Krishnakumar Devadas

Hemin Activation Ameliorates HIV-1 Infection via Heme Oxygenase-1 Induction

Hemin, a critical component of hemoglobin, is an active ingredient of a biologic therapeutic approved by the Food and Drug Administration for the treatment of acute porphyries. This report describes a biological function of this molecule in inducing host defense against HIV-1 infection via heme oxygenase-1 (HO-1) induction. Treatment of monocytes with hemin substantially inhibited HIV replication, as evident by nearly undetectable viral RNA and cell-free HIV-1 p24 protein in a dose-dependent manner. Hemin exposure of these cells before infection, at the time of infection, or after infection caused >90% reduction of HIV DNA with substantially low levels of HIV-1 p24 and HIV-associated cytopathic effects. In addition, hemin treatment significantly suppressed infection of both monocytes and T cells inoculated with R5, X4, R5X4 tropic strains, and reverse transcriptase-resistant, azidothymidine-resistant, ddC/ddI-resistant, nivirapine-resistant, and other clinical HIV isolates. Intraperitoneal administration of hemin 4 days after HIV infection reduced viral load in the serum of human PBMC-reconstituted nonobese diabetic SCID mice by >6-fold. Suppression of HIV replication in hemin-activated cells correlated with the induction of HO-1 and was attenuated by tin protoporphyrin (SnPP) IX, an inhibitor of HO-1 activity, suggesting a pivotal role of this endogenous enzyme in the regulation of HIV infection. Hemin-induced HO-1 induction in the CCR-5, CXCR-4, and CD4 coexpressing GHOST(3) cells was consistent with the inhibition of Tat-dependent activation of long terminal repeat promoter leading to reduced GFP expression. These findings suggest an important role of hemin-induced HO-1 activity as a host defense mechanism against HIV-1 infection.

Mechanisms for Macrophage-Mediated HIV-1 Induction

Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-α, IL-1 β, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-κB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1β, IL-6, IL-8, TNF-α, and TGF-β expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-κB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-α Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-κB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-κB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.

Absence of detectable xenotropic murine leukemia virus–related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus Type 1–infected blood donors or individuals in Africa

BACKGROUND: Since the identification of xenotropic murine leukemia virus–related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV‐1)‐infected individuals to gain evidence of XMRV infection in this geographic region.

Elevated Levels of Tumor Necrosis Factor Alpha (TNF-α) in Human Immunodeficiency Virus Type 1-Transgenic Mice: Prevention of Death by Antibody to TNF-α

ABSTRACT Homozygous human immunodeficiency virus type 1 (HIV-1)-transgenic mice (Tg26) appear normal at birth but die within 3 to 4 weeks. The skin of these animals shows diffuse scaling and high-level expression of both HIV-1 mRNA and gp120. Previous experiments showed that treatment with human chorionic gonadatropin (hCG) prevented death and the expression of HIV-1 mRNA and gp120. The present experiments were initiated to study the role of tumor necrosis factor alpha (TNF-α) in HIV-1-induced pathology. Examination of the sera of Tg26 mice revealed a 50-fold increase in TNF-α levels compared to those in nontransgenic mice. Treatment with antibody to TNF-α prevented death, resulted in near normal growth, and produced a marked decrease in skin lesions and a profound reduction in the expression of HIV-1 mRNA and gp120. Both TNF-α antibody and hCG reduced TNF-α levels in sera by approximately 75%. We conclude that TNF-α contributes in a major way to HIV-1-induced pathology in transgenic mice and that both hCG and antibody to TNF-α prevent the development of pathology by suppressing the level of TNF-α.

HIV-1 and HIV-2 infections induce autophagy in Jurkat and CD4+ T cells.

Autophagy plays important roles during innate and adaptive immune responses to pathogens, including virus infection. Viruses develop ways to subvert the pathway for their own benefit in order to escape restriction by autophagy, leading to increased viral replication and/or control over apoptosis of their host cells. The effects of HIV infection on the autophagic pathway in host cells have been little documented. Using the susceptible Jurkat cell line and CD4(+) T cells, we studied the relationship of HIV-1 and -2 infections with autophagy. We found that HIV infections significantly increase transcription of ULK1, a member of the autophagy-initiated complex. Two ubiquitin-like conjugation systems, the Atg12 conjugation system and the microtubule-associated protein L chain 3 (LC3) conjugation system that control the elongation of the autophore to form the autophagosome, were activated after HIV infection, with upregulation of Atg12-Atg5 complex and increased transcription of LC3, and formed more autophagosome in infected cells detected using an EM assay. We also found that HIV-1 induced more autophagic death in Jurkat cells relative to HIV-2, and the inhibition of autophagy with 3MA and Beclin-1 knockdown decreased HIV-1 replication significantly. The results indicate that HIV is able to induce the autophagic signaling pathway in HIV-infected host cells, which may be required for HIV infection-mediated apoptotic cell death.

Lipopolysaccharide suppresses HIV-1 replication in human monocytes by protein kinase C-dependent heme oxygenase-1 induction.

