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.

Frog virus 3 is the best characterized species within the genus Ranavirus, family Iridoviridae. FV3's large ( approximately 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 h 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 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.

The effect of synthetic fire ant venom alkaloid Solenopsin B (Sol B) on the human monocytic cell line U937 was examined to determine its ability to induce apoptosis and efficacy as a therapeutic agent. Sol B treated cells displayed a >50% reduction in viability along with DNA laddering, a hallmark of apoptosis. The apoptosis mechanism was further examined using DNA microarrays and quantitative RT‐PCR (qRT‐PCR). U937 cells were incubated in the presence of Sol B and total cellular RNA isolated. cDNA was synthesized, labeled with cy3/cy5 and hybridized to microarrays or used in qRT‐PCR RT2 Profiler(tm) PCR array analysis. Microarray analysis revealed that 661 genes and 620 ESTs were up regulated >1.5 fold, including several apoptosis and cell cycle genes. PCR arrays representing functional gene groupings of 90 apoptosis and 92 cell cycle genes showed up regulation of >90% and >70% respectively. Genes examined using both microarray and qRT‐PCR showed correlations of ~93% for apoptosis and ~79% for cell cycle genes. Further, transmission electron microscopy revealed Sol B treatment resulted in loss of cell membrane integrity further verifying the ability of Sol B to induce apoptosis. Collectively these findings indicate that Sol B induced programmed cell death in human cells by triggering the apoptotic pathway.

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.

A virus, designated Rana catesbeiana virus Z (RCV-Z), was isolated from the visceral tissue of moribund tadpoles of the North American bullfrog Rana catesbeiana. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of viral proteins and sequence analysis of the amino terminal end of the major capsid protein showed that RCV-Z was similar to frog virus 3 (FV3) and other ranaviruses isolated from anurans and fish. However, analysis of restriction fragment profiles following digestion of viral genomic DNA with XbaI and BamHI indicated that RCV-Z was markedly different from FV3. Moreover, in contrast to FV3, RCV-Z contained a full-length copy of the viral homolog of eukaryotic initiation factor 2 alpha (eIF-2 alpha). Experimental infection of bullfrog tadpoles with FV3 and RCV-Z demonstrated that RCV-Z was much more pathogenic than FV3, and that prior infection with FV3 protected them from subsequent RCV-Z induced mortality. Collectively, these results suggest that RCV-Z may represent a novel species of ranavirus capable of infecting frogs and that possession of a viral eIF-2 alpha homolog (vIF-2 alpha) correlates with enhanced virulence.

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIalpha triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.