A single-amino-acid change in murine norovirus NS1/2 is sufficient for colonic tropism and persistence

Abstract

Human norovirus (HuNoV) is the major cause of acute nonbacterial gastroenteritis worldwide but has no clear animal reservoir. HuNoV can persist after the resolution of symptoms, and this persistence may be essential for viral maintenance within the population. Many strains of the related murine norovirus (MNV) also persist, providing a tractable animal model for studying norovirus (NoV) persistence. We have used recombinant cDNA clones of representative persistent (CR6) and nonpersistent (CW3) strains to identify a domain within the nonstructural gene NS1/2 that is necessary and sufficient for persistence. Furthermore, we found that a single change of aspartic acid to glutamic acid in CW3 NS1/2 was sufficient for persistence. This same conservative change also caused increased growth of CW3 in the proximal colon, which we found to be a major tissue reservoir of MNV persistence, suggesting that NS1/2 determines viral tropism that is necessary for persistence. These findings represent the first identified function for NoV NS1/2 during infection and establish a novel model system for the study of enteric viral persistence.

Fig 1

CW3 and CR6 differ in quantity and persistence of viral shedding in stool. Stool was collected at the indicated days postinfection, and the genome copy number was determined by quantitative PCR. Data show the mean shedding of at least four mice per time point.

Fig 2

5′ Domain of NS1/2 is necessary and sufficient for persistence. (A) Diagram of the MNV genome and the genomes of MNV chimeras used in panels B to E to map the persistence trait. (B to E) Mice were inoculated with the indicated MNV strains. Stool was collected 35 days postinfection, and MNV genomes were detected by quantitative PCR. (B) Comparison of VP1-substituted strains. (C) A screen of additional single-gene substitutions. (D) Confirmation of CW3-NS1/2 persistence with three independent viral stocks. (E) Comparison of NS1/2 domain-substituted viruses. Data in panels C and D are from single experiments; those in panels B and E are combined from at least two experiments for each virus. 1/Ct indicates the inverse of the threshold cycle.

Fig 3

Change of aspartic acid to glutamic acid at position 94 of NS1/2 is sufficient for persistence. (A) Alignment of the first 117 amino acids of CW3 with CR6 and six additional persistent strains of MNV (accession numbers DQ223041, DQ223042, DQ223043, EU004672, EU004673, and EU004677 [15, 28]). Conserved amino acid residues are gray, and nonconserved residues are black. The arrow indicates a unique aspartic acid residue at position 94 of CW3. (B) Mice were inoculated with the indicated MNV strains. Stool was collected between 28 and 36 days postinfection, and MNV genomes were detected by quantitative PCR. Data are combined from at least two experiments for each virus.

Fig 4

Viral mutants with altered persistence phenotype have similar in vitro growth. RAW264.7 cells were infected at an MOI of either 0.05 or 5, and viral growth was measured by plaque assay at the indicated times postinfection. Data are combined from three experiments.

Fig 5

NS1/2 and amino acid 94 determine growth in the proximal colon, a major reservoir of persistent MNV. (A to E) Tissues were harvested from MNV-infected mice, and the genome copy number was measured by quantitative PCR. (A) Detection of CR6 in regions of the gastrointestinal tract 35 days postinfection. Data are combined from two experiments for a total of five mice. (B) A kinetic comparison of titers in spleen, proximal colon, and MLN following infection with either CW3 or CR6. Data are combined from three experiments for a total of nine mice per data point. (C and D) Detection of the indicated viruses in tissues at day seven postinfection. Data are combined from at least two experiments for a total of at least eight mice for each data point. CW3 and CR6 data from day seven in panel B are shown in panel C for reference. (E) Detection of CR6-VP1 in the indicated tissues at 3 or 35 days postinfection with 1 × 10⁶ PFU. Data are combined from two experiments for a total of six mice per data point.