Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. cpCh1, unusually for a virus, encodes a putative PCNA. Natrialba magadii, the only known host of cpCh1, also encodes a putative PCNA of high sequence similarity, differing in the presence of an alternate GUG start codon 5' of the AUG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behaviour, viral protein levels, and virus titer. Deletion of cpCh1 ORF59, encoding PCNAoCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. Complementation and overexpression with WT and mutant variants of ORF59 revealed that modification of the GTG start codon to ATG changes the life cycle in terms of production of progeny virus particles. The different variants have a broad range of variable effects on each of the phenotypes observed. Experiments with a halophilic betagalactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of cpCh1 modulated by the 5'GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the life cycle of the virus cpCh1. The 5' region upstream of the primary AUG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship.

Virus Phi-Ch1; Nucleotide-Sequence; Natrialba-Magadii; Dna; System; Phage; Transformation; Construction; Replication; Expression

application/pdf

Current Research in Biotechnology

12

4

428

438

Elsevier