HIV-1 Tat Upregulates TREM1 Expression in Human Microglia.

Because microglia are a reservoir for HIV and are resistant to the cytopathic effects of HIV infection, they are a roadblock for any HIV cure strategy. We have previously identified that triggering receptor expressed on myeloid cells 1 (TREM1) plays a key role in human macrophage resistance to HIV-mediated cytopathogenesis. In this article, we show that HIV-infected human microglia express increased levels of TREM1 and are resistant to HIV-induced apoptosis. Moreover, upon genetic inhibition of TREM1, HIV-infected microglia undergo cell death in the absence of increased viral or proinflammatory cytokine expression or the targeting of uninfected cells. We also show that the expression of TREM1 is mediated by HIV Tat through a TLR4, TICAM1, PG-endoperoxide synthase 2, PGE synthase, and PGE2-dependent manner. These findings highlight the potential of TREM1 as a therapeutic target to eradicate HIV-infected microglia without inducing a proinflammatory response.

IRAK1 inhibition blocks the HIV-1 RNA mediated pro-inflammatory cytokine response from microglia.

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are a common source of morbidity in people living with HIV (PLWH). Although antiretroviral therapy (ART) has lessened the severity of neurocognitive disorders, cognitive impairment still occurs in PLWH receiving ART. The pathogenesis of HAND is likely multifaceted, but common factors include the persistence of HIV transcription within the central nervous system, higher levels of pro-inflammatory cytokines in the cerebrospinal fluid, and the presence of activated microglia. Toll-like receptor (TLR) 7 and TLR8 are innate pathogen recognition receptors located in microglia and other immune and non-immune cells that can recognise HIV RNA and trigger pro-inflammatory responses. IL-1 receptor-associated kinase (IRAK) 1 is key to these signalling pathways. Here, we show that IRAK1 inhibition inhibits the TLR7 and TLR8-dependent pro-inflammatory response to HIV RNA. Using genetic and pharmacological inhibition, we demonstrate that inhibition of IRAK1 prevents IRAK1 phosphorylation and ubiquitination, and the subsequent recruitment of TRAF6 and the TAK1 complex to IRAK1, resulting in the inhibition of downstream signalling and the suppression of pro-inflammatory cytokine and chemokine release.

Pacritinib Inhibition of IRAK1 Blocks Aberrant TLR8 Signalling by SARS-CoV-2 and HIV-1-Derived RNA.

Macrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin (IL) 1ß (IL-1ß), tumour necrosis factor (TNF), and IL-6. One of the mechanisms through which cells sense pathogenic microorganisms is through Toll-like receptors (TLRs). IL-1 receptor-associated kinase (IRAK) 1, IRAK2, IRAK3, and IRAK4 are integral to TLR and IL-1 receptor signalling pathways. Recent studies suggest a role for aberrant TLR8 and NLRP3 inflammasome activation during both COVID-19 and HIV-1 infection. Here, we show that pacritinib inhibits the TLR8-dependent pro-inflammatory cytokine response elicited by GU-rich single-stranded RNA derived from SARS-CoV-2 and HIV-1. Using genetic and pharmacologic inhibition, we demonstrate that pacritinib inhibits IRAK1 phosphorylation and ubiquitination which then inhibits the recruitment of the TAK1 complex to IRAK1, thus inhibiting the activation of downstream signalling and the production of pro-inflammatory cytokines.

Genomics Links Inflammation With Neurocognitive Impairment in Children Living With Human Immunodeficiency Virus Type-1.

