An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses.

Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins.

The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

CRISPR knockout (KO) screens have identified host factors regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Here, we conducted a meta-analysis of these screens, which showed a high level of cell-type specificity of the identified hits, highlighting the necessity of additional models to uncover the full landscape of host factors. Thus, we performed genome-wide KO and activation screens in Calu-3 lung cells and KO screens in Caco-2 colorectal cells, followed by secondary screens in four human cell lines. This revealed host-dependency factors, including AP1G1 adaptin and ATP8B1 flippase, as well as inhibitors, including mucins. Interestingly, some of the identified genes also modulate Middle East respiratory syndrome coronavirus (MERS-CoV) and seasonal human coronavirus (HCoV) (HCoV-NL63 and HCoV-229E) replication. Moreover, most genes had an impact on viral entry, with AP1G1 likely regulating TMPRSS2 activity at the plasma membrane. These results demonstrate the value of multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential targets for therapeutic interventions.

Crystal structure of the TLDc domain of human NCOA7-AS.

The TLDc [Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic] domain is associated with oxidation-resistance related functions and is well conserved among eukaryotes. Seven proteins possess a TLDc domain in humans, notably proteins belonging to the oxidation resistance protein (OXR), nuclear receptor coactivator 7 (NCOA7) and TBC1 domain family member 24 (TBC1D24) families. Although the mechanism is unknown, a protective role of TLDc proteins against oxidative stress, notably in the brain, has been demonstrated. Neurobiological disorders caused by mutations in the TLDc domain have also been reported. The human NCOA7 gene encodes several mRNA isoforms; among these, isoform 4, named NCOA7-AS, is up-regulated by type 1 interferon in response to viral infection. NCOA7 and NCOA7-AS both interact with several subunits of the vacuolar proton pump V-ATPase, which leads to increased acidification of the endolysosomal system and consequently impairs infection by viruses that enter their host cells through the endosomal pathway, such as influenza A virus and hepatitis C virus. Similarly to full-length NCOA7, NCOA7-AS possesses a TLDc domain in its C-terminus. Structures of TLDc domains have been reported from zebrafish and fly but not from humans. Here, the expression, purification and crystallization of the TLDc domain from NCOA7 and NCOA7-AS is reported. The crystal structure solved at 1.8 Šresolution is compared with previously solved three-dimensional structures of TLDc domains.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to acute respiratory distress syndrome, and death in the most severe cases. Immune dysregulation with altered innate cytokine responses is thought to contribute to disease severity. Here, we characterized in depth host cell responses against SARS-CoV-2 in primary human airway epithelia (HAE) and immortalized cell lines. Our results demonstrate that primary HAE and model cells elicit a robust induction of type I and III interferons (IFNs). Importantly, we show for the first time that melanoma differentiation associated gene (MDA)-5 is the main sensor of SARS-CoV-2 in lung cells. IFN exposure strongly inhibited viral replication and de novo production of infectious virions. However, despite high levels of IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication in lung cells, contrary to what was previously reported in intestinal epithelial cells. Altogether, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.IMPORTANCE Mammalian cells express sensors able to detect specific features of pathogens and induce the interferon response, which is one of the first line of defenses against viruses and help controlling viral replication. The mechanisms and impact of SARS-CoV-2 sensing in lung epithelial cells remained to be deciphered. In this study, we report that despite a high production of type I and III interferons specifically induced by MDA-5-mediated sensing of SARS-CoV-2, primary and immortalized lung epithelial cells are unable to control viral replication. However, exogenous interferons potently inhibited replication, if provided early upon viral exposure. A better understanding of the ambiguous interplay between the interferon response and SARS-CoV-2 replication is essential to guide future therapeutical interventions.