An HIV-1 CRISPR-Cas9 membrane trafficking screen reveals a role for PICALM intersecting endolysosomes and immunity.

HIV-1 hijacks host proteins involved in membrane trafficking, endocytosis, and autophagy that are critical for virus replication. Molecular details are lacking but are essential to inform on the development of alternative antiviral strategies. Despite their potential as clinical targets, only a few membrane trafficking proteins have been functionally characterized in HIV-1 replication. To further elucidate roles in HIV-1 replication, we performed a CRISPR-Cas9 screen on 140 membrane trafficking proteins. We identified phosphatidylinositol-binding clathrin assembly protein (PICALM) that influences not only infection dynamics but also CD4+ SupT1 biology. The knockout (KO) of PICALM inhibited viral entry. In CD4+ SupT1 T cells, KO cells exhibited defects in intracellular trafficking and increased abundance of intracellular Gag and significant alterations in autophagy, immune checkpoint PD-1 levels, and differentiation markers. Thus, PICALM modulates a variety of pathways that ultimately affect HIV-1 replication, underscoring the potential of PICALM as a future target to control HIV-1.

Retinoic acid enhances HIV-1 reverse transcription and transcription in macrophages via mTOR-modulated mechanisms.

The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4+ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4+ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.

Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages.

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.