BACKGROUND: We identified host single-nucleotide variants (SNVs) associated with neurocognitive impairment (NCI) in perinatally HIV-infected (PHIV) children. METHODS: Whole-exome sequencing (WES) was performed on 217 PHIV with cognitive score for age (CSA) < 70 and 247 CSA ≥ 70 (discovery cohort [DC]). SNVs identified in DC were evaluated in 2 validation cohorts (VC). Logistic regression was used to estimate adjusted odds ratios (ORs) for NCI. A human microglia NLRP3 inflammasome assay characterized the role of identified genes. RESULTS: Twenty-nine SNVs in 24 genes reaching P ≤ .002 and OR ≥ 1.5 comparing CSA < 70 to CSA ≥ 70 were identified in the DC, of which 3 SNVs were identified in VCs for further study. Combining the 3 cohorts, SNV in CCRL2 (rs3204849) was associated with decreased odds of NCI (P < .0001); RETREG1/FAM134B (rs61733811) and YWHAH (rs73884247) were associated with increased risk of NCI (P < .0001 and P < .001, respectively). Knockdown of CCRL2 led to decreased microglial release of IL-1ß following exposure to ssRNA40 while knockdown of RETREG1 and YWHAH resulted in increased IL-1ß release. CONCLUSIONS: Using WES and 2 VCs, and gene silencing of microglia we identified 3 genetic variants associated with NCI and inflammation in HIV-infected children.

Trehalose Inhibits Human Immunodeficiency Virus Type 1 Infection in Primary Human Macrophages and CD4+ T Lymphocytes through Two Distinct Mechanisms.

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV-inhibitory effects of the MTOR-independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T cells through the downregulation of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages. Thus, we showed that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

Human immunodeficiency virus Type-1 single-stranded RNA activates the NLRP3 inflammasome and impairs autophagic clearance of damaged mitochondria in human microglia.

Despite the availability of antiretroviral therapy (ART) that fully suppresses human immunodeficiency virus type-1 (HIV), markers of inflammation and minor neurocognitive impairment are frequently identified in HIV-infected persons. Increasing data support that low-level replication defective viral RNA is made by infected cells despite the absence of infectious virus. Specific GU-rich single-stranded RNA from the HIV long terminal repeat region (ssRNA40) signaling through toll-like receptor (TLR)-7 and -8 has been shown to induce the secretion of interleukin-1ß (IL-1ß) in primary monocytes. Here, we examined the activation of microglial cells by HIV ssRNA40 and the potential subsequent neurotoxicity. Our findings show that exposure of human primary microglia to ssRNA40 activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Following exposure to ssRNA40, pro-inflammatory cytokines IL-1ß, IL-18, and neurotoxic cytokines TNF-α, IL-1α, and C1q expression and extracellular secretion are increased. The released cytokines are functional since culture supernatants from ssRNA40 exposed microglia-induced toxicity of human primary neurons. Moreover, inflammasome activation of microglia increased ROS generation with a loss of mitochondrial membrane potential and mitochondrial integrity. Treatment with ssRNA40 resulted in a blockade of autophagy/mitophagy mediated negative regulation of NLRP3 inflammasome activity with the release of inflammatory cytokines, caspase-1 activation, and pyroptotic microglial cell death. Thus, HIV ssRNA mediated activation of microglial cells can contribute to neurotoxicity and neurodegeneration via secretion of inflammatory and neurotoxic cytokines. These findings provide a potential mechanism that explains the frequent minor cognitive deficits and chronic inflammation that persist in HIV-infected persons despite treatment with suppressive ART.

Development and characterization of a human microglia cell model of HIV-1 infection.

Microglia cells are the major reservoir of HIV-1 (HIV) within the CNS. However, current models using transformed cell lines are not representative of primary microglia and fetal brain samples for isolation of primary human microglia (HMG) are increasingly difficult to obtain. Here, we describe a monocyte-derived microglia (MMG) cell model of HIV infection that recapitulates infection of primary HMG. CD14+ cells isolated from healthy donors were cultured with M-CSF, beta-nerve growth factor, GM-CSF, and CCL2, and compared to HMG. MMG and HMG cells were infected with HIV and viral replication was detected by p24 antigen. Both MMG and HMG cells were found to acquire spindle shape with few branched or unbranched processes at their ends during the second week in culture and both were found to be CD11b+/ CD11c+/ CD14+/ CD45+/ CD195+/ HLADRlow/ CD86low/ CD80+. Whereas hT-Hµglia and HMC3 transformed cell lines are deficient in human microglia signature genes (C1Q, GAS6, GPR34, MERTK, PROS1, and P2RY12), MMG cells expressed all of these genes. Additionally, MMG expressed all the microglia signature miRNA (miR-99a, miR125b-5p, and miR-342-3p). Both MMG and HMG produced ROS and phagocytosed labeled zymosan particles upon PMA stimulation. MMG and HMG infected with HIV produced equivalent levels of HIV p24 antigen in culture supernatants for 30 days post-infection. Thus, we have developed and characterized a microglia cell model of HIV infection derived from primary monocytes that recapitulates the phenotypic and molecular properties of HMG, is superior to transformed cell lines, and has similar HIV replication kinetics to HMG.

HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression.

HIV-1 efficiently hijacks host cellular machinery and exploits a plethora of host-viral interactions for its successful survival. Identifying host factors that affect susceptibility or resistance to HIV-1 may offer a promising therapeutic strategy against HIV-1. Previously, we have reported that heat shock proteins, HSP40 and HSP70 reciprocally regulate HIV-1 gene-expression and replication. In the present study, we have identified HSP70 binding protein 1 (HspBP1) as a host-intrinsic inhibitor of HIV-1. HspBP1 level was found to be significantly down modulated during HIV-1 infection and virus production inversely co-related with HspBP1 expression. Our results further demonstrate that HspBP1 inhibits HIV-1 long terminal repeat (LTR) promoter activity. Gel shift and chromatin immunoprecipitation assays revealed that HspBP1 was recruited on HIV-1 LTR at NF-κB enhancer region (κB sites). The binding of HspBP1 to κB sites obliterates the binding of NF-κB hetero-dimer (p50/p65) to the same region, leading to repression in NF-κB mediated activation of LTR-driven gene-expression. HspBP1 also plays an inhibitory role in the reactivation of latently infected cells, corroborating its repressive effect on NF-κB pathway. Thus, our results clearly show that HspBP1 acts as an endogenous negative regulator of HIV-1 gene-expression and replication by suppressing NF-κB-mediated activation of viral transcription.

Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration.

HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

Interleukin 23 produced by myeloid dendritic cells contributes to T-cell dysfunction in HIV type 1 infection by inducing SOCS1 expression.

The mechanism of myeloid dendritic cell (mDC)-mediated impaired T-cell function was investigated during human immunodeficiency virus type 1 (HIV-1) infection. HIV or gp120 were found to inhibit lipopolysaccharide-induced mDC maturation and cause defects in allogeneic T-cell proliferation, interleukin 2 and interferon γ (IFN-γ) production, and phosphorylated STAT1 expression. gp120-treated mDCs downregulated autologous T-cell proliferation and IFN-γ production against a peptide pool consisting of cytomegalovirus, Epstein-Barr virus, and influenza virus (CEF). These T-cell defects were associated with a decrease in production of the T-helper type 1-polarizing cytokine interleukin 12p70 and an increase in interleukin 23 (IL-23) production by gp120-treated mDCs. gp120-induced IL-23 upregulated suppressor of cytokine signaling 1 (SOCS1) protein in T cells, which inhibited IFN-γ production and killing of CEF-pulsed monocytes. These effector functions were recovered by silencing SOCS1 in T cells. Furthermore, we observed IL-23-induced SOCS1 binding to the IFN-γ transcription complex. These results identify SOCS1 as a novel target to improve the immune function in HIV-infected persons.

Epigenetic regulation of HIV-1 persistence and evolving strategies for virus eradication.

Despite the intense effort put by researchers globally to understand Human Immunodeficiency Virus (HIV-1) pathogenesis since its discovery 30 years ago, the acquired knowledge till date is not good enough to eradicate HIV-1 from an infected individual. HIV-1 infects cells of the human immune system and integrates into the host cell genome thereby leading to persistent infection in these cells. Based on the activation status of the cells, the infection could be productive or result in latent infection. The current regimen used to treat HIV-1 infection in an AIDS patient includes combination of antiretroviral drugs called Highly Active Anti-Retroviral Therapy (HAART). A major challenge for the success of HAART has been these latent reservoirs of HIV which remain hidden and pose major hurdle for the eradication of virus. Combination of HAART therapy with simultaneous activation of latent reservoirs of HIV-1 seems to be the future of anti-retroviral therapy; however, this will require a much better understanding of the mechanisms and regulation of HIV-1 latency. In this chapter, we have tried to elaborate on HIV-1 latency, highlighting the strategies employed by the virus to ensure persistence in the host with specific focus on epigenetic regulation of latency. A complete understanding of HIV-1 latency will be extremely essential for ultimate eradication of HIV-1 from the human host.