LPS is an important component of the Gram‐negative bacteria cell wall. It activates monocytes and induces multiple host immune and inflammatory responses. Interestingly, in spite of inducing host‐inflammatory responses, LPS also protects monocyte‐derived macrophages from infection by HIV‐1. In this report, we have shown that LPS treatment of human monocyte‐derived macrophages markedly suppressed HIV‐1 replication, even on addition to infected cells 24 h after infection. Inhibition of HIV‐1 replication was associated with PKC‐dependent induction of HO‐1, a cytoprotective enzyme known to catabolize heme. Pretreatment with the PKC inhibitor Go 6976 not only substantially inhibited LPS‐mediated induction of HO‐1 but also attenuated LPS‐induced suppression of HIV replication. Significant reduction of HIV replication by inhibitors of JNK, NF‐κB, and PI3K was independent of a LPS‐mediated anti‐HIV effect. Specificity of HO‐1 was confirmed by substantial reversal of LPS‐induced viral replication by pretreatment of cells with SnPP IX, an inhibitor of HO‐1 enzyme activity. These results demonstrate a previously undefined function of HO‐1 as a host defense mechanism in LPS‐mediated inhibition of HIV‐1 replication.

Effect of sex steroid hormones on replication and transmission of major HIV subtypes

BACKGROUND The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. METHODS Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. RESULTS Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. CONCLUSIONS Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV.

Some mechanisms of FLIP expression in inhibition of HIV‐1 replication in Jurkat cells, CD4+ T cells and PBMCs

HIV‐1 infection and replication are affected by host factors. Recent studies demonstrate that molecules from apoptotic pathways regulate HIV‐1 replication. Therefore, studies on effects of host factors that maintain host cell survival and influence HIV‐1 replication are critical to understanding the mechanisms of HIV‐1 replicative cycle. Using the susceptible Jurkat cell line, CD4+ T cells, and peripheral blood mononuclear cells (PBMCs), we studied the role of FLIP, an inhibitor of caspase‐8, in HIV‐1 production. Full length cellular FLIP (cFLIP) inhibited HIV‐1 replication in these cells. cFLIP upregulated the expression of viral restriction factors, such as TRIM5, Apobec3G, and Bst2/tetherin, decreased nuclear factor 1C expression and inactivated ERK and p38 induced by HIV‐1 in Jurkat cells. cFLIP blocked the trafficking of gp120 and Gag p24 capsid protein into lipid rafts with inhibition of Tsg101 and Alix in ESCRT signaling pathway. cFLIP also promoted Bst2/tetherin trafficking into lipid rafts. These results indicate that cFLIP may inhibit the HIV‐1 replication cycle at multiple steps, including viral RNA release, transcription, traffic and assembly. We also found that cFLIP expression downregulated Fas expression and inactivated FADD in the Fas‐mediated apoptotic pathway. The inactivated FADD also inhibited HIV‐1 replication. J. Cell. Physiol. 228: 2305–2313, 2013.

Absence of Detectable XMRV and Other MLV-Related Viruses in Healthy Blood Donors in the United States

Background Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern. Methodology/Principal Findings To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus. Conclusions/Significance Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.

HIV-1 Induced Nuclear Factor I-B (NF-IB) Expression Negatively Regulates HIV-1 Replication through Interaction with the Long Terminal Repeat Region

Background: Retroviruses rely on host factors for cell entry, replication, transcription, and other major steps during their life cycle. Human Immunodeficiency Virus-1 (HIV-1) is well known for utilizing a plethora of strategies to evade the host immune response, including the establishment of latent infection within a subpopulation of susceptible cells. HIV-1 also manipulates cellular factors in latently infected cells and persists for long periods of time, despite the presence of successful highly active antiretroviral therapy (HAART). Results: In this study we demonstrate that Nuclear Factor-IB (NF-IB) is induced during HIV-1 infection and its expression negatively impacts viral replication. During HIV-1 infection in peripheral blood mononuclear cells (PBMCs), and the T cell line, Jurkat or during induction of virus replication in latently infected cells, ACH2 and J1.1, we observed a time-dependent alteration in NF-IB expression pattern that correlated with HIV-1 viral expression. Using the Chip assay, we observed an association of NF-IB with the long terminal repeat region of HIV-1 (LTR) (-386 to -453 nt), and this association negatively correlated with HIV-1 transcription. Furthermore, knock-down of NF-IB levels in J1.1 cells resulted in an increase of HIV-1 levels. Knock-down of NF-IB levels in J-Lat-Tat-GFP (A1), (a Jurkat cell GFP reporter model for latent HIV-1 infection) resulted in an increase in GFP levels, indicating a potential negative regulatory role of NF-IB in HIV-1 replication. Conclusion: Overall, our results suggest that NF-IB may play a role in intrinsic antiretroviral defenses against HIV-1. These observations may offer new insights into the correlation of the latently infected host cell types and HIV-1, and help to define new therapeutic approaches for triggering the switch from latency to active replication thereby eliminating HIV-1 latent infection.