ABSTRACT
The prevalence of SARS-CoV-2 as a global health threat has called for population-wide vaccination to curb COVID-19. Hence, the World Health Organization (WHO) has approved several platforms of SARS-CoV-2 vaccines for emergency use. Therefore, a more comprehensive study on the immune response induced by vaccines in diverse individuals is still required. Here, we expressed a local variant of SARS-CoV-2 receptor-binding domain (RBD) and protease cleavage site (PCS), playing a vital role in binding and fusion in Rosetta (DE3). We then characterized it through SDS-PAGE analysis and western blotting. Moreover, we compared and monitored ChAdOx1 nCoV-19 vaccination-induced antibody response in convalescent and healthy vaccinated individuals after the first and second vaccine doses through serologic assay against RBD and PCS, which have not yet been compared. We investigated a cohort of 100 sera samples; based on our parameters, 25 serum samples were selected as convalescent samples and 25 serum samples as healthy samples for comparison. These findings demonstrate that most of the convalescent sera show more reactivity with PCS (80%) than with RBD (56%). Interestingly, IgG antibody response against PCS was more significant in both pre- and post-vaccination in convalescent individuals than in healthy individuals. Indeed, anti-RBD antibody titers were most significant in pre-vaccination and post-first vaccination in convalescent individuals than in healthy individuals and not in pre-vaccination and post-second vaccination. Besides monitoring IgG antibody response against COVID-19, these findings could shed light on the progress, assessment, and efficacy evaluation of SARS-CoV-2 vaccines.Communicated by Ramaswamy H. Sarma.
ABSTRACT
Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.