Insights into the SARS-CoV-2-Mediated Alteration in the Stress Granule Protein Regulatory Networks in Humans

The rapidly and constantly evolving coronavirus, SARS-CoV-2, imposes a great threat to human health causing severe lung disease and significant mortality. Cytoplasmic stress granules (SGs) exert anti-viral activities due to their involvement in translation inhibition and innate immune signaling. SARS-CoV-2 sequesters important SG nucleator proteins and impairs SG formation, thus evading the host response for efficient viral replication. However, the significance of SGs in COVID-19 infection remains elusive. In this study, we utilize a protein-protein interaction network approach to systematically dissect the crosstalk of human post-translational regulatory networks governed by SG proteins due to SARS-CoV-2 infection. We uncovered that 116 human SG proteins directly interact with SARS-CoV-2 proteins and are involved in 430 different brain disorders including COVID-19. Further, we performed gene set enrichment analysis to identify the drugs against three important key SG proteins (DYNC1H1, DCTN1, and LMNA) and also looked for potential microRNAs (miRNAs) targeting these proteins. We identified bexarotene as a potential drug molecule and miRNAs, hsa-miR-615-3p, hsa-miR-221-3p, and hsa-miR-124-3p as potential candidates for the treatment of COVID-19 and associated manifestations.

Disruption of the Interaction between ORF33 and the Conserved Carboxyl-Terminus of ORF45 Abolishes Progeny Virion Production of Kaposi Sarcoma-Associated Herpesvirus

The Open Reading Frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific, immediate-early, tegument protein required for efficient viral replication and virion production. We have previously shown that ORF45 interacts with the conserved herpesviral protein ORF33 through the highly conserved C-terminal 19 amino acids (C19) of ORF45. Because the deletion of C19 abolished ORF33 accumulation and viral production, we reasoned that this interaction could be critical for viral production and explored as an antiviral target for gammaherpesviruses. In work described in this article, we characterize this interaction in further detail, first by revealing that this interaction is conserved among gammaherpesviruses, then by identifying residues in C19 critical for its interaction with and stabilization of ORF33. More importantly, we show that disruption of the interaction, either by mutating key residues (W403A or W405A) in C19 or by using competing cell penetration peptide TAT-C19, dramatically reduce the yield of KSHV progeny viruses. Our results not only reveal critical roles of this interaction to viral production but also provide a proof of concept for targeting the ORF33-ORF45 interaction as a novel antiviral strategy against KSHV and other gammaherpesviruses.

Modeling Hepatotropic Viral Infections: Cells vs. Animals

The lack of an appropriate platform for a better understanding of the molecular basis of hepatitis viruses and the absence of reliable models to identify novel therapeutic agents for a targeted treatment are the two major obstacles for launching efficient clinical protocols in different types of viral hepatitis. Viruses are obligate intracellular parasites, and the development of model systems for efficient viral replication is necessary for basic and applied studies. Viral hepatitis is a major health issue and a leading cause of morbidity and mortality. Despite the extensive efforts that have been made on fundamental and translational research, traditional models are not effective in representing this viral infection in a laboratory. In this review, we discuss in vitro cell-based models and in vivo animal models, with their strengths and weaknesses. In addition, the most important findings that have been retrieved from each model are described.

Human liver organoid derived intra-hepatic bile duct cells support SARS-CoV-2 infection and replication and its comparison with SARS-CoV

BackgroundAlthough the main route of infection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory tract, liver injury is also commonly seen in many patients, as evidenced by deranged parenchymal liver enzymes. Furthermore, patients with severe liver disease have been shown to have higher mortality. Overall, the mechanism behind the liver injury remains unclear.Approach and resultsWe showed that intra-hepatic bile duct cells could be grown using a human liver organoid platform. The cholangiocytes were not only susceptible to SARS-CoV-2 infection, they also supported efficient viral replication. We also showed that SARS-CoV-2 replication was much higher than SARS-CoV.ConclusionOur findings suggested direct cytopathic viral damage being a mechanism for SARS-CoV-2 liver injury.

Protein S-Nitrosylation of Human Cytomegalovirus pp71 Inhibits Its Ability To Limit STING Antiviral Responses

