A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization

Abstract

Noroviruses cause epidemic outbreaks of acute viral gastroenteritis worldwide, and the number of reported outbreaks is increasing. Human norovirus strains do not grow in cell culture. However, murine norovirus (MNV) replicates in the RAW 264.7 macrophage cell line and thus provides a tractable model to investigate norovirus interactions with host cells. Epitopes recognized by monoclonal antibodies (MAbs) against the human norovirus strains Norwalk virus and Snow Mountain virus (SMV) identified regions in the P domain of major capsid protein VP1 important for interactions with putative cellular receptors. To determine if there was a relationship between domains of MNV VP1 and VP1 of human norovirus strains involved in cell binding, epitope mapping by phage display was performed with an MNV-1-neutralizing MAb, A6.2.1. A consensus peptide, GWWEDHGQL, was derived from 20 third-round phage clones. A synthetic peptide containing this sequence and constrained through a disulfide linkage reacted strongly with the A6.2.1 MAb, whereas the linear sequence did not. Four residues in the A6.2.1-selected peptide, G327, G333, Q334, and L335, aligned with amino acid residues in the P2 domain of MNV-1 VP1. This sequence is immediately adjacent to the epitope recognized by anti-SMV MAb 61.21. Neutralization escape mutants selected with MAb A6.2.1 contained a leucine-to-phenylalanine substitution at position 386 in the P2 domain. The predicted location of these residues on VP1 suggests that the phage peptide and the mutation in the neutralization-resistant viruses may be in close proximity to each other and to residues reported to be important for carbohydrate binding to VP1 of human norovirus strains.

FIG. 1.

Nonapeptides selected from the J404 phage library by anti-MNV-1 MAb. Shown are peptide sequences obtained from 20 third-round phage clones and the consensus sequence derived from the clones. Shaded residues indicate the most conserved residues, and the derived consensus sequence is shown below the alignment.

FIG. 2.

MAb A6.2.1 reacts with constrained but not linear consensus peptide by ELISA. (A) One hundred micrograms of constrained (black bars) or Tris(2-carboxyethyl)-phosphine-reduced (vertical stippled bars) peptide containing the consensus phage epitope was adsorbed onto microtiter plates in serial twofold dilutions and then probed with MAb A6.2.1 as described in Materials and Methods. Gray bars are wells containing serial dilutions of MNV-1. (B) Thirty-three micrograms of constrained phage epitope probed with A6.2.1 (black bar) in the ELISA format described in Materials and Methods. Irrelevant isotype control MAb 61.21 (vertical stippled bar), 33 μg of a constrained irrelevant peptide probed with A6.2.1 (diagonal stippled bar), MNV-1 probed with A6.2.1 (spotted bar), and primary and secondary antibody only (horizontal stippled bar) are shown.

FIG. 3.

MNV-1 passaged three times in the presence of MAb A6.2.1 is resistant to neutralization. (A) Titers of wild-type MNV-1 in the absence (left) and presence (right) of 20 μg of A6.2.1. (B) Mutant MNV-1 (MNV-1m) titers after three rounds of amplification in the presence of 20 μg A6.2.1 MAb. Plaque assays shown are in the absence (left) and presence (right) of the MAb.

FIG. 4.

Sequence alignments of the P2 domain of VP1 of MNV-1 and select human norovirus strains. Amino acid sequence alignment incorporating the variable P2 domain of VP1 of human noroviruses NV, SMV, VA387, and MNV-1 is shown. Conserved residues are indicated with asterisks. Red lines below the alignment indicate residues in MNV-1 VP1 defined by the phage peptide isolated with MAb A6.2.1 (G327, G333, Q334, and L335). The red dot below the alignment marks the L386F mutation in the escape mutants. Blue lines above the alignment mark SMV and putative NV epitopes described previously (24). Black dots mark residues suggested to be important for carbohydrate binding of gII human norovirus strain VA387 (5).

FIG. 5.

Putative location of MNV-1-neutralizing epitope. Shown is the VP1 monomer illustrating the location of amino acid residues of NV that align with the MNV-1 peptide selected from the phage library (red) and the escape mutant (yellow). The residues in yellow are NV amino acids W375, S377, and P378. The residues in red are D327, W328, and H329. The structure was drawn and analyzed with NCBI CN3D4.1.