TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Ubiquitination in viral entry and replication: Mechanisms and implications.

The ubiquitination process is a reversible posttranslational modification involved in many essential cellular functions, such as innate immunity, cell signaling, trafficking, protein stability, and protein degradation. Viruses can use the ubiquitin system to efficiently enter host cells, replicate and evade host immunity, ultimately enhancing viral pathogenesis. Emerging evidence indicates that enveloped viruses can carry free (unanchored) ubiquitin or covalently ubiquitinated viral structural proteins that can increase the efficiency of viral entry into host cells. Furthermore, viruses continuously evolve and adapt to take advantage of the host ubiquitin machinery, highlighting its importance during virus infection. This review discusses the battle between viruses and hosts, focusing on how viruses hijack the ubiquitination process at different steps of the replication cycle, with a specific emphasis on viral entry. We discuss how ubiquitination of viral proteins may affect tropism and explore emerging therapeutics strategies targeting the ubiquitin system for antiviral drug discovery.

Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

Prospects of Directly Reprogrammed Adult Human Neurons for Neurodegenerative Disease Modeling and Drug Discovery: iN vs. iPSCs Models.

A reliable disease model is critical to the study of specific disease mechanisms as well as for the discovery and development of new drugs. Despite providing crucial insights into the mechanisms of neurodegenerative diseases, translation of this information to develop therapeutics in clinical trials have been unsuccessful. Reprogramming technology to convert adult somatic cells to induced Pluripotent Stem Cells (iPSCs) or directly reprogramming adult somatic cells to induced Neurons (iN), has allowed for the creation of better models to understand the molecular mechanisms and design of new drugs. In recent times, iPSC technology has been commonly used for modeling neurodegenerative diseases and drug discovery. However, several technological challenges have limited the application of iN. As evidence suggests, iN for the modeling of neurodegenerative disorders is advantageous compared to those derived from iPSCs. In this review, we will compare iPSCs and iN models for neurodegenerative diseases and their potential applications in the future.

Signature molecules expressed differentially in a liver disease stage-specific manner by HIV-1 and HCV co-infection.

To elucidate HIV-1 co-infection-induced acceleration of HCV liver disease and identify stage-specific molecular signatures, we applied a new high-resolution molecular screen, the Affymetrix GeneChip Human Transcriptome Array (HTA2.0), to HCV-mono- and HIV/HCV-co-infected liver specimens from subjects with early and advanced disease. Out of 67,528 well-annotated genes, we have analyzed the functional and statistical significance of 75 and 28 genes expressed differentially between early and advanced stages of HCV mono- and HIV/HCV co-infected patient liver samples, respectively. We also evaluated the expression of 25 and 17 genes between early stages of mono- and co-infected liver tissues and between advanced stages of mono- and co-infected patient's samples, respectively. Based on our analysis of fold-change in gene expression as a function of disease stage (i.e., early vs. advanced), coupled with consideration of the known relevant functions of these genes, we focused on four candidate genes, ACSL4, GNMT, IFI27, and miR122, which are expressed stage-specifically in HCV mono- and HIV-1/HCV co-infective liver disease and are known to play a pivotal role in regulating HCV-mediated hepatocellular carcinoma (HCC). Our qRT-PCR analysis of the four genes in patient liver specimens supported the microarray data. Protein products of each gene were detected in the endoplasmic reticulum (ER) where HCV replication takes place, and the genes' expression significantly altered replicability of HCV in the subgenomic replicon harboring regulatory genes of the JFH1 strain of HCV in Huh7.5.1. With respect to three well-known transferrable HIV-1 viral elements-Env, Nef, and Tat-Nef uniquely augmented replicon expression, while Tat, but not the others, substantially modulated expression of the candidate genes in hepatocytic cells. Combinatorial expression of these cellular and viral genes in the replicon cells further altered replicon expression. Taken together, these results showed that HIV-1 viral proteins can exacerbate liver pathology in the co-infected patients by disparate molecular mechanisms-directly or indirectly dysregulating HCV replication, even if lack of association of HCV load and end-stage liver disease in hemophilic patients were reported, and modulating expression of hepatocellular genes critical for disease progression. These findings also provide major insights into development of stage-specific hepatocellular biomarkers for improved diagnosis and prognosis of HCV-mediated liver disease.