Reciprocal regulation of human immunodeficiency virus-1 gene expression and replication by heat shock proteins 40 and 70.

Cellular heat shock proteins (Hsps) are induced upon heat shock, UV irradiation and microbial or viral infection. They are also known to be involved in apoptosis and immune response in addition to their chaperone function. Although some literature exists regarding the role of Hsps in human immunodeficiency virus (HIV)-1 infection, a clear understanding of their role remains elusive. Previously, we have shown that Hsp40, a co-chaperone of Hsp70, interacts with HIV-1 negative regulatory factor (Nef) and is required for Nef-mediated increase in viral gene expression and replication. We now show that Hsp70 is also present in the Nef-Hsp40 complex reported earlier. Furthermore, Hsp70 inhibits viral gene expression and replication; however, Hsp40 can rescue this down regulation of viral gene expression induced by Hsp70. We also show that HIV-1 viral protein R is required for this inhibitory effect of Hsp70 on viral replication. Our data further show that Hsp40 is consistently up regulated in HIV-1 infection, whereas Hsp70 is down regulated after initial up regulation favoring viral replication. Finally, Hsp70 expression inhibits the phosphorylation of cyclin-dependent kinase 9 required for high-affinity binding of HIV-1 transactivator of transcription-positive transcription elongation factor b complex to transactivation response RNA, whereas Hsp40 seems to induce it. Thus, Hsp40 and Hsp70, both closely associated in their chaperone function, seem to act contrary to each other in regulating viral gene expression. It seems that Hsp70 favors the host by inhibiting viral replication, whereas Hsp40 works in favor of the virus by inducing its replication. Thus, differential expression of Hsp40 and Hsp70 reciprocally regulates viral gene expression and replication in HIV-1 infection.

Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways.

Human immunodeficiency virus-1 (HIV-1) infection leads to changes in cellular gene expression, which in turn tend to modulate viral gene expression and replication. Cellular heat shock proteins (HSPs) are induced upon heat shock, UV irradiation and microbial or viral infections. We have reported earlier Nef-dependent induction of HSP40 leading to increased HIV-1 gene expression; however, the mechanism of induction remained to be elucidated. As expression of HSPs is regulated by heat shock factors (HSFs), we have now studied the role of HSF1 not only in Nef-dependent HSP40 induction but also in HIV-1 gene expression. Our results show that HSF1 is also induced during HIV-1 infection and it positively regulates HIV-1 gene expression by two distinct pathways. First, along with Nef it activates HSP40 promoter which in turn leads to increased HIV-1 gene expression. Second, HSF1 directly interacts with newly identified HSF1 binding sequence on HIV-1 LTR promoter and induces viral gene expression and replication. Thus, the present work not only identifies a molecular basis for HSF1-mediated enhancement of viral replication but also provides another example of how HIV-1 uses host cell machinery for its successful replication in the host.

Human cellular microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1 replication.

BACKGROUND: Cellular miRNAs play an important role in the regulation of gene expression in eukaryotes. Recently, miRNAs have also been shown to be able to target and inhibit viral gene expression. Computational predictions revealed earlier that the HIV-1 genome includes regions that may be potentially targeted by human miRNAs. Here we report the functionality of predicted miR-29a target site in the HIV-1 nef gene. RESULTS: We find that the human miRNAs hsa-miR-29a and 29b are expressed in human peripheral blood mononuclear cells. Expression of a luciferase reporter bearing the nef miR-29a target site was decreased compared to the luciferase construct without the target site. Locked nucleic acid modified anti-miRNAs targeted against hsa-miR-29a and 29b specifically reversed the inhibitory effect mediated by cellular miRNAs on the target site. Ectopic expression of the miRNA results in repression of the target Nef protein and reduction of virus levels. CONCLUSION: Our results show that the cellular miRNA hsa-miR29a downregulates the expression of Nef protein and interferes with HIV-1 replication.