Alternative expression of a chitosanase gene produces two different proteins in cells infected with Chlorella virus CVK2.

Several Chlorella virus CVK2 proteins had chitosanase and/or chitinase activities. A gene coding for an ORF of 328 amino acids (aa) with a predicted molecular mass of 36,769 Da was cloned from the viral genome. The predicted amino acid sequence of an N'-portion (174 aa) of this gene product (vChta-1) showed 22 to 25% identity with various bacterial chitosanases. A glutathione S-transferase (GST)-vChta-1 fusion protein had strong chitosanase activity. Western blot analysis with antisera raised against the vChta-1 protein identified two proteins of 37 and 65 kDa in virus-infected Chlorella cells beginning at 240 min postinfection and continuing until cell lysis. The larger protein was packaged in the virion, while the smaller one remained in the cell lysate. Both chitosanase proteins were produced from the single gene, vChta-1, by a mechanism of alternative gene expression.

Proteolytic processing of Chlorella virus CVK2 capsid proteins.

Chlorella virus CVK2 particles contain several capsid proteins that are selectively released by treatment with 4 M urea. Seven of them (Vp52, Vp45, Vp41, Vp25, Vp20.5, Vp20, and Vp16.2) have been identified based on amino acid sequences at the N-terminus. Comparison of the amino acid sequences with the corresponding ORFs deduced from the nucleotide sequence of PBCV-1, the prototype member of Phycodnaviridae, revealed that the CVK2 proteins were processed at the N-terminal region by two different proteolytic activities.

Characterization of DNA-binding proteins and protein kinase activities in Chlorella virus CVK2.

Several capsid proteins were selectively released from the viral core of Chlorella virus CVK2 by treatment with 4M urea. Among the viral core proteins, seven species (Vp154, Vp73, Vp63, Vp52, Vp48, Vp42, and Vp25) were shown to have DNA-binding activities by Southwestern blot analysis. Except for Vp154 and Vp25, these DNA-binding proteins showed a specific affinity for the viral genomic DNA. The viral core also contained three proteins with protein kinase activity (Vp73, Vp60, and Vp37); Vp73 seemed to have both DNA-binding and protein kinase activities. Antisera raised against Vp73 were used to screen a lambda-CVK2 expression library for the gene encoding Vp73. Three different clones (Vp73-3, Vp73-29, and Vp73-42) were obtained and analyzed. ORFs found in these clones all contained characteristic proline-rich motifs. The Vp73-42 ORF showed a strong similarity with histone H1 of various organisms and the Vp73-29 ORF contained two regions with leucine-zipper motifs. All three genes were expressed late in infection.

Large deletions in the genome of Chlorella virus CVK1.

Large (30-45 kbp) deletions were induced in the Chlorella virus CVK1 genome by UV irradiation. Restriction endonuclease maps of the mutant genomes showed that these deletions occurred in a region located from 1.5 kbp to 47 kbp from the left DNA end. The nucleotide sequences determined around the deletion boundaries indicate that the deletion process took place by both homologous and nonhomologous recombinations. In one case, the recombination site was within a region of about 600 bp, consisting of 40 tandem repetitions of a 15-bp sequence element. The deleted region may contain several multigene families. Northern blot analyses with probes including the genes for translational elongation factor 3 and DNA polymerase showed no discernible aberrancy in the gene expression patterns in the mutants. However, at least two protein bands were missing from the mutant virions.

Horizontal transmission of group-I ribozymes: viruses as a carrier of the introns.

The nucleotide sequences of two self-splicing group I introns found in viruses that infect the eukaryotic green alga Chlorella were compared with those of 19 nuclear group I introns of 11 green algae, 1 red alga, 2 yeasts and 1 protist. The similarity ranging from 69.5% to 89.0% in any comparison strongly suggested a close relatedness of these introns. Since this kind of viruses infect algal cells residing in protists such as Paramecium, they may serve as a mediator to spread group I introns horizontally among organisms of different phylogenetic kingdoms.

Self-splicing group I introns in eukaryotic viruses.

We report the occurrence of self-splicing group I introns in viruses that infect the eukaryotic green alga Chlorella. The introns contained all the conserved features of primary sequence and secondary structure previously described for the group IB introns. The Chlorella viral introns (approximately 400 nt) self-spliced in vitro, yielding the typical group I intron splicing intermediates and products. Contrasting to eukaryotic nuclear group I introns, all of which are located in the rRNA genes, these introns were inserted in genes encoding proteins. In one case, the exons encoded a protein showing significant homology to the eukaryotic transcription factor SII (TFIIS), which may be important for viral gene expression. In another case, the gene for the open reading frame (ORF) of a 14.2 kDa polypeptide with unknown functions contained the intron. Scattered distribution of these introns among the viral species and their structural similarity to the group I introns of algae and protists indicated horizontal intron transmission. These eukaryotic viral introns offer an opportunity to understand how group I introns reach organisms of different phylogenetic kingdoms.

Expression of the gene encoding a translational elongation factor 3 homolog of Chlorella virus CVK2.

A gene encoding a putative translational elongation factor 3 (EF-3) on the genome of Chlorella virus CVK2 has been cloned and sequenced. It encodes for a predicted polypeptide of 1120 amino acids (aa) with a molecular mass of 127 kDa. The overall amino acid sequence of CVK2 EF-3 (vEF-3) showed 36.1% identity and 83.6% similarity to that of Saccharomyces cerevisiae EF-3. Functional domains including two sets of ATP-binding motifs were extremely well conserved between vEF-3 and yeast EF-3; 63.6% identity and 92.4% similarity in total 330-aa portions. Northern blot analysis indicated that the vEF-3 gene was transcribed in the host cells early, at 20 min postinfection (p.i.), as well as late, 3-4 hr p.i. Western blot analyses with anti-vEF-3 antibody detected the 120 kDa vEF-3 protein product after 40 min p.i. It was present until the final stages of infection but absent in the virion. The vEF-3 gene was highly conserved among all Chlorella viruses isolated in Japan.