Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.

Phenotypic Lentivirus Screens to Identify Antiviral Single Domain Antibodies.

Our understanding of infection biology is based on experiments in which pathogen or host proteins are perturbed by small compound inhibitors, mutation, or depletion. This approach has been remarkably successful, as, for example, demonstrated by the independent identification of the endosomal membrane protein Niemann-Pick C1 as an essential factor for Ebola virus infection in both small compound and insertional mutagenesis screens (Côté, Nature 477:344-348, 2011; Carette et al., Nature 477:340-343, 2011). However, many aspects of host-pathogen interactions are poorly understood because we cannot target all of the involved molecules with small molecules, or because we cannot deplete essential proteins. Single domain antibody fragments expressed in the cytosol or other organelles constitute a versatile alternative to perturb the function of any given protein by masking protein-protein interaction interfaces, by stabilizing distinct conformations, or by directly interfering with enzymatic activities. The variable domains of heavy chain-only antibodies (VHHs) from camelid species can be cloned from blood samples of animals immunized with the desired target molecules. We can thus exploit the ability of the camelid immune system to generate affinity-matured single domain antibody fragments to obtain highly specific tools. Interesting VHH candidates are typically identified based on their affinity toward immobilized antigens using techniques such as phage display.The phenotypical screening approach described here allows the direct identification of VHHs that prevent infection of cells with influenza A virus (IAV) or other pathogens. The VHH repertoire is cloned into a lentiviral vector, which is used to generate pseudo-typed lentivirus particles. Target cells are transduced with the lentivirus, so that every cell inducibly expresses a different VHH. This cell collection is then challenged with a lethal dose of virus. Only the cells which express a VHH that prevents infection by targeting virus proteins or host cell components essential for infection will survive. We can thus identify critical target molecules including vulnerable epitopes and conformations, render target molecules accessible to informative perturbation studies, and stabilize intermediates of virus entry for detailed analysis.

The intra- and extracellular functions of ASC specks.

Inflammasomes are the central signaling hubs of the inflammatory response. They process cytosolic evidence of infection, cell damage, or metabolic disturbances, and elicit a pro-inflammatory response mediated by members of the interleukin-1 family of cytokines and pyroptotoic cell death. On the molecular level, this is accomplished by the sensor-nucleated recruitment and oligomerization of the adapter protein ASC. Once a tunable threshold is reached, cooperative assembly of ASC into linear filaments and their condensation into macromolecular ASC specks promotes an all-or-none response. These structures are highly regulated and provide a unique signaling platform or compartment to control the activity of caspase-1 and likely other effectors. Emerging evidence indicates that ASC specks are also released from inflammasome-activated cells and accumulate in inflamed tissues, where they can continue to mature cytokines or be internalized by surrounding cells to further nucleate ASC specks in their cytosol. Little is known about the mechanisms governing ASC speck release, uptake, and endosomal escape, as well as its contribution to inflammation and disease. Here, we describe the different outcomes of inflammasome activation and discuss the potential function of extracellular ASC specks. We highlight gaps in our understanding of this central process of inflammation, which may have direct consequences on the modulation of host responses and chronic inflammation.

siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor.

Poxvirus genome uncoating is a two-step process. First, cytoplasmic viral cores are activated and early viral genes are expressed. Next, cores are disassembled and the genomes released. This second step depends on an early viral factor(s) that has eluded identification for over 40 years. We used a large-scale, high-throughput RNAi screen directed against vaccinia virus (VACV) to identify the VACV AAA+ ATPase D5 as the poxvirus uncoating factor. We show that the ATPase activity of D5 is required for uncoating. Superresolution microscopy suggests that D5 acts directly at viral cores for genome release. Thus, the putative helicase D5 is a multifunctional protein required for genome uncoating and replication. Additionally, in vivo delivery of anti-D5 siRNAs reduced virus production in a mouse model of VACV infection. These results demonstrate the use of virus-targeting RNAi libraries to investigate viral gene function and suggest therapeutic avenues.

Vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector VH1 from lateral bodies.

Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-γ-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors.

Poxvirus host cell entry.

Poxviruses are characterized by their large size, complex composition, and cytoplasmic life cycle. They produce two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). Both MVs and EVs of vaccinia virus, the model poxvirus, take advantage of host cell endocytosis for internalization: they activate macropinocytosis-the most suitable form of endocytosis for large particles. Although largely dependent on the same cellular machinery, MV and EV entry differs with regard to the mechanisms used to trigger macropinocytosis and to undergo fusion. While EVs have to shed an additional membrane to expose the fusion complex, MV fusion requires the inactivation of fusion inhibitory proteins absent in EVs. This review highlights recent advances in the understanding of poxvirus MV and EV cell entry.

Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture.

Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.