SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS To Evade the Innate Immune Response

The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

The Role of Coronavirus RNA-Processing Enzymes in Innate Immune Evasion

Viral RNA sensing triggers innate antiviral responses in humans by stimulating signaling pathways that include crucial antiviral genes such as interferon. RNA viruses have evolved strategies to inhibit or escape these mechanisms. Coronaviruses use multiple enzymes to synthesize, modify, and process their genomic RNA and sub-genomic RNAs. These include Nsp15 and Nsp16, whose respective roles in RNA capping and dsRNA degradation play a crucial role in coronavirus escape from immune surveillance. Evolutionary studies on coronaviruses demonstrate that genome expansion in Nidoviruses was promoted by the emergence of Nsp14-ExoN activity and led to the acquisition of Nsp15- and Nsp16-RNA-processing activities. In this review, we discuss the main RNA-sensing mechanisms in humans as well as recent structural, functional, and evolutionary insights into coronavirus Nsp15 and Nsp16 with a view to potential antiviral strategies.

TcpC inhibits neutrophil extracellular trap formation by enhancing ubiquitination mediated degradation of peptidylarginine deiminase 4

TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.

Guinea Pig Cytomegalovirus Protective T Cell Antigen GP83 Is a Functional pp65 Homolog for Innate Immune Evasion and Pentamer-Dependent Virus Tropism

ABSTRACT The guinea pig is the only small animal model for congenital cytomegalovirus (CMV) but requires species-specific guinea pig cytomegalovirus (GPCMV). Tegument protein GP83 is the presumed homolog of HCMV pp65, but gene duplication in the UL82-UL84 homolog locus in various animal CMVs made it unclear if GP83 was a functional homolog. A GP83 null deletion mutant GPCMV (GP83dPC+) generated in the backdrop of glycoprotein pentamer complex (PC)-positive virus, required for nonfibroblast infection, had normal growth kinetics on fibroblasts but was highly impaired on epithelial and trophoblast cells. GP83dPC+ virus was highly sensitive to type I interferon (IFN-I), suggesting that GP83 had an innate immune evasion function. GP83 interacted with cellular DNA sensors guinea pig IFI16 and cGAS, indicating a role in the cGAS/STING pathway. Ectopically expressed GP83 in trophoblast cells restored GP83dPC+ virus growth. Additionally, mutant virus growth was restored in epithelial cells by expression of bovine viral diarrhea virus (BVDV) Npro protein targeting IRF3 as part of the cGAS/STING pathway, or alternatively, by expression of the fibroblast cell receptor platelet-derived growth factor receptor alpha (PDGFRA). HCMV pp65 is a T cell target antigen, and a recombinant adenovirus encoding GP83 was evaluated as a vaccine. In GPCMV challenge studies, vaccinated animals had various levels of protection against wild-type virus, with a protective response against the 22122 prototype strain but little protection against a novel clinical strain of GPCMV (TAMYC), despite 100% identity in GP83 protein sequences. Overall, GP83 is a functional pp65 homolog with novel importance for epithelial cell infection, but a GP83 T cell response provides limited vaccine efficacy. IMPORTANCE Congenital CMV (cCMV) is a leading cause of cognitive impairment and deafness in newborns, and a vaccine is a high priority. The guinea pig is the only small animal model for cCMV but requires guinea pig cytomegalovirus (GPCMV). The translational impact of GPCMV research is potentially reduced if the virus does not encode functional HCMV homolog proteins. This study demonstrates that tegument protein GP83 (pp65 homolog) is involved in innate immune evasion and highly important for infection of nonfibroblast cells via the viral glycoprotein pentamer complex (PC)-dependent endocytic entry pathway. The PC pathway is highly significant for virus dissemination and disease in the host, including cCMV. A GP83 candidate adenovirus (Ad) vaccine strategy in animals induced a cell-mediated response but failed to provide cross-strain protection against a novel clinical strain of GPCMV. Results suggest that the pp65 antigen provides very limited efficacy as a stand-alone vaccine, especially in cross-strain protection.

Innate immune evasion revealed in a colorectal zebrafish xenograft model

Cancer immunoediting is a dynamic process of crosstalk between tumor cells and the immune system. Herein, we explore the fast zebrafish xenograft model to investigate the innate immune contribution to this process. Using multiple breast and colorectal cancer cell lines and zAvatars, we find that some are cleared (regressors) while others engraft (progressors) in zebrafish xenografts. We focus on two human colorectal cancer cells derived from the same patient that show contrasting engraftment/clearance profiles. Using polyclonal xenografts to mimic intra-tumor heterogeneity, we demonstrate that SW620_progressors can block clearance of SW480_regressors. SW480_regressors recruit macrophages and neutrophils more efficiently than SW620_progressors; SW620_progressors however, modulate macrophages towards a pro-tumoral phenotype. Genetic and chemical suppression of myeloid cells indicates that macrophages and neutrophils play a crucial role in clearance. Single-cell-transcriptome analysis shows a fast subclonal selection, with clearance of regressor subclones associated with IFN/Notch signaling and escaper-expanded subclones with enrichment of IL10 pathway. Overall, our work opens the possibility of using zebrafish xenografts as living biomarkers of the tumor microenvironment.