Generation and characterization of Autographa californica nucleopolyhedrovirus mutants defective in pcna gene homologue

Two Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) mutants defective in conserved pcna 1 and pcna 2 sequences and in substantial portion of the C-terminal region of pcna open reading frame were generated. Biological characterization revealed that these pcna-defective mutants were both viable in the cell culture and the kinetics of viral DNA replication, viral structural protein synthesis, budded virion yield and polyhedrin production in the pcna-defective AcMNPV-infected cells were comparable to those in the cells infected with wild-type (wt) AcMNPV. Localization experiments showed that the amount of cellular PCNA in the nuclear fraction was significantly higher in the cells infected with pcna-defective AcMNPVs than in the cells infected with wt AcMNPV from 8 to 16 h postinfection. These results indicate that AcMNPV PCNA is not essential not only for viral DNA replication but also for the yield of progeny virions and suggest that cellular PCNA functions in the viral DNA replication in the cells infected with pcna-defective AcMNPV mutants. On the basis of our data from cellular PCNA localization experiments and the fact that pcna-missing NPVs are able to multiply to high titers in the cell culture, it is also suggested that virus-encoded PCNA is crucial for viral DNA replication only in the cells that provide an environment unfavorable for DNA replication.