Murine norovirus protein NS1/2 aspartate to glutamate mutation, sufficient for persistence, reorients side chain of surface exposed tryptophan within a novel structured domain

Abstract

Compact viral genomes such as those found in noroviruses, which cause significant enteric disease in humans, often encode only a few proteins, but affect a wide range of processes in their hosts and ensure efficient propagation of the virus. Both human and mouse noroviruses persistently replicate and are shed in stool, a highly effective strategy for spreading between hosts. For mouse norovirus (MNV), the presence of a glutamate rather than an aspartate at position 94 of the NS1/2 protein was previously shown to be essential for persistent replication and shedding. Here, we analyze these critical sequences of NS1/2 at the structural level. Using solution NMR methods we determined folded NS1/2 domain structures from a nonpersistent murine norovirus strain CW3, a persistent strain CR6, and a persistent mutant strain CW3D94E. We found an unstructured PEST-like domain followed by a novel folded domain in the N-terminus of NS1/2. All three forms of the domain are stable and monomeric in solution. Residue 94, critical for determining persistence, is located in a reverse turn following an α-helix in the folded domain. The longer sidechain of glutamate, but not aspartate, allows interaction with the indole group of the nearby tryptophan, reshaping the surface of the domain. The discrimination between glutamyl and aspartyl residue is imposed by the stable tertiary conformation. These structural requirements correlate with the in vivo function of NS1/2 in persistence, a key element of norovirus biology and infection.

Advances in Norovirus Biology

“…The nonstructural protein NS1/2 - and specifically aspartic acid to glutamic acid change at position 94 of NS1/2 -enhances colonic replication early after infection and allows establishment of life-long persistent fecal shedding (Figure 1) (Nice et al, 2013). This single amino acid change is accompanied by a significant structural rearrangement of the N-terminal domain of the protein (Borin et al, 2013). Unanswered questions relating to MuNoV persistence include elucidating the mechanism by which NS1/2 regulates colonic tropism and persistence; and determining whether the VF1 protein, previously shown to antagonize the innate immune response in MuNoV-1 (McFadden et al, 2011), contributes to viral persistence.…”

Recent advances in understanding norovirus pathogenesis

“…For example, a single glutamic acid at position 94 in the 5′ domain of the nonstructural protein NS1/2 is sufficient to enhance MNV‐1 replication and persistence in the colon. This residue, D94, which is conserved in all persistent murine norovirus strains identified to date [Nice et al, ], regulates the tertiary structure of the protein [Borin et al, ]. Notably, the human norovirus NS1/2 protein colocalizes with Golgi markers and impairs host protein secretory pathways in transfection experiments [Ettayebi and Hardy, ; Fernandez‐Vega et al, ].…”

Interferon-λ cures persistent murine norovirus infection in the absence of adaptive immunity

“…S2). The capacity of strain CW3 to infect systemic organs maps to the protruding domain of the viral capsid protein (11, 22), whereas a single coding change (Asp 94 →Glu 94 , hereafter D94E) in the NS1–2 protein confers the capacity for enteric persistence upon CW3 (11, 23). In chimeric viruses, the presence of the entire CW3 capsid gene or the protruding domain of the CW3 capsid gene correlated with IFN-β and IFN-λ induction (fig.…”