Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex.

Ted Hackstadt,Emmie De Wit,Elizabeth R Fischer,Cheryl A Dooley,Forrest H Hoyt,Sonja M Best,Paul A Beare,Brandi N Williamson,Abhilash I Chiramel

doi:10.3390/v13091798

Abstract

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

Highlights

Multiple immunolabeling techniques incorporating both light and electron microscopy were utilized to examine the cellular origins of the Severe Acute RespiratorySyndrome (SARS)-CoV-2 replication organelle
Host membranes associated with the coronavirus replication complex are frequently identified as ER in origin based upon ultrastructure alone [30]
The means of viral dispersal of the Golgi apparatus is unknown, but the mechanism appears critical to the virus as dispersal of the Golgi with a chemical inhibitor, brefeldin A, is inhibitory to viral replication

Summary

Introduction

Coronaviruses are widespread in nature with thousands of genomes being annotated. A very few are known to be pathogenic toward humans and had been associated primarily with mild upper respiratory infections such as common colds [1]. In 2002, SARS-coronavirus apparently crossed species barriers to cause Severe Acute Respiratory. The disease had a high mortality, symptoms appeared prior to patients becoming highly infectious dissemination was inefficient enough that public health measures silenced the outbreak. A similar scenario occurred in 2012 with Middle East Respiratory Syndrome (MERS) coronavirus, which showed a high mortality rate but was again contained by public health measures. SARS is no longer epidemic in humans and it is unclear whether the virus remains in animal reservoirs

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