Abstract
ABSTRACTThe 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.
Highlights
The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown
In this report, we define the importance of the VAMP-associated protein A (VAPA) host protein and its interaction with viral nonstructural protein NS1/2 in replication of murine norovirus (MNoV)
Using a variety of approaches, including analysis of the interaction of the proteins in vitro and in cells, we delineated the structural basis for the interaction between VAPA and NS1/2 and used these data to test for the importance of specific amino acids in NS1/2 for viral replication and for the interaction between VAPA and NS1/2
Summary
The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. 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. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for 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. Noroviruses (NoVs) are nonenveloped positive-sense single-stranded RNA viruses that primarily infect the gastrointestinal tract They are a leading cause of gastroenteritis worldwide [1,2,3]. The role of VAPA interactions with NS1/2 during viral replication has not been defined
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