Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.

DEAD-box helicases are a large family of conserved RNA-binding proteins that belong to the broader group of cellular DExD/H helicases. Members of the DEAD-box helicase family have roles throughout cellular RNA metabolism from biogenesis to decay. Moreover, there is emerging evidence that cellular RNA helicases, including DEAD-box helicases, play roles in the recognition of foreign nucleic acids and the modulation of viral infection. As intracellular parasites, viruses must evade detection by innate immune sensing mechanisms and degradation by cellular machinery while also manipulating host cell processes to facilitate replication. The ability of DEAD-box helicases to recognize RNA in a sequence-independent manner, as well as the breadth of cellular functions carried out by members of this family, lead them to influence innate recognition and viral infections in multiple ways. Indeed, DEAD-box helicases have been shown to contribute to intracellular immune sensing, act as antiviral effectors, and even to be coopted by viruses to promote their replication. However, our understanding of the mechanisms underlying these interactions, as well as the cellular roles of DEAD-box helicases themselves, is limited in many cases. We will discuss the diverse roles that members of the DEAD-box helicase family play during viral infections.Viruses 2020, 12, 181 2 of 15 helicases to particular RNAs or proteins; the diversity of these regions accounts for the breadth of function observed within this protein family [7]. As a result of their diversity of binding and function, DEAD-box RNA helicases are involved in all aspects of cellular RNA metabolism, including transcription, splicing, microRNA biogenesis, ribosomal RNA processing, RNA export, translation, RNP granule formation, and decay [10]. Moreover, they are also involved in cellular stress responses, contributing to double-strand break (DSB) repair [11], stress granule formation, and antimicrobial responses [12]. DEAD-Box Helicases in Canonical Innate Immune SensingInnate immune defenses depend upon the recognition of invading pathogens by the host, and viral infections, including RNA viruses, are largely sensed through nucleic acid recognition. RNA viruses present a host cell with various foreign RNA features, such as unique ends, structured RNA elements, or dsRNA replication intermediates, which can be recognized as hallmarks of infection. RNA binding proteins thus play important roles in the sensing of viral infection, and DExD/H helicases are well-suited to recognize structural features characteristic of viral RNAs.The canonical cytoplasmic sensors of viral RNA, the RIG-I-like receptors (RLRs) RIG-I and MDA-5, contain DExD/H-box helicase domains [13]. RIG-I recognizes uncapped viral RNAsand short dsRNAs containing a 5 triphosphate, while MDA-5 binds longer dsRNA replication intermediates [13][14][15]. In addition to the helicase core domain, these proteins contain caspase activation and recruitment domains (CARDs).RNA recognition by these RLRs leads to oligomerization of ...

Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

The mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV), is a highly successful pathogen for which there are no vaccines or therapeutics. Translational arrest is a common antiviral strategy used by hosts. In response, RVFV inhibits two well-known antiviral pathways that attenuate translation during infection, PKR and type I IFN signaling. Despite this, translational arrest occurs during RVFV infection by unknown mechanisms. Here, we find that RVFV infection triggers the decay of core translation machinery mRNAs that possess a 5′-terminal oligopyrimidine (5′-TOP) motif in their 5′-UTR, including mRNAs encoding ribosomal proteins, which leads to a decrease in overall ribosomal protein levels. We find that the RNA decapping enzyme NUDT16 selectively degrades 5′-TOP mRNAs during RVFV infection and this decay is triggered in response to mTOR attenuation via the translational repressor 4EBP1/2 axis. Translational arrest of 5′-TOPs via 4EBP1/2 restricts RVFV replication, and this increased RNA decay results in the loss of visible RNA granules, including P bodies and stress granules. Because RVFV capsnatches in RNA granules, the increased level of 5′-TOP mRNAs in this compartment leads to snatching of these targets, which are translationally suppressed during infection. Therefore, translation of RVFV mRNAs is compromised by multiple mechanisms during infection. Together, these data present a previously unknown mechanism for translational shutdown in response to viral infection and identify mTOR attenuation as a potential therapeutic avenue against bunyaviral infection.translational arrest | 5′-TOP mRNA | RNA decay | Rift Valley fever virus |