Chronic neuroinflammation is observed in HIV+ individuals on suppressive combination antiretroviral therapy (cART) and is thought to cause HIV-associated neurocognitive disorders. We have recently reported that expression of HIV intron-containing RNA (icRNA) in productively infected monocyte-derived macrophages induces pro-inflammatory responses. Microglia, yolk sac-derived brain-resident tissue macrophages, are the primary HIV-1 infected cell type in the central nervous system (CNS). In this study, we tested the hypothesis that persistent expression of HIV icRNA in primary human microglia induces innate immune activation. We established multiple orthogonal primary human microglia-like cell cultures including peripheral blood monocyte-derived microglia (MDMG) and induced pluripotent stem cell (iPSC)-derived microglia. Unlike MDMG, human iPSC-derived microglia (hiMG), which phenotypically mimic primary CNS microglia, were robustly infected with replication competent HIV-1, and establishment of productive HIV-1 infection and de novo viral gene expression led to pro-inflammatory cytokine production. Blocking of HIV-1 icRNA expression, but not multiply spliced viral RNA, either via infection with virus expressing a Rev-mutant deficient for HIV icRNA nuclear export or infection in the presence of small molecule inhibitor of CRM1-mediated viral icRNA nuclear export pathway, attenuated induction of innate immune responses. These studies suggest that Rev–CRM1-dependent nuclear export and cytosolic sensing of HIV-1 icRNA induces pro-inflammatory responses in productively infected microglia. Novel strategies targeting HIV icRNA expression specifically are needed to suppress HIV-induced neuroinflammation.

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169-mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and subgenomic RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral genomic and subgenomic RNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1β) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.

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Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1β) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.Author SummaryOver-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.

The SARS-CoV-2 pandemic has caused widespread illness, loss of life, and socioeconomic disruption that is unlikely to resolve until vaccines are widely adopted, and effective therapeutic treatments become established. Here, a well curated and annotated library of 6710 clinical and preclinical molecules, covering diverse chemical scaffolds and known host targets was evaluated for inhibition of SARS-CoV-2 infection in multiple infection models. Multi-concentration, high-content immunocytofluorescence-based screening identified 172 strongly active small molecules, including 52 with submicromolar potencies. The active molecules were extensively triaged by in vitro mechanistic assays, including human primary cell models of infection and the most promising, obatoclax, was tested for in vivo efficacy. Structural and mechanistic classification of compounds revealed known and novel chemotypes and potential host targets involved in each step of the virus replication cycle including BET proteins, microtubule function, mTOR, ER kinases, protein synthesis and ion channel function. In the mouse disease model obatoclax effectively reduced lung virus load by 10-fold. Overall, this work provides an important, publicly accessible, foundation for development of novel treatments for COVID-19, establishes human primary cell-based pharmacological models for evaluation of therapeutics and identifies new insights into SARS-CoV-2 infection mechanisms.