-independent pathway

In HIV-infected patients, increased levels of IL-10, mainly produced by virally infected monocytes, were reported to be associated with impaired cell-mediated immune responses. In this study, we investigated how HIV-1 induces IL-10 production in human monocytes. We found that CD14(+) monocytes infected by either HIV-1(213) (X4) or HIV-1(BaL) (R5) produced IL-10, IL-6, tumor necrosis factor-alpha (TNF-alpha), and to a lesser extent, IFN-gamma. However, the capacity of HIV-1 to induce these cytokines was not dependent on virus replication since UV-inactivated HIV-1 induced similar levels of these cytokines. In addition, soluble HIV-1 gp160 could induce CD14(+) monocytes to produce IL-10 but at lower levels. Cross-linking CD4 molecules (XLCD4) with anti-CD4 mAbs and goat anti-mouse IgG (GAM) resulted in high levels of IL-6, TNF-alpha and IFN-gamma but no IL-10 production by CD14(+) monocytes. Interestingly, neither anti-CD4 mAbs nor recombinant soluble CD4 (sCD4) receptor could block IL-10 secretion induced by HIV-1(213), HIV-1(BaL) or HIV-1 gp160 in CD14(+) monocytes, whereas anti-CD4 mAb or sCD4 almost completely blocked the secretion of the other cytokines. Furthermore, HIV-1(213) could induce IL-10 mRNA expression in CD14(+) monocytes while XLCD4 by anti-CD4 mAb and GAM failed to do so. As with IL-10 protein levels, HIV-1(213)-induced IL-10 mRNA expression in CD14(+) monocytes could not be inhibited by anti-CD4 mAb or sCD4. Taken together, HIV-1 binding to CD14(+) monocytes can induce CD4-independent IL-10 production at both mRNA and protein levels. This finding suggests that HIV induces the immunosuppressive IL-10 production in monocytes and is not dependent on CD4 molecules and that interference with HIV entry through CD4 molecules may have no impact on counteracting the effects of IL-10 during HIV infection.     

Role of IL-15 in HIV-1-associated hypergammaglobulinaemia

IL-15 is a novel cytokine, produced by monocytes/macrophages, with biological activities similar to IL-2 but with no significant sequence homology. IL-15 also stimulates human B cells to proliferation and immunoglobulin secretion. We measured serum levels of IL-15 in 84 HIV-1-infected individuals at different stages of disease in reference to 41 healthy blood donors. Our results show a marked elevation of IL-15 serum levels during HIV-1 infection. Moreover, we found that this increase correlated with serum levels of IgG (r=0.376, P<0.0001), and partly with serum IgM (r=0.265, P<0.0015). A significant increase of IL-15 production by cultured peripheral blood mononuclear cells (PBMC) and purified monocytes in the presence of HIV-1 virus suggests that monocytes/macrophages may be a source of higher IL-15 serum levels in HIV-1-infected individuals. These findings indicate a participation of IL-15 in the hypergammaglobulinaemia frequently associated with HIV-1 infection.     

IL-12、IL-18基因对HIV-1外膜蛋白基因疫苗诱导的免疫应答的影响

目的 研究IL-12、IL-18基因对HIV-1外膜蛋白基因疫苗诱导免疫应答的影响，以探求HIV-1核酸疫苗的新策略。方法 pVAX1GP120联合IL-12、IL-18基因或者pVAX1GP120单独免疫BALB／c小鼠，采用ELISA检测免疫小鼠的特异性抗体和IFN-γ水平，以NIT比色法检测免疫小鼠脾淋巴细脯增殖，用乳酸脱氢酶（LDH）试验检测小鼠特异性细胞毒性T淋巴细胞（CIL）的应答。结果 与pVAX1GP120免疫组比较，pVAX1GP120联合IL-12、IL-18基因免疫组小鼠血清的抗HIV-1 gp120抗体滴度升高，IFN-γ升高，小鼠的脾淋巴细胞增殖实验刺激指数（SI）以及特异性CTL活性均高，差异均有统计学意义（P〈0．01）。结论 IL-12、IL-18基因联合HIV-1核酸疫苗免疫小鼠，有可能增强特异性TH1细胞和CTL反应，IL-12、IL-18基因对体液免疫可能有抑制作用。因此，IL-12、IL-18基因对于治疗性HIV-1核酸疫苗可能是具有较好应用前景的免疫佐剂。     

