Self-assembly of Severe Acute Respiratory Syndrome Coronavirus Membrane Protein

Chin-Tien Wang,Amber I-Ru Lee,Ying-Tzu Tseng,Shiu-Mei Wang,Chien-Cheng Chiang,Kuo-Jung Huang

doi:10.1074/jbc.m109.030270

Abstract

Coronavirus membrane (M) protein can form virus-like particles (VLPs) when coexpressed with nucleocapsid (N) or envelope (E) proteins, suggesting a pivotal role for M in virion assembly. Here we demonstrate the self-assembly and release of severe acute respiratory syndrome coronavirus (SARS-CoV) M protein in medium in the form of membrane-enveloped vesicles with densities lower than those of VLPs formed by M plus N. Although efficient N-N interactions require the presence of RNA, we found that M-M interactions were RNA-independent. SARS-CoV M was observed in both the Golgi area and plasma membranes of a variety of cells. Blocking M glycosylation does not appear to significantly affect M plasma membrane labeling intensity, M-containing vesicle release, or VLP formation. Results from a genetic analysis indicate involvement of the third transmembrane domain of M in plasma membrane-targeting signal. Fusion proteins containing M amino-terminal 50 residues encompassing the first transmembrane domain were found to be sufficient for membrane binding, multimerization, and Golgi retention. Surprisingly, we found that fusion proteins lacking all three transmembrane domains were still capable of membrane binding, Golgi retention, and interacting with M. The data suggest that multiple SARS-CoV M regions are involved in M self-assembly and subcellular localization.

Highlights

Coronaviruses are enveloped, positive-stranded RNA viruses that cause common respiratory and enteric diseases in humans and domesticated animals [1]
This explanation is compatible with studies demonstrating M carboxyl-terminal region involvement in M-N interaction in SARS-CoV [28, 41], mouse hepatitis virus (MHV) [14], and transmissible gastroenteritis virus [26]
Our results indicate that extracellular M became undetectable following treatment with protease and Triton X-100, suggesting that released M proteins were enveloped in lipid bilayers

Summary

Introduction

Coronaviruses are enveloped, positive-stranded RNA viruses that cause common respiratory and enteric diseases in humans and domesticated animals [1]. S and E are translated on membranebound polysomes, inserted into ER, and transported to the Golgi complex This complex is where E and M proteins interact and trigger virion budding, with nucleocapsids enclosed [7, 15]. SARS-CoV M self-assembly involves both amino- and carboxyl-terminal regions along the M sequence, amino-terminal 50 residues containing the first transmembrane domain are sufficient for conferring M selfassociation, membrane affinity, and Golgi retention. These findings for SARS-CoV M plasma membrane localization and secretion in medium indicate an undefined trafficking pathway in coronavirus assembly and budding

Methods

Results

Conclusion