Elucidation of Frog Virus 3 Gene Function and Pathways of Virion Formation

Frog virus 3 is a nucleo-cytoplasmic large DNA virus (NCLDV) that encodes ∼100 putative proteins. Approximately 33% of FV3 open reading frames (ORFs) encode proteins with a known or putative function, whereas the remaining 67% encode proteins with no known function. Interestingly, despite the absence of known homologues in other systems, most of these genes are conserved within other iridoviruses suggesting that they encode proteins important for virus replication and survival. Because the roles these genes play have yet to be elucidated, we sought to address their function through the use of antisense technologies and temperature sensitive (ts) mutants. In an attempt to elucidate gene function we designed antisense morpholino oligonucleotides (asMOs) and short-interfering RNAs (siRNAs) that targeted four FV3 genes: (1) the major capsid protein (MCP), a major structural component of the virus capsid; (2) ICP-18, 18 kDa immediate-early protein that may function as a putative viral regulatory protein; (3) the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha), an enzyme believed to be responsible for the synthesis of late viral genes; and (4) a cytosine DNA-methyltransferase (DMT), a virus-encoded enzyme that methylates viral DNA found within CpG motifs. Using asMOs, we inhibited translation of the MCP, vPol-IIalpha, and ICP-18 messages and found that treatment with MCP and vPol-IIalpha asMOs resulted in marked phenotypic changes along with striking reductions in virus replication suggesting that these two genes were essential for replication in vitro. In contrast, ICP-18 was not required for replication in vitro and was likely not a key regulatory protein. In addition, we targeted the MCP, vPol-IIalpha, and DMT genes using siRNAs, and established that siRNA-mediated gene silencing led to a marked reduction in viral gene expression that was inversely proportional to the multiplicity of infection. In the final study designed to detect viral assembly intermediates, FV3 ts mutants were characterized by transmission electron microscopy. Analysis of mutants representing eight complementation groups identified viral assembly intermediates and determined that FV3 virion assembly was strikingly similar to that of African swine fever virus. Collectively, the above work expanded our understanding of FV3 replicative strategies and demonstrated the usefulness of antisense and is approaches in elucidating events in viral biogenesis.