FcepsilonRI-FcgammaRII coaggregation inhibits IL-16 production from human Langerhans-like dendritic cells

Langerhans-like dendritic cells (LLDC) express the high-affinity IgE receptor FcepsilonRI form that lacks the beta-chain, and may play an important role in allergic inflammation via production of IL-16. Secretion of mediators by human mast cells and basophils is mediated through FcepsilonRI and is decreased by coaggregating these receptors to the low-affinity IgG receptor, FcgammaRII. We used a recently described human Ig fusion protein (GE2), which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to investigate its ability to inhibit IL-16 production from FcepsilonRI-positive Langerhans-like dendritic cells through coaggregation of FcgammaRII and FcepsilonRI. Unstimulated LLDC-derived from CD14-positive monocytes from atopic donors were shown to express FcgammaRII, an ITIM-containing receptor, but not FcepsilonRI or FcgammaRIII which are activating (ITAM) receptors. When passively sensitized with antigen-specific, human IgE and then challenged with antigen, LLDC were stimulated to produce IL-16. However, when FcepsilonRI and FcgammaRII were coaggregated with GE2, IL-16 production was significantly inhibited. Exposure of LLDCs to GE2 alone did not induce IL-16 production. Our results further extend our studies demonstrating the ability of GE2 to inhibit FcepsilonRI-mediated responses through coaggregation with FcgammaRIIB and at the same time show that human LDCC can be modulated in a fashion similar to mast cells and basophils.     

血必净对活化诱导T细胞凋亡的调节

目的 观察活化诱导对脾脏T淋巴细胞凋亡、凋亡相关基因mRNA表达及caspase3活性的影响,以及活血化瘀中药的调节作用.方法 提取BALB/c小鼠脾脏T淋巴细胞并培养,以Con A+IL-2诱导T细胞活化凋亡,MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡率,RT-PCR检测Fas、FasL、Bcl-2、Bax、IL-2 mRNA表达水平,分光光度法测定caspase3酶活性,并观察活血化瘀中药对上述各项指标的影响.结果 活化T淋巴细胞于诱导18h后凋亡率明显增加,于诱导6h时未见FasL、Bax mRNA表达,Fas、Bcl-2 mRNA表达无明显变化;于诱导18 h后Fas、FasL、Bax mRNA表达升高,Bel-2 mRNA表达下降,caspase3活性增高.活血化瘀中药可降低T细胞凋亡,并可分别降低Fas、FasL、Bax mRNA表达,提高Bcl-2 mRNA表达,减轻easpase3酶活性.在活化诱导早期(6 h)促进T淋巴细胞内IL-2 mRNA表达,在晚期(18 h)减少IL-2 mRNA表达.结论 过度活化是脾脏T淋巴细胞异常凋亡的诱发因素,而凋亡的发生与Fas、FasL、Bax、Bcl-2 mRNA表达的改变有关.活血化瘀中药可通过调节IL-2及凋亡相关基因mRNA表达而减轻脾脏T淋巴细胞凋亡,同时可以促进T淋巴细胞的增殖活性.     

Relationship between IL-2 receptor expression and proliferative responses in lymphocytes from HIV-1 seropositive homosexual men.

Previous studies have shown that exogenous IL-2 does not correct the reduction in phytohaemagglutinin (PHA)-induced proliferation of lymphocytes from HIV-1 infected (HIV+) individuals. We investigated the mechanism of this reduction to determine if reduced expression of the complete IL-2 receptor (IL-2R) was responsible. In a series of experiments, PHA-stimulated lymphocytes from a total of 89 HIV- and 93 HIV+ homosexual men from the Baltimore Multicentre AIDS Cohort Study (MACS) were studied to determine the expression of messages for the alpha and beta subunits of the IL-2R, the binding of 125I-IL-2 to high affinity IL-2R, and the effect of IL-2 on cell proliferation. Compared to HIV- donors, PHA-stimulated lymphocytes from most HIV+ donors demonstrated (i) a reduction in high affinity IL-2R expression that correlated with the reduction in the IL-2-induced proliferative response; and (ii) a reduction in expression of both IL-2R alpha- and beta-chain mRNA which may be responsible for decreased high affinity IL-2R expression. However, lymphocytes from some HIV+ individuals had borderline low IL-2-induced proliferation despite normal or elevated expression of high affinity IL-2R. These results suggest that decreased expression of IL-2R may account, at least in part, for the lower proliferative response of cells from HIV+ donors.     

