Frog virus 3 is the best characterized species within the genus Ranavirus, family Iridoviridae. FV3's large (∼105 kbp) dsDNA genome encodes 98 putative open reading frames (ORFs) that are expressed in a coordinated fashion leading to the sequential appearance of immediate early (IE), delayed early (DE) and late (L) viral transcripts. As a step toward elucidating molecular events in FV3 replication, we sought to identify the temporal class of viral messages. To accomplish this objective an oligonucleotide microarray containing 70-mer probes corresponding to each of the 98 FV3 ORFs was designed and used to examine viral gene expression. Viral transcription was initially monitored during the course of a productive replication cycle at 2, 4 and 9 hours after infection. To confirm results of the time course assay, viral gene expression was also monitored in the presence of cycloheximide (CHX), which limits expression to only IE genes, and following infection with a temperature sensitive (ts) mutant which at non-permissive temperatures is defective in viral DNA synthesis and blocked in late gene expression. Subsequently, microarray analyses were validated by RT-PCR and qRT-PCR. Using these approaches we identified 33 IE genes, 22 DE genes and 36 L viral genes. The temporal class of the 7 remaining genes could not be determined. Comparison of putative protein function with temporal class indicated that, in general, genes encoding putative regulatory factors, or proteins that played a part in nucleic acid metabolism and immune evasion, were classified as IE and DE genes, whereas those involved in DNA packaging and virion assembly were considered L genes. Information on temporal class will provide the basis for determining whether members of the same temporal class contain common upstream regulatory regions and perhaps allow us to identify virion-associated and virus-induced proteins that control viral gene expression.

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

No abstract

Viruses of the genus Ranavirus, family Iridoviridae, are a group of large, icosahedral dsDNA viruses which have emerged as new pathogens of poikilothermic vertebrates. To confront their spread, it is vital to understand the mechanisms which control virus replication, host‐range, and pathogenesis. Therefore, Frog virus 3 (FV3), the ranavirus type species, was chosen as the model system. The FV3 genome has been completely sequenced and ~25% of the genes have recognized functions whereas the remaining ~75% have no known function. An antisense approach using RNA interference (RNAi) and antisense morpholinos (asMOs) was chosen to identify gene function and determine the requirements for replication in vitro and pathogenesis. The major capsid protein (MCP), the viral homologue of RNA Polymerase II (vPol‐IIα}), myristolated membrane protein (MMP), ICP‐46 (46K), and the cytosine DNA‐methyltransferase (DMT) were targeted using gene specific siRNAs or asMOs. Reductions of >80% in gene expression and >90% in viral titers were observed along with reductions in cytopathic effect. Transmission electron microscopy revealed reductions in virion assembly and production of atypical elements. These results support antisense techniques as a way of identifying and elucidating the function of genes involved in viral replication and pathogenesis.