A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication.

Andrea Mikulasova,Alice M Trenerry,Turgut E Aktepe,Jason M Mackenzie,Susann Liebscher,Leah K Gillespie,Rebecca L Ambrose

doi:10.3389/fcell.2021.655606

Abstract

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Highlights

Replication of flaviviruses, like all positive-stranded RNA viruses, is associated with specialized cytoplasmic membrane structures that wrap around the active replication complexes (RC), providing a favorable microenvironment that facilitates efficient RNA synthesis
We have observed that the biogenesis of the vesicle packets (VP) appears to occur on a membrane platform derived from the endoplasmic reticulum (ER), and recruitment of both host and viral proteins occurs at a pre-Golgi step (Gillespie et al, 2010)
We observed that eGFP-erlin-2 and dsRNA were dispersed in visibly smaller foci throughout entire cytoplasm of the cell and revealed reduced co-localization in cells replicating the Y/S mutant (Figures 4Ai–l). These results suggested that there was an impairment of the West Nile virus strain Kunjin virus (WNVKUN) Y/S mutant to localize to lipid microdomains in the ER to establish biogenesis of the RC, an impairment that correlates with attenuation of replication efficiency (Figure 2)

Summary

Introduction

Replication of flaviviruses, like all positive-stranded RNA viruses, is associated with specialized cytoplasmic membrane structures that wrap around the active replication complexes (RC), providing a favorable microenvironment that facilitates efficient RNA synthesis. It has been observed that dengue virus replication alters lipid homeostasis (Perera et al, 2012), but is dependent on cholesterol, sphingolipid and continual fatty acid synthesis (Rothwell et al, 2009; Heaton et al, 2010; Martin-Acebes et al, 2014). These complementary studies have emphasized the strict requirement of cellular lipids in providing a membrane platform for efficient flavivirus replication

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