Hrs inhibits citron kinase-mediated HIV-1 budding via its FYVE domain

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a key component of the endosomal sorting complexes required for transport and has been demonstrated to play a regulatory role in endocytosis/exocytosis and the accumulation of internal vesicles in multivesicular bodies. Citron kinase is a Ser/The kinase that we previously reported to enhance human immunodeficiency virus type 1 (HIV-1) virion production. However, the relationship between Hrs and citron kinase in HIV-1 production remains elusive. Here, we report that Hrs interacts with citron kinase via its FYVE domain. Overexpression of Hrs or the FYVE domain resulted in a significant decrease in HIV-1 virion production. Depletion of Hrs by RNA interference in HEK293T cells increased HIV-1 virion production and enhanced the activity of citron kinase. These data suggest that Hrs inhibits HIV-1 production by inhibiting citron kinase-mediated exocytosis.     

Blockage of HIV-1 production through inhibition of proviral DNA synthesis by N,O-didecanoyl serinal dimethylacetal

Six serinal derivatives were synthesized and tested for their anti-human immunodeficiency virus type-1 (HIV-1) activity against HIV-1-infected cells. Of the 6 serinal derivatives tested, only N,O-didecanoyl serinal dimethylacetal (DDSD) was found to strongly suppress progeny virus production from acute HIV-1-infected CEM cells, while not suppressing the HIV-1 p24 production from latent HIV-1-infected ACH-2 cells after stimulation with phorbol 12-myristate 13-acetate. DDSD also inhibited the synthesis of HIV-1 proviral DNA at 20-50 microM, not only 1 h but also 24 h after HIV-1 infection. Taken together, DDSD is a potent inhibitor of HIV-1 production, and may become a unique leading compound for chemotherapy of acquired immunodeficiency syndrome.     

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.     

HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.     

Impairment of human immunodeficiency virus type 1 (HIV-1) entry into Jurkat T cells by constitutive expression of the HIV-1 vpr protein: role of CD4 down-modulation

Jurkat T-cell clones, stably expressing the human immunodeficiency virus type 1 (HIV-1) Vpr protein, exhibited an impaired susceptibility to HIV-1 infection. A marked down-modulation of surface CD4 receptors was detected in Vpr-expressing clones with respect to control cells. Likewise, a reduced CD4 expression was also observed in parental Jurkat cells infected with wild-type but not with Vpr-mutant HIV-1. Notably, Vpr-expressing clones were fully susceptible to infection with a vesicular stomatitis virus G protein-pseudotyped HIV-1 virus, indicating that a block at the level of viral entry was responsible for the inhibition of viral replication. The effect exerted by Vpr on HIV replication and CD4 expression suggests that this protein can regulate both the establishment of a productive HIV-1 infection and CD4-mediated T-cell functions.     

The IFITM proteins inhibit HIV-1 infection

Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.     

Sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type 1 (HIV-1) infection and dendritic-cell-directed HIV-1 transfer

Both endocytic uptake and viral fusion can lead to human immunodeficiency virus type 1 (HIV-1) transfer to CD4+ lymphocytes, either through directional regurgitation (infectious transfer in trans [I-IT]) or through de novo viral production in dendritic cells (DCs) resulting in a second-phase transfer to CD4+ lymphocytes (infectious second-phase transfer [I-SPT]). We have evaluated in immature monocyte-derived DCs both pathways of transfer with regard to their susceptibilities to being blocked by potential microbicidal compounds, including cyanovirin (CNV); the plant lectins Hippeastrum hybrid agglutinin, Galanthus nivalis agglutinin, Urtica dioica agglutinin, and Cymbidium hybrid agglutinin; and the glycan mannan. I-IT was a relatively inefficient means of viral transfer compared to I-SPT at both high and low levels of the viral inoculum. CNV was able to completely block I-IT at 15 microg/ml. All other compounds except mannan could inhibit I-IT by at least 90% when used at doses of 15 microg/ml. In contrast, efficient inhibition of I-SPT was remarkably harder to achieve, as 50% effective concentration levels for plant lectins and CNV to suppress this mode of HIV-1 transfer increased significantly. Thus, our findings indicate that I-SPT may be more elusive to targeting by antiviral drugs and stress the need for drugs affecting the pronounced inhibition of the infection of DCs by HIV-1.     

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.     

Autophagy: An overlooked mechanism of HIV-1 pathogenesis and NeuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4⁺ lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4+ T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.

Addendum to: Zhou D, Spector SA. Human immunodeficiency virus type-1 infection inhibits autophagy. Aids 2008;22:695-9.     

Role of HIV-1 Vpu protein for virus spread and pathogenesis

Vpu is an accessory viral protein almost unique to HIV-1 among primate immunodeficiency viruses, and has two major functions: degradation of the CD4 molecule in endoplasmic reticulum and enhancement of virion release from cells. Recent identification of a novel host restriction factor, tetherin, as a Vpu-antagonist suggests that Vpu contributes to virus spread by facilitating progeny virion production. This review focuses on the two distinct functions of Vpu and summarizes current knowledge on its virological role in the HIV-1 life cycle.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.