ABSTRACT
Four spontaneously derived, antigenic variants of chlorella virus PBCV-1 contained 27- to 37-kb deletions in the left end of the 330-kb genome. Two of the mutants, which were serologically identical, had deletions that began from map position 4.9 or 16 and ended at position 42.2 kb. In total, the two deleted regions encoded 28 putative functional open reading frames (ORFs); these deletions probably arose from homologous recombination. The other two mutants, which were serologically identical but distinct from the first two mutants, lacked the entire left terminal 37 kb of the PBCV-1 genome, including an identical 2.2-kb inverted terminal repeat region present at both ends of the wild-type genome. The deleted left end region was replaced by the transposition of an inverted 7.7- or 18.5-kb copy of the right end of the PBCV-1 genome. The region deleted in these two viruses encoded 26 single-copy ORFs, of which 23 were common to those deleted in the first two mutant viruses. The junctions of the deletions/transpositions probably arose from nonhomologous recombination. Taken together, the results indicate that 40.1 kb of single-copy DNA encoding 31 ORFs at the left end of the genome are unnecessary for PBCV-1 replication in Chlorella strain NC64A in the laboratory. The results also indicate that the size of the inverted terminal repeat region in this virus can be highly variable and that the PBCV-1 DNA packaging process tolerates large changes in genome size.
Subject(s)
Antigenic Variation/genetics , Chlorophyta/virology , Gene Deletion , Phycodnaviridae/genetics , Base Sequence , Binding Sites , DNA, Viral/genetics , Molecular Sequence Data , Phycodnaviridae/immunology , Phycodnaviridae/isolation & purification , Recombination, Genetic , Restriction Mapping , Viral Proteins/geneticsABSTRACT
A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells. R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences. However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation. The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined. These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease. R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.
Subject(s)
Chlorella/enzymology , Deoxyribonucleases, Type II Site-Specific/genetics , Genes, Viral/genetics , Methyltransferases/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Viral Proteins/genetics , Viral Structural Proteins/genetics , Amino Acid Sequence , Base Sequence , Chlorella/genetics , Cloning, Molecular , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Escherichia coli/genetics , Genomic Library , Methyltransferases/chemistry , Methyltransferases/metabolism , Molecular Sequence Data , Site-Specific DNA-Methyltransferase (Adenine-Specific)/chemistry , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Viruses/enzymology , Viruses/geneticsABSTRACT
The majority, if not the entire life cycle, of the large dsDNA-containing algal virus PBCV-1 occurs in localized regions in the cytoplasm. Thirteen drugs that disrupt the cytoskeleton had no effect on PBCV-1 replication at concentrations which inhibited host growth. Therefore, host cytoskeletal elements do not appear to be important in PBCV-1 morphogenesis.
