Adaptation of Puumala hantavirus to cell culture is associated with point mutations in the coding region of the L segment and in the noncoding regions of the S segment

Abstract

We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells. With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed. The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053. Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding. Nucleotide substitutions observed in individual L cDNA clones, most of them A-->G and U-->C transitions, suggested that the population of L RNA molecules is represented by quasispecies. The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies. Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions. However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.

FIG. 1.

Schematic structure of hantaviral L segment. Black arrows indicate the locations of the differences found between the wild-type (WT) and Vero E6-I variants in the nucleotide and amino acid sequences. The three black lines indicate three fragments of the L segment that were amplified and sequenced separately, and numbers mark the position of each fragment in the nucleotide sequence. The gray bar below represents the L protein and its conserved regions, with numbers showing the location of each region in the amino acid sequence. The black stripe in the C terminus shows the site where the Ser2053Phe mutation resides. Alignment of hantaviral L protein sequences for the region surrounding the amino acid replacement Ser2053Phe is shown below, and nonhomologous amino acid substitutions observed in this region are shown in bold. Hantaviral sequences used for the alignment and retrieved from the GenBank include Hantaan virus strain 76-118 (X55901) (38), Seoul virus strain 80-39 (X56492) (2), Sin Nombre virus strain NM H10 (L37901) (4), Puumala virus strains CG1820 (M63194) (40) and Sotkamo (Z66548) (30), Tula virus strain Tula/Moravia/5302v (AJ005637) (23), Dobrava virus strain Ano-Poroia/9Af/99 (AJ410617) (29), and Saaremaa virus strain Saaremaa/160V (AJ410618) (29).

FIG. 2.

Folding of L viral RNA of wild-type (A and C) and Vero E6 cell-adapted (B and D) variants of Puumala virus. The positions of the nucleotide differences between the wild-type and Vero E6-I variants are marked by arrows. Note that the C1053U and C6194U substitutions observed in cDNA clones correspond to substitutions G5498A and G357A in the L viral RNA, respectively.

FIG. 2.

Folding of L viral RNA of wild-type (A and C) and Vero E6 cell-adapted (B and D) variants of Puumala virus. The positions of the nucleotide differences between the wild-type and Vero E6-I variants are marked by arrows. Note that the C1053U and C6194U substitutions observed in cDNA clones correspond to substitutions G5498A and G357A in the L viral RNA, respectively.

FIG. 2.

Folding of L viral RNA of wild-type (A and C) and Vero E6 cell-adapted (B and D) variants of Puumala virus. The positions of the nucleotide differences between the wild-type and Vero E6-I variants are marked by arrows. Note that the C1053U and C6194U substitutions observed in cDNA clones correspond to substitutions G5498A and G357A in the L viral RNA, respectively.

FIG. 2.

Folding of L viral RNA of wild-type (A and C) and Vero E6 cell-adapted (B and D) variants of Puumala virus. The positions of the nucleotide differences between the wild-type and Vero E6-I variants are marked by arrows. Note that the C1053U and C6194U substitutions observed in cDNA clones correspond to substitutions G5498A and G357A in the L viral RNA, respectively.

FIG. 3.

Mutant spectra of S segment sequences of the Vero E6-I variant. The numbers in parentheses indicate the number of clones with a particular sequence. Solid arrowheads mark mutations in the NCRs identified earlier (24). Open arrowheads mark mutations described in this study. wt, wild type.