Virion-Associated Restriction Endonucleases of Chloroviruses

Abstract

Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is Paramecium bursaria chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.Chloroviruses are large, icosahedral, plaque-forming, genetically diverse but morphologically similar, linear, doublestranded-DNA (dsDNA) viruses (315 to 380 kb) that belong to the family Phycodnaviridae and the genus Chlorovirus (8, 22). The prototype chlorovirus, Paramecium bursaria chlorella virus 1 (PBCV-1), has a 331-kb genome that contains 366 proteinencoding genes and a polycistronic tRNA gene encoding 11 tRNAs. The chloroviruses infect certain freshwater, unicellular, chlorella-like green algae (e.g., Chlorella strain NC64A or Chlorella strain Pbi), which normally exist as endosymbionts in the protozoan Paramecium bursaria. Viruses that infect Chlorella NC64A (NC64A viruses) are serologically different from viruses that infect Chlorella Pbi (Pbi viruses). NC64A viruses neither infect nor attach to Chlorella Pbi, and vice versa (16).One distinctive feature of the chloroviruses is that their genomes contain methylated nucleotides: 5-methylcytosine (m5C) levels range from 0.1% to 47% of the total cytosines, and N 6 -methyladenine (m6A) levels vary from 0% to 37% of the total adenines (25, 26). The discovery that m5C...

Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystisturfacea Chlorella Virus 1

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Chlorovirus-Mediated Membrane Depolarization ofChlorellaAlters Secondary Active Transport of Solutes

“…The growth of the host Chlorella strain NC64A on bold basal medium (BBM) modified by the addition of 0.5% sucrose and 0.1% peptone (MBBM) and the production and purification of viruses PBCV-1 and NY-2A have been previously described elsewhere (1,43).…”

The Chlorella variabilis NC64A Genome Reveals Adaptation to Photosymbiosis, Coevolution with Viruses, and Cryptic Sex

“…Pulse field gel electrophoresis studies were performed according to Agarkova et al (2006). Chromosomal DNAs were separated in a CHEF-DR II (Bio-Rad) unit in a 0.8% agarose gel.…”