Analysis of lymphocyte cell death and apoptosis in HIV-2-infected patients.

Recent evidence suggests that T cell apoptosis could be involved in the pathogenesis of HIV-1 infection. As the progression of HIV-2 associated disease appears to be slower than that of HIV-1, we investigated whether there were differences in the degree of T cell death and apoptosis in peripheral blood mononuclear cell (PBMC) cultures from patients with HIV-1 or HIV-2 infection. PBMC from healthy controls (n = 28) and patients infected with HIV-1 (n = 26: asymptomatic (ASY)/persistent generalized lymphadenopathy (PGL), n = 16; and AIDS-related complex (ARC)/AIDS n = 10) or HIV-2 (n = 30: ASY/PGL, n = 16; ARC/AIDS, n = 14) were cultured in the absence or presence of mitogens (PHA, PWM) or superantigen (SEB). After 48 h, cell death (CD) was assessed by trypan blue exclusion and in some patients programmed cell death (PCD) was quantified in flow cytometry by measuring the percentage of hypodiploid nuclei corresponding to fragmented DNA, after treating the cells with a propidium iodide hypotonic solution. HIV-1 and HIV-2 ARC/AIDS patients and ASY/PGL HIV-1+ patients had significant increases in cell death percentages compared with controls, both in unstimulated and stimulated lymphocyte cultures. However, HIV-2+ ASY/PGL patients did not exhibit significant increases of cell death in unstimulated cultures. In addition, the comparison between HIV-1 and HIV-2 infected subjects in similar stages of disease, showed no significant differences in CD in the ARC/AIDS patients, although ASY/PGL HIV-2 infected subjects had lower levels of CD than the HIV-1+ ASY/PGL (3.4% +/- 0.6 s.e.m. versus 6.8% +/- 1.1 s.e.m., P < 0.01). PCD was significantly increased both in ASY/PGL (14.3% +/- 2.2 s.e.m., n = 8, P < 0.005) and in ARC/AIDS (25.3% +/- 4.5 s.e.m., n = 9, P < 0.001) HIV-1+ patients compared with healthy controls (5.8% +/- 1.7 s.e.m., n = 11). This contrasts with HIV-2 infected subjects where the ASY/PGL patients (10.0% +/- 2.8 s.e.m., n = 6) did not differ significantly from healthy controls, although ARC/AIDS patients (27.2% +/- 4.2 s.e.m., n = 9, P < 0.001) had significantly increased levels of PCD. In conclusion, this is the first report describing the occurrence of spontaneous and activation-induced lymphocyte death by apoptosis in HIV-1 infected subjects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence against a direct antiviral activity of the proteasome during the early steps of HIV-1 replication

The infectivity of HIV-1 virions can be enhanced by inhibition of the proteasome in target cells, leading to the hypothesis that the proteasome degrades incoming virions as part of the intracellular antiviral defense. Here, several lines of evidence suggest instead that proteasome inhibition renders target cells more susceptible to infection via an indirect effect on the cellular environment: (1) proteasome inhibition increased infectivity more effectively when target cells were exposed to the inhibitors before exposure to virions, rather than when the inhibitors and virions were present simultaneously; (2) increased infectivity correlated directly with the duration of pre-exposure of cells to the inhibitors; (3) although increased infectivity was induced by as little as 30 min of pretreatment of target cells, binding of virions to target cells before the addition of inhibitor abolished the effect; and (4) increased infectivity persisted after removal of the inhibitors and the recovery of proteasome activity within the target cells. Cell cycle analyses revealed that an increased fraction of cells in G2/M may correlate with increased efficiency of infection. These data suggest that rather than relieving a target cell restriction based on the degradation of incoming virions, proteasome inhibitors likely increase infectivity either via their effects on the cell cycle or by increasing the expression of a host cell factor that facilitates infection.     

