Analysis of coronavirus nonstructructural proteins in virus-indued membrane modifications

Abstract

Coronaviruses are positive-sense RNA viruses that cause significant diseases in humans. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) caused a pandemic in 2002-2003, and Middle East Respiratory Syndrome coronavirus (MERS-CoV) is currently circulating in Saudi Arabia, causing severe respiratory infection with high mortality. Murine hepatitis virus is a well-established model system to study coronavirus replication. Coronavirus replication occurs in the host cell cytoplasm on cell membranes. Coronaviruses induce many membrane modifications, including double membrane vesicles (DMVs) and convoluted membranes. Three coronavirus replicase nonstructural proteins - nsp3, nsp4, and nsp6 - are required for modification of host membranes. The specific mechanisms and determinants by which these proteins interact to modify membranes are unknown. This study uses genetic approaches to introduce designed mutations in nsp3 and nsp4 in order to determine the effect during virus infection. The results of the experiments identify amino acid residues in nsp3 and nsp4 that are important for viral replication, DMV formation, and virus fitness. The data further demonstrate that intact wildtype DMV formation or morphology is not required for virus viability, but likely serves purposes in the timing and efficiency of replication. Thus nsp3-6 and the modified membranes may be novel targets for inhibition of virus replication.