Induction of Endoplasmic Reticulum-Derived Replication-Competent Membrane Structures by West Nile Virus Non-Structural Protein 4B

Pakieli H Kaufusi,Richard Yanagihara,Vivek R Nerurkar,James F Kelley,Houssam Attoui

doi:10.1371/journal.pone.0084040

Pakieli H Kaufusi, Richard Yanagihara + Show 3 more

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https://doi.org/10.1371/journal.pone.0084040

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Journal: PloS one	Publication Date: Jan 20, 2014
Citations: 73	License type: CC BY 4.0

Affiliation: University of Hawaiʻi at Mānoa

Abstract

Replication of flaviviruses (family Flaviviridae) occurs in specialized virus-induced membrane structures (IMS). The cellular composition of these IMS varies for different flaviviruses implying different organelle origins for IMS biogenesis. The role of flavivirus non-structural (NS) proteins for the alteration of IMS remains controversial. In this report, we demonstrate that West Nile virus strain New York 99 (WNVNY99) remodels the endoplasmic reticulum (ER) membrane to generate specialized IMS. Within these structures, we observed an element of the cis-Golgi, viral double-stranded RNA, and viral-envelope, NS1, NS4A and NS4B proteins using confocal immunofluorescence microscopy. Biochemical analysis and microscopy revealed that NS4B lacking the 2K-signal peptide associates with the ER membrane where it initiates IMS formation in WNV-infected cells. Co-transfection studies indicated that NS4A and NS4B always remain co-localized in the IMS and are associated with the same membrane fractions, suggesting that these proteins function cooperatively in virus replication and may be an ideal target for antiviral drug discovery.

Highlights

The West Nile virus (WNV) genome consists of a singlestranded, positive-sense RNA of approximately 11 kb that encodes a single polyprotein precursor, which is processed by cellular and viral-encoded proteases into three structural proteins and seven non-structural (NS) proteins
Previous studies suggest that the intracellular membranes for induced membrane structures (IMS) biogenesis appear to differ across flaviviruses [4,19,20] and the discrete virus-IMS are readily identifiable by confocal immunofluorescence microscopy (IFM) [4,13,20]
There was no apparent co-localization of the mannose-6-phosphate receptor (M6PR), a marker for early endosome [28], with NS1 (Fig. 1A, n–p) indicating that the membrane for West Nile virus strain New York 99 (WNVNY99) IMS formation was not derived from this organelle

Summary

Introduction

The West Nile virus (WNV) genome consists of a singlestranded, positive-sense RNA of approximately 11 kb that encodes a single polyprotein precursor, which is processed by cellular and viral-encoded proteases into three structural proteins and seven non-structural (NS) proteins. The roles of NS proteins in the WNV life cycle are known [1], except for NS4B, the largest of the small hydrophobic NS proteins of flaviviruses, which consists of three endoplasmic reticulum (ER) membrane-spanning segments. The precise role of NS proteins during IMS formation in flavivirus-infected cells remains poorly understood. It is unclear which cellular organelle membranes are exploited by viral proteins during IMS biogenesis. For DENV-2 and WNVKUN, it has been speculated that the proteins within the polyprotein NS4A-2K-NS4B are responsible for remodeling infected cell membranes [4], and that regulated processing of NS4A-2K-NS4B to release NS4A and NS4B proteins is critical for IMS formation [2,4]. NS4A may initiate IMS formation from different cellular organelles or sub-organelles, depending on the flavivirus species

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