Algal viruses with distinct intraspecies host specificities include identical intein elements

Abstract

Heterosigma akashiwo virus (HaV) is a large double-stranded DNA virus infecting the single-cell bloom-forming raphidophyte (golden brown alga) H. akashiwo. A molecular phylogenetic sequence analysis of HaV DNA polymerase showed that it forms a sister group with Phycodnaviridae algal viruses. All 10 examined HaV strains, which had distinct intraspecies host specificities, included an intein (protein intron) in their DNA polymerase genes. The 232-amino-acid inteins differed from each other by no more than a single nucleotide change. All inteins were present at the same conserved position, coding for an active-site motif, which also includes inteins in mimivirus (a very large double-stranded DNA virus of amoebae) and in several archaeal DNA polymerase genes. The HaV intein is closely related to the mimivirus intein, and both are apparently monophyletic to the archaeal inteins. These observations suggest the occurrence of horizontal transfers of inteins between viruses of different families and between archaea and viruses and reveal that viruses might be reservoirs and intermediates in horizontal transmissions of inteins. The homing endonuclease domain of the HaV intein alleles is mostly deleted. The mechanism keeping their sequences basically identical in HaV strains specific for different hosts is yet unknown. One possibility is that rapid and local changes in the HaV genome change its host specificity. This is the first report of inteins found in viruses infecting eukaryotic algae.

FIG. 1.

Genome sizes of viruses, as determined by pulsed-field gel electrophoresis. Lane M, molecular weight size standard (lambda concatemers); lane HaV01, Heterosigma akashiwo virus 01 (28); lane HcV10, Heterocapsa circularisquama virus 10 (46).

FIG. 2.

Partial nucleotide sequence of the HaV01 DNA polymerase gene and the 200 bases preceding the putative start codon (boxed ATG). Potential TATA sequences are underlined. The region 5′ of the coding region is numbered relative to it. The coding region is numbered by codons.

FIG. 3.

Amino acid alignment of B-family DNA polymerase genes of a Heterosigma akashiwo virus (HaV01), Chlorella viruses (PBCV-1 and NY-2A), a Feldmania species virus (FsV), and an Ectocaupus silliculosus virus (EsV-1). Highly conserved regions are shaded. The intein region of HaV01 is shown as a black square, and its amino acid sequence is shown in Fig. 5. Arrows indicate the relative positions of the primers used for the present experiments (see Materials and Methods).

FIG. 4.

Phylogenetic neighbor-joining trees calculated from confidently aligned regions of the amino acid sequences of viral DNA polymerases. (A) Relationship of HaV to other dsDNA viruses infecting algae. The phylogenetic tree was computed from 198 amino acid positions in the DNA polymerase region determined for many alga-infecting virus species (shown in bold). Nodes with bootstrap values below 70% were collapsed. (B) Relationship of HaV to diverse dsDNA viruses. The phylogenetic tree was computed from 216 amino acid positions from fully sequenced DNA polymerases of dsDNA viruses. Nodes with bootstrap values below 50% were collapsed. Both trees were rooted by using human DNA polymerase delta. Numbers at thenodes indicate bootstrap values. The databank accession numbers of the sequences are provided in Materials and Methods. Bar, 0.1 fixed mutations per amino acid position. Corresponding topologies were also found by using maximum-likelihood methods.

FIG. 5.

Amino acid sequence of the HaV DNA polymerase intein and its immediate flanks. Extein flanks are boxed and numbered by their positions in the precursor DNA polymerase protein. The intein is numbered relative to its first residue (Ser647 of the precursor). Sites corresponding to intein motifs (38) are underlined and named. The intein Met34-Thr change due to the one single nucleotide polymorphism in 2 of the 10 HaV strains is marked above that position.

FIG. 6.

Phylogenetic neighbor-joining tree calculated from confidently aligned regions of the amino acid sequences of type B DNA polymerase intein alleles. The tree was rooted by using the Drosophila hedgehog HINT domain. A total of 75 amino acid sites were used to construct the tree. Numbers at the nodes indicate bootstrap values from 1,000 samples. Nodes with bootstrap values below 70% were collapsed. The DNA polymerase integration point (a, b, or c) is shown in parentheses for each intein. The GenBank accession numbers of all sequences are provided in Materials and Methods. Bar, 0.1 fixed mutations per amino acid position. Corresponding topologies were also found by using maximum-likelihood methods.

FIG. 7.

Alignment of intein amino acid sequences of mimivirus and HaV01. Note the high degree of similarity in the distal regions and the deletion of 131 amino acids from the central homing-endonuclease domain of the HaV intein.

FIG. 8.

RT-PCR analysis demonstrating constitutive transcription of the DNA polymerase gene within virus-infected cells. HaV01 DNA polymerase fragments amplified by RT-PCR from the mRNAs of a HaV01-inoculated Heterosigma akashiwo H93616 culture by use of the DPC-F and DPC-R primers were electrophoresed in a 1% agarose gel. Lanes 1 to 8 show the RT-PCR fragments amplified from cDNAs at 0, 1, 3, 5, 7, 9, 12, and 24 h postinoculation, respectively, and lane 9 shows the PCR fragments amplified from HaV01 DNA (positive control).