Novel Sulfolobus Fuselloviruses with Extensive Genomic Variations

Abstract

Fuselloviruses are among the most widespread and best-characterized archaeal viruses. They exhibit remarkable diversity, as the list of members of this family is rapidly growing. However, it has yet to be shown how a fuselloviral genome may undergo variation at the levels of both single nucleotides and sequence stretches. Here, we report the isolation and characterization of four novel spindle-shaped viruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22), from a hot spring in the Philippines. SSV19 is a member of the genus Alphafusellovirus, whereas SSV20-22 belong to the genus Betafusellovirus The genomes of SSV20-SSV22 are identical except for the presence of two large variable regions, as well as numerous sites of single-nucleotide polymorphisms (SNPs) unevenly distributed throughout the genomes and enriched in certain regions, including the gene encoding the putative end filament protein VP4. We show that coinfection of the host with SSV20 and SSV22 led to the formation of an SSV21-like virus, presumably through homologous recombination. In addition, large numbers of SNPs were identified in DNA sequences retrieved by PCR amplification targeting the SSV20-22 vp4 gene from the original enrichment culture, indicating the enormous diversity of SSV20-22-like viruses in the environment. The high variability of VP4 is consistent with its potential role in host recognition and binding by the virus.IMPORTANCE How a virus survives in the arms race with its host is an intriguing question. In this study, we isolated and characterized four novel fuselloviruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22). Interestingly, SSV20-22 differ primarily in two genomic regions and are apparently convertible through homologous recombination during coinfection. Moreover, sites of single-nucleotide polymorphism (SNP) were identified throughout the genomes of SSV20-22 and, notably, enriched in certain regions, including the gene encoding the putative end filament protein VP4, which is believed to be involved in host recognition and binding by the virus.

Keywords: fuselloviruses; genomic diversity; homologous recombination; single-nucleotide polymorphism; virus-host interaction.

FIG 1

Electron micrographs of SSV19 and SSV20-22. (A and B) SSV19 virions. (C and D) SSV20-22 virions. The end filaments are indicated by arrows.

FIG 2

Genome maps of SSV19-22. A number in each ORF indicates the number of known fuselloviruses that possess a homologue of the ORF. The arrows of the outer ring indicate the putative transcripts, as predicted based on the previous analysis of transcription of the SSV1 genome (16, 49).

FIG 3

Comparison between members of SSV19 to SSV22. (A) Synteny analysis of proteins encoded by SSV19-22 using BLASTP (coverage, ≥50%; identity, ≥30%). (B) Pairwise alignments of genomes of SSV20-22. The vertical lines indicate sites of sequence variation. Red boxes show regions I and II.

FIG 4

Distribution of SNPs and the sites of amino acid variation in VP4 of SSV20-22. (A) Thirteen SNPs in SSV20-22. The vp4 gene of SSV22 is used as the reference sequence, and the position and frequency of each SNP are indicated. (B) Amino acid variation in VP4 from SSV20-22. Amino acid residues at each site and their frequencies in the three viruses are shown. (C) Variable amino acid residues in VP4, as predicted from SNPs in the vp4 gene sequences from the enrichment culture for the isolation of SSV20-22. The bar diagram indicates the site of amino acid variation and the proportion of different amino acid residues at the site.

FIG 5

Protein and lipid analyses of SSV19 and SSV20-22 virions. (A and B) Tricine–SDS-PAGE and glycoprotein staining of the virions of SSV19 (A) and SSV20-22 (B). Purified SSV20-22 virions were subjected to electrophoresis in a 14% tricine–SDS-polyacrylamide gel. The gel was stained with Coomassie brilliant blue (right) or with a glycoprotein staining kit (left). Glycosylated proteins were stained pink. +, positive control (horseradish peroxidase provided by the manufacturer); −, negative control (soybean trypsin inhibitor provided by the manufacturer); M, molecular mass markers. (C) Thin-layer chromatography of lipids extracted from SSV20-22 virions and the host cells. Differences between the lipids from SSV20-22 and those from the host cells are indicated by arrows. (D) Sequence alignment of the C-terminal regions of VP2-like proteins. Homologues of SSV19-VP2 with an e value of ≤10⁻³ were selected and aligned with Muscle. The C-terminal regions of SSV19 and SMF1 VP2 with similar HK repeats are shown in a red box.

FIG 6

Phylogenetic analysis of viral hosts and sequence alignment of the sites of integration. (A) A 16S rRNA gene-based phylogenetic tree of Sulfolobus sp. strain E5-1-F, Sulfolobus sp. strain E11-6, and 17 additional Sulfolobus strains constructed by using the neighbor-joining method (45). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (50). The evolutionary distances are in units of the number of base substitutions per site. (B) Sequence alignment of the sites of integration of SSV19-SSV22 with the 3′ terminal part of the tRNA genes, which contain the potential sites of viral integration, from their hosts.

FIG 7

Detection of the integration of SSV19-22 DNA into their host genomes. (A) A diagram showing the sites of integration for SSV19-22 as well as the positions of primers used in the analysis of viral integration. The tRNA genes, in which integration occurred, and primer pairs flanking the attP sites are indicated. (B) Occupancy of the integration sites on the host chromosomes. DNA extracted from virus-infected host cells was amplified using specific primer pairs for integrated and unoccupied sites. PCR products were loaded onto a 1.0% agarose gel. After electrophoresis, the gel was photographed under UV light. The expected sizes of the PCR products were 784 bp (1F/1R), 1,030 bp (2F/2R), 1,033 bp (1F/2R), 876 bp (9F/9R), 874 bp (10F/10R), 758 bp (9F/10R), 848 bp (11F/11R), 679 bp (12F/12R), 860 bp (11F12R), and 1,828 bp (11F/13R), respectively. M, molecular weight standards.

FIG 8

Infection of Sulfolobus sp. strain E5-1-F by SSV20-22. Strain E5-1 was grown to the exponential phase (OD₆₀₀ of ∼0.2). The cells were infected with SSV20 (A), SSV21 (B), or SSV22 (C) at an MOI of ∼5. The OD₆₀₀ of the culture was monitored. DNAs were extracted from the culture and the harvested cells. The viral copy number was determined by qPCR. Each data point represents an average from three independent measurements with the means and standard deviations indicated.

FIG 9

Coinfection of Sulfolobus sp. strain E5-1-F by SSV20-22. (A) A diagram showing proposed crossovers between the three viruses. Primer pairs designed to detect the HR events are indicated. (B) Agarose gel electrophoresis of the products of recombination between SSV20 to SSV22. Strain E5-1 was grown to the exponential phase (OD₆₀₀ of ∼0.2). The cells were infected or coinfected with different viruses at an MOI of ∼5. Lanes 1 to 8: SSV20, SSV21, SSV22, SSV20 plus SSV21, SSV20 plus SSV22, SSV21 plus SSV22, SSV20 plus SSV21 plus SSV22, and a no virus control. After incubation for 48 h, total DNA was extracted from the infected culture. PCRs were performed with indicated primer pairs using the total DNA as the template. PCR products were subjected to electrophoresis in 1.0% agarose gel. Recombination products are shown in green boxes. (C) Estimation of the frequency of recombination between SSV20 and SSV22. The total DNA extracted from the coinfected culture was serially diluted by 5¹- to 5⁵-fold. PCRs targeting SSV21 and SSV23 were performed using the serially diluted total DNA as the template. Undiluted DNA is indicated by a zero; M, molecular weight standards.