Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein

Investigating the Role of Cholesterol during Murine Norovirus Infection

From the Washington University Senior Honors Thesis Abstracts (WUSHTA), Spring 2015. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Stacy Ross, Editor; Kristin G. Sobotka, Undergraduate Research Coordinator; Jennifer Kohl. Mentor: Herbert Virgi

Investigating the Role of Cholesterol During Murine Norovirus Infection

Human norovirus (HNV) is the leading cause of acute non-bacterial epidemic gastroenteritis worldwide and affects millions of people every year. HNV does not grow in tissue culture so murine norovirus (MNV), which does have a culture system, is a useful model to study the lifecycle and virus-host interactions of noroviruses. Previous research in the Virgin lab has shown that the MNV protein NS1/2 interacts with the host protein vesicle associated membrane protein (VAMP)-associated-protein A (Vapa) which is an integral endoplasmic reticulum (ER) membrane protein that is involved in cholesterol homoeostasis. In addition, we observed that fluorescently tagged NS1/2 and Vapa co-localized when overexpressed in 293T cells. Because of this, we hypothesize that cholesterol is important for MNV infection, and Vapa mediates this relationship. Through multiple measures, MNV infection does not lead to a change in cholesterol levels in RAW 264.7 cells. Surprisingly, depleting cholesterol by growing cells in lipoprotein depleted serum (LPDS) media showed no effect on cell viability or viral titers. However, Vapa-knockout cells showed increased cell viability during infection, further suggesting that Vapa is important during MNV infection