TRIM32 inhibits Venezuelan Equine Encephalitis Virus Infection by targeting a late step in viral entry.

Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.

Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo.

The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spikes from multiple SARS-CoV-2 variants and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for the delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrated protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses. IMPORTANCE: The rapid evolution of SARS-CoV-2 variants poses a challenge for immune recognition and antibody therapies. However, the virus is constrained by the requirement that it recognizes a human host receptor protein. A recombinant ACE2 could protect against SARS-CoV-2 infection by functioning as a soluble decoy receptor. We designed a mutant version of ACE2 with impaired catalytic activity to enable the purification of the protein using a single affinity purification step. This protein can be nebulized and retains the ability to bind the relevant domains from SARS-CoV-1 and SARS-CoV-2. Moreover, this protein inhibits viral infection against a panel of coronaviruses in cells. Finally, we developed an aerosolized delivery system for animal studies and show the modified ACE2 offers protection in an animal model of COVID-19. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2.

IRF7 from the black flying fox induces a STAT1-independent ISG response in unstimulated cell lines that protects against diverse RNA viruses.

Bats are considered unique in their ability to harbor large numbers of viruses and serve as reservoirs for zoonotic viruses that have the potential to spill over into humans. However, these animals appear relatively resistant to the pathogenic effects of many viruses. Mounting evidence suggests that bats may tolerate viral infections due to unique immune features. These include evolutionary innovations in inflammatory pathways and in the molecules involved in viral sensing, interferon induction, and downstream interferon-induced antiviral effectors. We sought to determine whether interferon-stimulated genes (ISGs) from the black flying fox ( Pteropus alecto ) encoded proteins with unique antiviral activity relative to their human orthologs. Accordingly, we compared the antiviral activity of over 50 ISG human-bat ortholog pairs to identify differences in individual effector functions. We identified IRF7 from Pteropus alecto (Pa.IRF7) as a potent and broad-acting antiviral molecule that provides robust antiviral protection without prior activation. We show that Pa.IRF7 uniquely induces a subset of protective ISGs independent of canonical IFN signaling, which leads to protection from alphaviruses, a flavivirus, a rhabdovirus, and a paramyxovirus. In uninfected cells, Pa.IRF7 partially localizes to the nucleus and can directly bind interferon-sensitive regulatory elements (ISREs). Compared to human IRF7, Pa.IRF7 also has additional serines in its C terminal domain that contribute to antiviral activity and may serve as unique phosphorylation hubs for activation. These properties constitute major differences between bat and human IRF7 that offer additional insight into the potential uniqueness of the black flying fox immune system.

IFIT1 is rapidly evolving and exhibits disparate antiviral activities across 11 mammalian orders.

Mammalian mRNAs possess an N7-methylguanosine (m7G) cap and 2'O methylation of the initiating nucleotide at their 5' end, whereas certain viral RNAs lack these characteristic features. The human antiviral restriction factor IFIT1 recognizes and binds to specific viral RNAs that lack the 5' features of host mRNAs, resulting in targeted suppression of viral RNA translation. This interaction imposes a significant host-driven evolutionary pressure on viruses, and many viruses have evolved mechanisms to evade the antiviral action of human IFIT1. However, less is known about the virus-driven pressures that may have shaped the antiviral activity of IFIT1 genes across mammals. Here, we take an evolution-guided approach to show that the IFIT1 gene is rapidly evolving in multiple mammalian clades, with positive selection acting upon several residues in distinct regions of the protein. In functional assays with 39 IFIT1s spanning diverse mammals, we demonstrate that IFIT1 exhibits a range of antiviral phenotypes, with many orthologs lacking antiviral activity against viruses that are strongly suppressed by other IFIT1s. We further show that IFIT1s from human and a bat, the black flying fox, inhibit Venezuelan equine encephalitis virus (VEEV) and strongly bind to Cap0 RNAs. Unexpectedly, chimpanzee IFIT1, which differs from human IFIT1 by only 8 amino acids, does not inhibit VEEV infection and exhibits minimal Cap0 RNA-binding. In mutagenesis studies, we determine that amino acids 364 and 366, the latter of which is undergoing positive selection, are sufficient to confer the differential anti-VEEV activity between human and chimpanzee IFIT1. These data suggest that virus-host genetic conflicts have influenced the antiviral specificity of IFIT1 across diverse mammalian orders.

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo.

The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spike from multiple SARS-CoV-2 variants, and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrate protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.

LY6E is a pan-coronavirus restriction factor in the respiratory tract.

LY6E is an antiviral restriction factor that inhibits coronavirus spike-mediated fusion, but the cell types in vivo that require LY6E for protection from respiratory coronavirus infection are unknown. Here we used a panel of seven conditional Ly6e knockout mice to define which Ly6e-expressing cells confer control of airway infection by murine coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loss of Ly6e in Lyz2-expressing cells, radioresistant Vav1-expressing cells and non-haematopoietic cells increased susceptibility to murine coronavirus. Global conditional loss of Ly6e expression resulted in clinical disease and higher viral burden after SARS-CoV-2 infection, but little evidence of immunopathology. We show that Ly6e expression protected secretory club and ciliated cells from SARS-CoV-2 infection and prevented virus-induced loss of an epithelial cell transcriptomic signature in the lung. Our study demonstrates that lineage confined rather than broad expression of Ly6e sufficiently confers resistance to disease caused by murine and human coronaviruses.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes.

Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors-ZAP, IFIT3, and IFIT1-together constitute the majority of interferon-mediated restriction of VEEV, while accounting for < 0.5% of the interferon-induced transcriptome. Together, our data suggest a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.

FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms.

The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes.

Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors - ZAP, IFIT3, and IFIT1 - together constitute the majority of interferon-mediated restriction of VEEV, while accounting for less than 0.5% of the interferon-induced transcriptome. Together, our data suggests a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.

LY6E protects mice from pathogenic effects of murine coronavirus and SARS-CoV-2.

LY6E is an antiviral protein that inhibits coronavirus entry. Its expression in immune cells allows mice to control murine coronavirus infection. However, it is not known which immune cell subsets mediate this control or whether LY6E protects mice from SARS-CoV-2. In this study, we used tissue-specific Cre recombinase expression to ablate Ly6e in distinct immune compartments or in all epiblast-derived cells, and bone marrow chimeras to target Ly6e in a subset of radioresistant cells. Mice lacking Ly6e in Lyz2 -expressing cells and radioresistant Vav1 -expressing cells were more susceptible to lethal murine coronavirus infection. Mice lacking Ly6e globally developed clinical disease when challenged with the Gamma (P.1) variant of SARS-CoV-2. By contrast, wildtype mice and mice lacking type I and type III interferon signaling had no clinical symptoms after SARS-CoV-2 infection. Transcriptomic profiling of lungs from SARS-CoV-2-infected wildtype and Ly6e knockout mice revealed a striking reduction of secretory cell-associated genes in infected knockout mice, including Muc5b , an airway mucin-encoding gene that may protect against SARS-CoV-2-inflicted respiratory disease. Collectively, our study reveals distinct cellular compartments in which Ly6e confers cell intrinsic antiviral effects, thereby conferring resistance to disease caused by murine coronavirus and SARS-CoV-2.

Genome-Scale CRISPR Screening Reveals Host Factors Required for Ribosome Formation and Viral Replication.

Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss-of-function CRISPR screen, we show that synthesis of a flavivirus-encoded fluorescent reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in rRNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeast Drg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication. IMPORTANCE Viruses are well known for their ability to co-opt host ribosomes to synthesize viral proteins. The specific factors involved in translation of viral RNAs are not fully described. In this study, we implemented a unique genome-scale CRISPR screen to identify previously uncharacterized host factors that are important for the synthesis of virally encoded protein. We found that multiple genes involved in 60S ribosome biogenesis were required for viral RNA translation. Loss of these factors severely impaired viral replication. Mechanistic studies on the AAA ATPase SPATA5 indicate that this host factor is required for a late step in ribosome formation. These findings reveal insight into the identity and function of specific ribosome biogenesis proteins that are critical for viral infections.

CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.

During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.

Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection.

Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PM's sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, and this depletion is sustained for long periods of time through 25HC-mediated suppression of SREBPs and continued activation of ACAT. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.

The Use of Mebendazole in COVID-19 Patients: An Observational Retrospective Single Center Study.

Background: An in-silico screen identified mebendazole with potential antiviral activity that could be a repurposed drug against SARS-CoV-2. Mebendazole is a well-tolerated and cheap antihelminthic agent that is readily available worldwide and thus could be a therapeutic tool in the fight against COVID-19. Methods: This is an observational retrospective study of PCR-confirmed COVID-19 patients who received mebendazole with the intention-to-treat. The study included an inpatient cohort (157 inpatients) and an outpatient cohort (185 outpatients). Of the 157 inpatients and 185 outpatients, 68 (43.3%) and 94 (50.8%) received mebendazole, respectively. Patients who presented within the same timeframe but did not receive mebendazole were used as controls. Patients received standard-of-care treatment including remdesivir, dexamethasone, and anticoagulants as deemed necessary by the treating physician. The following clinical outcomes were evaluated: for the inpatient cohort, length of stay (LOS) at the hospital, need for ventilation (combined invasive and noninvasive), and mortality; for the outpatient cohort, time to symptom resolution, need for hospitalization, and mortality. Results: For the inpatient cohort, the median age did not differ between the treatment and control groups; 62 (56, 67) vs. 62 (56, 68), P, and there was a comparable proportion of males in both groups; 43 (63%) vs. 55 (62%), P=0.85. The hospital LOS was 3.5 days shorter in the treatment group compared to the control group (P < 0.001). There were fewer patients who required invasive or noninvasive ventilation in the treatment group, 2 (2.9%) vs. 7 (7.9%), and the mortality rate is lower in the treatment group, 3 (4.4%) vs. 8 (9.0%), though the differences did not reach statistical significance. For the outpatient cohort, the median age was lower in the treatment group compared with the control group; 40 (34, 48) vs. 48 (41, 54), P < 0.001. There was a comparable proportion of males between both groups; 50 (53%) vs. 52 (57%), P=0.59. Patients in the treatment group were 3.3 days closer to symptom resolution (P < 0.001). There were numerically fewer patients requiring hospitalization in the treatment group compared with the control group, 3 (3.2%) vs. 6 (6.6%), though this did not reach statistical significance (P=0.33). Conclusion: In this retrospective observational study, the use of mebendazole in COVID-19 patients was associated with shorter hospitalizations in the inpatient cohort and shorter durations of symptom resolution in the outpatient cohort. The findings from this small observational study are hypothesis-generating and preclude drawing conclusions about clinical efficacy. Further studies are needed to examine the role of mebendazole in the treatment of COVID-19 patients.

