Genetic diversity in chlorella viruses flanking kcv, a gene that encodes a potassium ion channel protein

Abstract

The chlorella virus PBCV-1 encodes a 94-amino acid protein named Kcv that produces a K+-selective and slightly voltage-sensitive conductance when expressed in heterologous systems. As reported herein, (i) Northern analysis of kcv expression in PBCV-1-infected cells revealed a complicated pattern suggesting that the gene might be transcribed as a di- or tri-cistronic mRNA both at early and late times after virus infection. (ii) The protein kinase inhibitors H-89, A3, and staurosporine inhibited PBCV-1 Kcv activity in Xenopus oocytes, suggesting that Kcv activity might be controlled by phosphorylation or dephosphorylation. (iii) The PBCV-1 genomic sequence revealed a gene encoding a putative protein kinase (pkx) adjacent to kcv. These findings prompted us to examine the kcv flanking regions in 16 additional chlorella viruses and transcription in two of these viruses, as well as the effect of the three protein kinase inhibitors on two Kcv homologs in Xenopus oocytes. The results indicate (i) pkx is always located 5' to kcv, but the spacing between the two genes varies from 31 to 1588 nucleotides. More variation occurs in the kcv 3' flanking region of the 16 viruses. (ii) The kcv gene is expressed as a late mono-cistronic mRNA. (iii) Unlike the affect on PBCV-1 Kcv, the three protein kinase inhibitors have little or no effect on the activity of the two Kcv homologs in oocytes. (iv) A comparison of the kcv 5' upstream sequences from the 16 viruses identified a highly conserved 10-nucleotide sequence that is present in the promoter region of all of the viruses.

Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

“…In addition, some probes not only hybridize to mRNAs of the expected size, but they also hybridize to larger mRNAs at other times in the virus replication cycle. These complex patterns occur even with single-stranded DNA probes (e.g., the potassium ion channel CDS, kcv) (13). One difference between the previous Northern analyses and the microarray results is that hybridization to total RNA was used in the Northern analyses and in the research described in the present study the cDNAs were synthesized from poly(A) ϩ RNAs.…”

“…The combined sizes of them with the RNase III CDS are compatible with either a dicistronic or even a tricistronic mRNA. The other example is the potassium ion channel CDS (kcv, A250R) that Northern analysis indicated has a complex expression pattern, at early times A250R is expressed as a large message and at late stages as a monocistronic mRNA (13). However, when the early transcript for this CDS was mapped, the start and the stop sites were within the adjacent CDSs, so A250R is clearly not a polycistronic mRNA.…”

Chlorella viruses prevent multiple infections by depolarizing the host membrane

“…In this sense, Kcvs have a functional analogy with the RexB protein from phage l (Snyder & McWilliams, 1989); RexB protein induces a rapid depolarization of the host cell membrane that is an effective mechanism for achieving l exclusion. Since the chlorella virus Kcvs are apparently essential for virus-triggered membrane depolarization, their genes are probably under high selective pressure (Kang et al, 2004b). The fitness of the virus is probably enhanced by any mutation which modifies the channel in such a way that it increases membrane depolarization and/or decreases sensitivity to potential channel blockers that might exist in the environment.…”

Epigenetic transcriptional repression of cellular genes by a viral SET protein

“…The SET gene was isolated from chlorella viruses MA-1D, NY-2A and NY-2B using an established protocol 33. The DNA bands that hybridized weakly with the PBCV-1 vSET probe were excised from the gel, recovered with a QIAEX II gel extraction kit (Qiagen) and cloned into pGEM-7Zf + .…”