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.

FIG. 1.

Kinetics of DNA degradation of Chlorella NC64A cells upon infection with PBCV-1 virus. (A) No CHX; (B) 5 μg/ml CHX. Times (min p.i.) are listed in the figures. Electrophoresis conditions were as follows: 100 V with pulses of 250 to 900 s for 60 h at 14°C in 1× Tris-acetate-EDTA (TAE) buffer.

FIG. 2.

Effect of UV treatment on PBCV-1 virus. (A) Effect of UV radiation dose on PBCV-1 virus replication. (B) UV radiation dose and ability of PBCV-1 virus to cause Chlorella NC64A chromatin degradation. Lanes: 1, mock-infected NC64A cells at 30 min p.i.; 2, PBCV-1 virus DNA; 3, NC64A cells infected with PBCV-1 virus at 30 min p.i.; 4 to 13, NC64A cells at 30 min p.i. with UV-treated PBCV-1 virus receiving the following doses (Joule/m²): 4, 0; 5, 0.5 × 10³; 6, 1.2 × 10³; 7, 2.5 × 10³; 8, 4 × 10³; 9, 5 × 10³; 10, 7.5 × 10³; 11, 10 × 10³; 12, 15 × 10³; and 13, 20 × 10³. Electrophoresis conditions were as follows: 100 V with pulses of 250 to 900 s for 60 h at 14°C in 1× TAE buffer.

FIG. 3.

Nuclease activities of virion-associated proteins. (A and D) SDS-DNA-PAGE gel assay; (B and C) REase activities of virion-associated proteins. (A) SDS-DNA-PAGE gel assay of PBCV-1 virion-associated proteins. (B) Heparin-Sepharose column chromatography of enzyme extracts prepared from PBCV-1 virions eluted with potassium acetate. The fractions were assayed on DNA amplicon B. (C) R.CviAII activity of disrupted PBCV-1 supernatant fraction (S-15) assayed on DNA amplicon C. DNA fragments were electrophoresed in a 1% agarose gel in 0.5× TBE buffer at 5.5 V/cm. (D) SDS-DNA-PAGE assay of virion-associated proteins from different chlorella viruses.

FIG. 4.

Kinetics of DNA degradation of Chlorella Pbi cells upon infection with MT325 virus. Electrophoresis conditions were as follows: 100 V with pulses of 250 to 900 s for 60 h at 14°C in 1× TAE buffer.

FIG. 5.

Digestion of viral and Chlorella DNAs with virion-associated nucleases. (A) PBCV-1 and Chlorella NC64A DNAs treated with MboI (an isoschizomer of R.CviAI), R.CviAII, and MboI plus R.CviAII. (B) PBCV-1 DNA in agarose plugs (1 μg/plug) treated with different concentrations of sonicated PBCV-1 virus extract. (C) Digestion of viral DNAs in agarose plugs (1 μg/plug) with sonicated virus extracts. c, untreated viral DNA (control); v, viral DNA treated with sonicated virus (150 μg/treatment) in the presence of 10 mM Mg²⁺. Electrophoresis conditions were as follows: 200 V with pulses of 5 to 30 s for 25 h at 14°C in 0.5× TBE buffer.