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SARS‐CoV‐2 infection causes broad‐spectrum immunopathological disease, exacerbated by inflammatory co‐morbidities. A better understanding of mechanisms underpinning virus‐associated inflammation is required to develop effective therapeutics. Here, we discover that SARS‐CoV‐2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG‐I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS‐CoV‐2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS‐CoV‐2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation‐associated COVID‐19. SYNOPSIS: SARS‐CoV‐2 induces a robust, delayed innate immune response in airway epithelial cells, driven by activation of RNA sensors, which propagates inflammation through macrophage activation. SARS‐CoV‐2 activates RNA sensors and consequent inflammatory responses in lung epithelial cells.Epithelial RNA sensing responses drive pro‐inflammatory macrophage activation.Exogenous inflammatory stimuli exacerbate responses to SARS‐CoV‐2 in both epithelial cells and macrophages.Immunomodulators inhibit RNA sensing responses and consequent macrophage inflammation. [ABSTRACT FROM AUTHOR]

Detection of viral DNA by cyclic GMP‐AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV‐1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV‐1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild‐type HIV‐1, infection with cleavage defective HIV‐1 triggered an IFN response in THP‐1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF‐74 also induced a cGAS‐dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid‐ and protease‐targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo. Synopsis: Viruses must avoid or block activation of an interferon response in order to replicate. HIV‐1 uses its capsid to physically protect viral reverse transcripts from detection by the DNA sensor cGAS, thus disruption of capsid integrity/stability enhances innate immune sensing that may be harnessed therapeutically. Treatment of HIV‐1 with protease inhibitors or mutation of protease cleavage sites in gag leads to defective Gag cleavage, reduced particle infectivity and diminished capacity to saturate restriction factor TRIM5α.Gag cleavage defective viruses, unlike wild‐type HIV‐1, activate a potent interferon response in THP‐1 cells and primary macrophages.Innate immune activation is dependent on viral reverse transcription and the cellular DNA sensor cGAS.Treatment of HIV‐1 with capsid destabiliser PF‐74 also leads to a potent cGAS‐dependent interferon response.The HIV‐1 capsid physically masks viral DNA from innate immune detection. [ABSTRACT FROM AUTHOR]

Abstract: Endogenous retroviruses (ERVs) have accumulated in vertebrate genomes and contribute to the complexity of gene regulation. KAP1 represses ERVs during development by its recruitment to their repetitive sequences through KRAB zinc‐finger proteins (KZNFs), but little is known about the regulation of ERVs in adult tissues. We observed that KAP1 repression of HERVK14C was conserved in differentiated human cells and performed KAP1 knockout to obtain an overview of KAP1 function. Our results show that KAP1 represses ERVs (including HERV‐T and HERV‐S) and ZNF genes, both of which overlap with KAP1 binding sites and H3K9me3 in multiple cell types. Furthermore, this pathway is functionally conserved in adult human peripheral blood mononuclear cells. Cytosine methylation that acts on KAP1 regulated loci is necessary to prevent an interferon response, and KAP1‐depletion leads to activation of some interferon‐stimulated genes. Finally, loss of KAP1 leads to a decrease in H3K9me3 enrichment at ERVs and ZNF genes and an RNA‐sensing response mediated through MAVS signaling. These data indicate that the KAP1‐KZNF pathway contributes to genome stability and innate immune control in adult human cells. [ABSTRACT FROM AUTHOR]

We report that DNA damage induced by topoisomerase inhibitors, including etoposide ( ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages ( MDM). SAMHD1 suppresses viral reverse transcription ( RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2 LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM. Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post- RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs. [ABSTRACT FROM AUTHOR]

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/ MEK/ ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo. Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions. [ABSTRACT FROM AUTHOR]

TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved. [ABSTRACT FROM AUTHOR]

SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1−/− cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism. [ABSTRACT FROM AUTHOR]

The mouse gene Fv1 encodes a saturable restriction factor that selectively blocks infection by N-tropic or B-tropic murine leukemia virus (MLV) strains. Despite the absence of an Fv1 gene, a similar activity is present in humans that blocks N-MLV infection (Ref1). Moreover, some non-human primate cell tines express a potentially related inhibitor of HIV-1 and/or SIVmac infection (Lv1). Here, we examine the spectrum of retrovirus-restricting activities expressed by human and African green monkey cell tines. Human cells restrict N-MLV and equine infectious anemia virus (EIAV), but not HIV- 1, HIV-2, SlVmac or SIVagm, whilst AGM cells restrict N-MLV, EIAV, HIV-1, HIV-2 and SlVmac. Remarkably, in each example examined, restriction of infection by a given retrovirus can be abrogated at least partially by saturation with another retrovirus, provided that it is also restricted but regardless of whether it is closely related. These data suggest that restriction factors in human and non-human primate cells are able to recognize and block infection by multiple, widely divergent retroviruses and that the factors themselves may be related. [ABSTRACT FROM AUTHOR]