Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Virol
2011 Nov 01;8521:11131-8. doi: 10.1128/JVI.05589-11.
Show Gene links
Show Anatomy links
Improved knockout methodology reveals that frog virus 3 mutants lacking either the 18K immediate-early gene or the truncated vIF-2alpha gene are defective for replication and growth in vivo.
Chen G
,
Ward BM
,
Yu KH
,
Chinchar VG
,
Robert J
.
???displayArticle.abstract???
To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.
Abbate,
Bifunctional protein conferring enhanced green fluorescence and puromycin resistance.
2001, Pubmed
Abbate,
Bifunctional protein conferring enhanced green fluorescence and puromycin resistance.
2001,
Pubmed
Chinchar,
Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization.
1986,
Pubmed
Chinchar,
Family Iridoviridae: poor viral relations no longer.
2009,
Pubmed
Duffus,
Frog virus 3-like infections in aquatic amphibian communities.
2008,
Pubmed
Eaton,
Comparative genomic analysis of the family Iridoviridae: re-annotating and defining the core set of iridovirus genes.
2007,
Pubmed
Essbauer,
Comparison of the eIF-2alpha homologous proteins of seven ranaviruses (Iridoviridae).
2001,
Pubmed
Gantress,
Development and characterization of a model system to study amphibian immune responses to iridoviruses.
2003,
Pubmed
,
Xenbase
Goorha,
Macromolecular synthesis in cells infected by frog virus 3. VIII. The nucleus is a site of frog virus 3 DNA and RNA synthesis.
1978,
Pubmed
Granoff,
The isolation and properties of viruses from Rana pipiens: their possible relationship to the renal adenocarcinoma of the leopard frog.
1965,
Pubmed
Gray,
Ecology and pathology of amphibian ranaviruses.
2009,
Pubmed
,
Xenbase
Huang,
Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.
2011,
Pubmed
Huang,
Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae.
2009,
Pubmed
Jancovich,
Innate immune evasion mediated by the Ambystoma tigrinum virus eukaryotic translation initiation factor 2alpha homologue.
2011,
Pubmed
Keesing,
Impacts of biodiversity on the emergence and transmission of infectious diseases.
2010,
Pubmed
Langland,
Inhibition of PKR by RNA and DNA viruses.
2006,
Pubmed
Majji,
Rana catesbeiana virus Z (RCV-Z): a novel pathogenic ranavirus.
2006,
Pubmed
Majji,
Transcriptome analysis of Frog virus 3, the type species of the genus Ranavirus, family Iridoviridae.
2009,
Pubmed
Mazzoni,
Mass mortality associated with a frog virus 3-like Ranavirus infection in farmed tadpoles Rana catesbeiana from Brazil.
2009,
Pubmed
Pallister,
Bohle iridovirus as a vector for heterologous gene expression.
2007,
Pubmed
Rothenburg,
Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.
2011,
Pubmed
Sadler,
Interferon-inducible antiviral effectors.
2008,
Pubmed
Sample,
Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II.
2007,
Pubmed
Schloegel,
Two amphibian diseases, chytridiomycosis and ranaviral disease, are now globally notifiable to the World Organization for Animal Health (OIE): an assessment.
2010,
Pubmed
Song,
Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis.
2004,
Pubmed
Tan,
Comparative genomic analyses of frog virus 3, type species of the genus Ranavirus (family Iridoviridae).
2004,
Pubmed
Whitley,
Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro.
2010,
Pubmed
Willis,
trans activation of an immediate-early frog virus 3 promoter by a virion protein.
1985,
Pubmed
Yarden,
Characterization of sINR, a strict version of the Initiator core promoter element.
2009,
Pubmed