An ultrastructural observation of negatively-stained human immunodeficiency virus type 1 (HIV-1) cores

Cores of human immunodeficiency virus type 1 (HIV-1) were observed using negative staining for electron microscopy. On the surfaces of cores isolated from viral particles with a Nonidet P40 and glutaraldehyde mixture, mortar-like units were detected in the wide and narrow margins. In some of the cores which were burst with distilled water, the units were linked together revealing a beaded structure.     

IL-18和HIV-1 gag-gp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答

目的 :探讨IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫小鼠的免疫应答。方法 :构建含IL 18的真核表达质粒pVAXIL18,将他与表达HIV 1gag gp12 0嵌合基因的核酸疫苗质粒pVAXGE共同肌注免疫BALB/c小鼠 ,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体滴度。结果 :联合免疫组小鼠脾特异性CTL杀伤活性和血清抗体水平均显著高于单独免疫组 (P <0 .0 5 ) ,空白质粒对照组 (P <0 .0 1)和PBS对照组 (P <0 .0 1)。结论 :IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫可诱导小鼠产生特异性细胞和体液免疫 ,且IL 18发挥了免疫佐剂的作用。     

TNFR1 delivers pro-survival signals that are required for limiting TNFR2-dependent activation-induced cell death (AICD) in CD8+ T cells

Members of the TNF and TNF receptor (TNFR) superfamily play important roles in the maintenance of homeostasis of the immune system. Furthermore, several members of the TNFR family participate in T-cell activation and sustaining T-cell responses. We have shown that TNFR2 regulates T-cell activation by lowering the activation threshold and providing costimulatory signaling. Furthermore, activated TNFR2(-/-) CD8(+) T cells are highly resistant to activation-induced cell death (AICD). Here, we showed that using anti-TNFR2 antibodies to block TNFR2 on activated WT CD8(+) T cells rendered them resistant to AICD. This resistance of activated TNFR2(-/-) CD8(+) T cells to AICD correlated with the accumulation of TNF receptor-associated factor 2 (TRAF2). Overexpression of TRAF2 by retroviral transfection and knockdown of TRAF2 by small interfering RNA also support this conclusion. Furthermore, neutralizing TNF-α reduced TRAF2 accumulation in activated TNFR2(-/-) CD8(+) T cells and increased their susceptibility to AICD. AICD-resistant TNFR2(-/-) CD8(+) T cells expressed elevated levels of phosphorylated IκBα and higher DNA-binding activity of the p65 NK-κB subunit and neutralization of TNF-α blocked this increase. Therefore, in activated TNFR2(-/-) CD8(+) T cells, TNFR1 functions as a survival receptor by utilizing high intracellular levels of TRAF2 to promote IκBα phosphorylation and NF-κB activation.     

Polymorphism in HIV-1 dependency factor PDE8A affects mRNA level and HIV-1 replication in primary macrophages

Four genome-wide RNAi screens have recently identified hundreds of HIV-1 dependency factors (HDFs). Previously, we reported a large variation in the ability of HIV-1 to replicate in monocyte-derived macrophages (MDM) derived from > 400 healthy seronegative blood donors. Here we determined whether SNPs in genes encoding newly identified HDFs were associated with this variation in HIV-1 replication. We found a significant association between the minor allele of SNP rs2304418 in phosphodiesterase 8A (PDE8A) and lower HIV-1 replication (p = 2.4 × 10⁻⁶). The minor allele of SNP rs2304418 was also significantly associated with lower PDE8A mRNA levels in MDM (p = 8.3 × 10⁻⁵). In accordance with this, overexpression of PDE8A in HEK293T cells resulted in increased HIV-1 replication, while subsequent knock-down of PDE8A decreased replication. This study links host genetic variation in a newly identified HDF to variation in HIV-1 replication in a relevant primary target cell for HIV-1 and may provide new leads for treatment of this infection.