Abstract

ABSTRACTBetacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.

Highlights

  • Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals

  • When using the V5-specific antibody, HA-nsp3 was coimmunoprecipitated when nsp4-V5 was present. These findings demonstrated that these two MERS-CoV proteins interact and further supported the notion that this is a common feature of coronaviruses

  • In this study, focusing on betacoronaviruses, we sought to identify the viral proteins required to induce the formation of double-membrane vesicles (DMVs), the most prominent membrane structure formed during coronavirus infection

Read more

Summary

Introduction

Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Coronaviruses, like all positive stranded RNA viruses of eukaryotes, hijack intracellular membranes to form their replication organelles (ROs) [14,15,16,17,18] These generally reside in the perinuclear region of the cell and are assumed to constitute microenvironments that promote viral RNA synthesis while possibly shielding replicative intermediates, double-stranded RNA, from detection by the innate immune system. They form an elaborate reticulovesicular network (RVN), for which the ER probably serves as the membrane donor [19]

Objectives
Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call