A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors.

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.

Atovaquone for treatment of COVID-19: A prospective randomized, double-blind, placebo-controlled clinical trial.

Background: An in silico screen was performed to identify FDA approved drugs that inhibit SARS-CoV-2 main protease (Mpro), followed by in vitro viral replication assays, and in vivo pharmacokinetic studies in mice. These studies identified atovaquone as a promising candidate for inhibiting viral replication. Methods: A 2-center, randomized, double-blind, placebo-controlled trial was performed among patients hospitalized with COVID-19 infection. Enrolled patients were randomized 2:1 to atovaquone 1500 mg BID versus matched placebo. Patients received standard of care treatment including remdesivir, dexamethasone, or convalescent plasma as deemed necessary by the treating team. Saliva was collected at baseline and twice per day for up to 10 days for RNA extraction for SARS-CoV-2 viral load measurement by quantitative reverse-transcriptase PCR. The primary outcome was the between group difference in log-transformed viral load (copies/mL) using a generalized linear mixed-effect models of repeated measures from all samples. Results: Of the 61 patients enrolled; 41 received atovaquone and 19 received placebo. Overall, the population was predominately male (63%) and Hispanic (70%), with a mean age of 51 years, enrolled a mean of 5 days from symptom onset. The log10 viral load was 5.25 copies/mL vs. 4.79 copies/mL at baseline in the atovaquone vs. placebo group. Change in viral load did not differ over time between the atovaquone plus standard of care arm versus the placebo plus standard of care arm. Pharmacokinetic (PK) studies of atovaquone plasma concentration demonstrated a wide variation in atovaquone levels, with an inverse correlation between BMI and atovaquone levels, (Rho -0.45, p = 0.02). In post hoc analysis, an inverse correlation was observed between atovaquone levels and viral load (Rho -0.54, p = 0.005). Conclusion: In this prospective, randomized, placebo-controlled trial, atovaquone did not demonstrate evidence of enhanced SARS-CoV-2 viral clearance compared with placebo. However, based on the observed inverse correlation between atovaquone levels and viral load, additional PK-guided studies may be warranted to examine the antiviral effect of atovaquone in COVID-19 patients.

Enterovirus 3C Protease Cleaves TRIM7 To Dampen Its Antiviral Activity.

Mammalian TRIM7 is an antiviral protein that inhibits multiple human enteroviruses by degrading the viral 2BC protein. Whether TRIM7 is reciprocally targeted by enteroviruses is not known. Here, we report that the 3C protease (3Cpro) from two enteroviruses, coxsackievirus B3 (CVB3) and poliovirus, targets TRIM7 for cleavage. CVB3 3Cpro cleaves TRIM7 at glutamine 24 (Q24), resulting in a truncated TRIM7 that fails to inhibit CVB3 due to dampened E3 ubiquitin ligase activity. TRIM7 Q24 is highly conserved across mammals, except in marsupials, which instead have a naturally occurring histidine (H24) that is not subject to 3Cpro cleavage. Marsupials also express two isoforms of TRIM7, and the two proteins from koalas have distinct antiviral activities. The longer isoform contains an additional exon due to alternate splice site usage. This additional exon contains a unique 3Cpro cleavage site, suggesting that certain enteroviruses may have evolved to target marsupial TRIM7 even if the canonical Q24 is missing. Combined with computational analyses indicating that TRIM7 is rapidly evolving, our data raise the possibility that TRIM7 may be targeted by enterovirus evasion strategies and that evolution of TRIM7 across mammals may have conferred unique antiviral properties. IMPORTANCE Enteroviruses are significant human pathogens that cause viral myocarditis, pancreatitis, and meningitis. Knowing how the host controls these viruses and how the viruses may evade host restriction is important for understanding fundamental concepts in antiviral immunity and for informing potential therapeutic interventions. In this study, we demonstrate that coxsackievirus B3 uses its virally encoded protease to target the host antiviral protein TRIM7 for cleavage, suggesting a potential mechanism of viral immune evasion. We additionally show that TRIM7 has evolved in certain mammalian lineages to express protein variants with distinct antiviral activities and susceptibilities to viral protease-mediated cleavage.