Evolution of human calicivirus RNA in vivo: accumulation of mutations in the protruding P2 domain of the capsid leads to structural changes and possibly a new phenotype

Abstract

In the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype. FUT2 gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor(+), a finding in accordance with the hypothesis of FUT2 gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with the FUT2 gene characterization, may lead to a better understanding of calicivirus pathogenesis.

FIG. 1.

Chronicexcretion of norovirus. Monthly distribution of norovirus positive specimens.

FIG. 2.

Amino acid sequence and substitutions in ORF2 of 12 isolates. Underlined sequence refer to the protruding P2 subdomain of the capsid.

FIG. 2.

Amino acid sequence and substitutions in ORF2 of 12 isolates. Underlined sequence refer to the protruding P2 subdomain of the capsid.

FIG. 3.

Sporadic and permanent amino acid microheterogenicities in ORF2.

FIG. 4.

Schematic illustration of norovirus capsid protein. The numbers refer to amino acids. The S domain represents the most conserved region of the sequence. P refers to the protruding domain, with P2 being the most variable. The numbers on top of the line refer to 11 accumulated amino acid changes, with 8 of 11 (boxed) accumulating within the P2 domain.

FIG. 5.

Schematic representations of secondary structure predictions of the deduced proteins from isolates showing the disappearance of the helix structure in the P2 region. Both panels depict the amino-terminal region (amino acids 281 to 548) of the proteins, including the variable P2 region. Boxed regions indicate the disappearance of a helix structure within the P2 region.

FIG. 6.

FUT2 gene polymorphism characterization. The PCR-SSP pattern from saliva shows that the patient is Secretor⁺, since the pattern indicates heterozygous mutation at nucleotide 428 and no mutations at positions 385 and 571. For allele characterization the following primers were used: allele 385 (primers 385wt [wt] and 385A→T [mt]), allele 428 (primers 428wt [wt] and 428G→A [mt]), and allele 571 (primers 571wt [wt] and 571C→T [mt]), as indicated in the figure. M, Molecular weight markers (φX174). The arrow indicates an internal PCR gene control (human growth hormone) of 428 bp. The right side of the panel (homozygot⁴²⁸) shows a non-Secretor control being homozygously mutated at nucleotide 428 of FUT2. PCR products were separated on agarose gels (1%) and stained with ethidium bromide.