Electrostatic Interaction Between NS1 and Negatively Charged Lipids Contributes to Flavivirus Replication Organelles Formation.

Yali Ci,Lei Shi,Cheng-Feng Qin,Yang Yang,Caimin Xu

doi:10.3389/fmicb.2021.641059

Abstract

Flavivirus replication occurs in membranous replication compartments, also known as replication organelles (ROs) derived from the host ER membrane. Our previous study showed that the non-structural (NS) protein 1 (NS1) is the essential factor for RO creation by hydrophobic insertion into the ER membrane. Here, we found that the association of NS1 with the membrane can be facilitated by the electrostatic interaction between NS1 and negatively charged lipids. NS1 binds to a series of negatively charged lipids, including PI4P, and a positively charged residue, R31, located on the membrane-binding face of NS1, plays important roles in this interaction. The NS1 R31E mutation significantly impairs NS1 association with negatively charged membrane and its ER remodeling ability in the cells. To interfere with the electrostatic interaction between NS1 and negatively charged lipids, intracellular phosphatidylinositol phosphates (PIPs) level was downregulated by the overexpression of Sac1 or treatment with PI3K and PI4K inhibitors to attenuate flavivirus replication. Our findings emphasize the importance of electrostatic interaction between NS1 and negatively charged lipids in flavivirus RO formation.

Highlights

Positive strand RNA viruses, including flavivirus, coronavirus, and many others, are tightly associated with host cells’ membrane systems throughout their whole lifecycles, ranging from virus entry, fusion to replication, virions packaging, and secretion
Single-membrane invaginated vesicles of flavivirus and double-membrane vesicles (DMVs) of hepatitis C virus (HCV) and coronavirus stem from the endoplasmic reticulum (ER) (Knoops et al, 2008; Romero-Brey et al, 2012), whereas the DMVs or multilamellar vesicles of enterovirus originate from the ER and Golgi (Schlegel et al, 1996; Rust et al, 2001; Hsu et al, 2010; Limpens et al, 2011)
Purified NS1 proteins were incubated with liposomes with (w/) or without (w/o) PI4P, and the NS1-liposome mixture was submitted to Nycodenz gradient centrifugation

Summary

Introduction

Positive strand RNA viruses, including flavivirus, coronavirus, and many others, are tightly associated with host cells’ membrane systems throughout their whole lifecycles, ranging from virus entry, fusion to replication, virions packaging, and secretion. These viruses replicate in the host membranous structure, which provides a unique environment and ensures that viral replication proceeds successfully without invoking the possible offensive from the host immune system (den Boon and Ahlquist, 2010). Positive-strand RNA viruses create membranous replication organelles (ROs) by reorganizing host cell organelles. NS1 remodels the host ER through its hydrophobic insertion into the ER membrane, creating invaginated vesicles that provide the environment for viral replication

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Conclusion