ABSTRACT Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that has coevolved with its host and, in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems, including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. To this end, we took an unbiased proteomics approach to identify the highly reversible, stress-induced, posttranslational modification (PTM) protein S-nitrosylation on viral proteins to determine the biological impact on viral replication. We identified protein S-nitrosylation of 13 viral proteins during infection of highly permissive fibroblasts. One of these proteins, pp71, is critical for efficient viral replication, as it undermines host antiviral responses, including stimulator of interferon genes (STING) activation. By exploiting site-directed mutagenesis of the specific amino acids we identified in pp71 as protein S-nitrosylated, we found this pp71 PTM diminishes its ability to undermine antiviral responses induced by the STING pathway. Our results suggest a model in which protein S-nitrosylation may function as a host response to viral infection that limits viral spread. IMPORTANCE In order for a pathogen to establish a successful infection, it must undermine the host cell responses inhibitory to the pathogen. As such, herpesviruses encode multiple viral proteins that antagonize each host antiviral response, thereby allowing for efficient viral replication. Human Cytomegalovirus encodes several factors that limit host countermeasures to infection, including pp71. Herein, we identified a previously unreported posttranslational modification of pp71, protein S-nitrosylation. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71 posttranslational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this posttranslational modification is to render a viral protein with diminished abilities to block host responses to infection. This novel work supports a model in which protein S-nitrosylation may be an additional mechanism in which a cell inhibits a pathogen during the course of infection.

Murine cytomegaloviruses m139 targets DDX3 to curtail interferon production and promote viral replication

Cytomegaloviruses (CMV) infect many different cell types and tissues in their respective hosts. Monocytes and macrophages play an important role in CMV dissemination from the site of infection to target organs. Moreover, macrophages are specialized in pathogen sensing and respond to infection by secreting cytokines and interferons. In murine cytomegalovirus (MCMV), a model for human cytomegalovirus, several genes required for efficient replication in macrophages have been identified, but their specific functions remain poorly understood. Here we show that MCMV m139, a gene of the conserved US22 gene family, encodes a protein that interacts with the DEAD box helicase DDX3, a protein involved in pathogen sensing and interferon (IFN) induction, and the E3 ubiquitin ligase UBR5. DDX3 and UBR5 also participate in the transcription, processing, and translation of a subset of cellular mRNAs. We show that m139 inhibits DDX3-mediated IFN-β induction and is necessary for efficient viral replication in bone-marrow derived macrophages. In vivo, m139 is crucial for viral dissemination to local lymph nodes and to the salivary glands. An m139-deficient MCMV also replicated to lower titers in SVEC4-10 endothelial cells. This replication defect was not accompanied by increased IFN-β transcription, but was rescued by knockout of either DDX3 or UBR5. Moreover, m139 co-localized with DDX3 and UBR5 in viral replication compartments in the cell nucleus. These results suggest that m139 inhibits DDX3-mediated IFN-β production in macrophages and antagonizes DDX3 and UBR5-dependent functions related to RNA metabolism in endothelial cells.Author SummaryHuman cytomegalovirus is an opportunistic pathogen that causes severe infections in immunocompromised individuals. The virus infects certain types, such as macrophages and endothelial cells, to ensure its dissemination within the body. Little is known about the viral factors that promote a productive infection of these cell types. The identification of critical viral factors and the molecular pathways they target can lead to the development of novel antiviral treatment strategies. Using the mouse cytomegalovirus as a model, we studied the viral m139 gene, which is important for virus replication in macrophages and endothelial cells and for dissemination in the mouse. This gene encodes a protein that interacts with the host proteins DDX3 and UBR5. Both proteins are involved in gene expression, and the RNA helicase DDX3 also participates in mounting an innate antiviral response. By interacting with DDX3 and UBR5, m139 ensures efficient viral replication in endothelial cells. Importantly, we identify m139 as a new viral DDX3 inhibitor, which curtails the production of interferon in macrophages.

Protein S-nitrosylation of Human Cytomegalovirus pp71 inhibits its ability to limit STING antiviral responses

ABSTRACTHuman Cytomegalovirus (HCMV) is a ubiquitous pathogen that has co-evolved with its host and in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. To this end, we took an unbiased proteomics approach to identify the highly reversible, stress induced, post-translational modification (PTM), protein S-nitrosylation, on viral proteins to determine the biological impact on viral replication.We identified protein S-nitrosylation of 13 viral proteins during infection of highly permissive fibroblasts. One of these proteins, pp71, is critical for efficient viral replication, as it undermines host antiviral responses, including STING activation. By exploiting site-directed mutagenesis of the specific amino acids we identified in pp71 as protein S-nitrosylated, we found this pp71 PTM diminishes its ability to undermine antiviral responses induced by the STING pathway. Our results suggest a model in which protein S-nitrosylation may function as a host response to viral infection that limits viral spread.IMPORTANCEIn order for a pathogen to establish a successful infection, it must undermine the host cell responses inhibitory to the pathogen. As such, herpesviruses encode multiple viral proteins that antagonize each host antiviral response, thereby allowing for efficient viral replication. Human Cytomegalovirus encodes several factors that limit host countermeasures to infection, including pp71. We identified a previously unreported modification of pp71 residues within the protein are protein S-nitrosylated. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71 post-translational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this post-translational modification is to render a viral protein with diminished abilities to block host responses to infection. This novel work supports a model in which protein S-nitrosylation may be an additional mechanism in which a cell inhibits a pathogen during the course of infection.