Genetic Inactivation of COPI Coatomer Separately Inhibits Vesicular Stomatitis Virus Entry and Gene Expression

Abstract

Viruses coopt cellular membrane transport to invade cells, establish intracellular sites of replication, and release progeny virions. Recent genome-wide RNA interference (RNAi) screens revealed that genetically divergent viruses require biosynthetic membrane transport by the COPI coatomer complex for efficient replication. Here we found that disrupting COPI function by RNAi inhibited an early stage of vesicular stomatitis virus (VSV) replication. To dissect which replication stage(s) was affected by coatomer inactivation, we used visual and biochemical assays to independently measure the efficiency of viral entry and gene expression in hamster (ldlF) cells depleted of the temperature-sensitive -COP subunit. We show that -COP depletion for 12 h caused a primary block to virus internalization and a secondary defect in viral gene expression. Using brefeldin A (BFA), a chemical inhibitor of COPI function, we demonstrate that short-term (1-h) BFA treatments inhibit VSV gene expression, while only long-term (12-h) treatments block virus entry. We conclude that prolonged coatomer inactivation perturbs cellular endocytic transport and thereby indirectly impairs VSV entry. Our results offer an explanation of why COPI coatomer is frequently identified in screens for cellular factors that support cell invasion by microbial pathogens.

Dissecting the Role of COPI Complexes in Influenza Virus Infection

“…Cureton and colleagues showed that COPI depletion also leads to defects in VSV entry (25), although there are differences between the two viruses in which particular steps are perturbed. Cureton and colleagues found that COPI depletion results in a decrease in viral fusion due to an ϳ40% decrease in VSV binding and a decreased rate of VSV internalization.…”

“…The results from Cureton and colleagues suggest that indirect effects caused by long-term inactivation of COPI lead to general defects in the clathrin-mediated endocytosis pathway (25). VSV exclusively uses clathrin-mediated endocytosis for infection (27)(28)(29)(30)(31).…”

“…Recently, Cureton and colleagues reported that disruption of COPI complexes differentially affects vesicular stomatitis virus (VSV) entry versus viral gene expression (25). The authors used a temperature-sensitive CHO cell line (IdlF), which degrades ε-COPI at 40°C (23), to show that ε-COPI depletion inhibits virus binding and internalization as well as transferrin (Tfn) uptake.…”

Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression

“…Recently, we reported through a genome-wide siRNA screen the requirement of several host factors for VSV replication at various stages of the virus infection cycle (33). Among these, alanine deaminase-like (ADAL) protein was shown to be required for the VSV assembly/budding step, whereas GBF1, ARF1, and the proteins of the coatomer I (COPI) complex were required for VSV genome transcription and replication (33,48). Therefore, we examined if hnRNP K silencing affects the expression of some of these cellular factors.…”

Antiviral drug discovery: broad-spectrum drugs from nature

“…Targeting lysobisphosphatidic acid (LBPA), which plays a role in cargo trafficking within endosomes, cholesterol mobilization and the formation of multivesicular bodies 86 has also been shown to inhibit influenza virus, vesicular stomatitis virus (VSV), Lassa Fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) 61,[87][88][89] . Depletion of components of the coat protein complex I (COPI) affects the entry of influenza virus and VSV and the endocytosis and vesicular transport of HCV and HIV [90][91][92] .…”