Biology subjects

Doerger, E.

Publications and source records attributed to Doerger, E..

2 recordsLinked to original sources

bioRxiv2021

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS-CoV is adept at antagonizing early innate immune pathways - interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L) - generated in response to viral double-stranded (ds)RNA generated during genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication. ImportanceMiddle East Respiratory Syndrome Coronavirus (MERS-CoV) causes highly lethal respiratory disease. MERS-CoV encodes several innate immune antagonists, accessory proteins NS4a and NS4b unique to the merbeco lineage and the nsp15 protein endoribonuclease (EndoU), conserved among all coronaviruses. While mutation of each antagonist protein alone has little effect on innate immunity, infections with recombinant MERS-CoVs with mutations of EndoU in combination with either NS4a or NS4b, activate innate signaling pathways and are attenuated for replication. Our data indicate that EndoU and accessory proteins NS4a and NS4b together suppress innate immunity during MERS-CoV infection, to optimize viral replication. This is in contrast to SARS-CoV-2 which activates these pathways and consistent with greater mortality observed during MERS-CoV infection compared to SARS-CoV-2.

microbiology↗

bioRxiv2021

An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-{beta} transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics. IMPORTANCEIn the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate, and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within non-structural protein 3. It has received significant attention as a potential drug target as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the various roles and functions of Mac1 during infection remain largely unknown. Here, utilizing recombinant Mac1 mutant viruses, we have determined that different biochemical functions of Mac1 have distinct roles in the replication of MHV, a model CoV. These results indicate that Mac1 is more important for CoV replication than previously appreciated, and could help guide the development of inhibitory compounds that target unique regions of this protein domain.

microbiology↗