RING finger proteins of infectious spleen and kidney necrosis virus (ISKNV) function as ubiquitin ligase enzymes.
Infectious spleen and kidney necrosis virus (ISKNV) is the etiological agent that causes a pandemic and severe disease in fish characteristic of enlarged and damaged spleen and kidney. To identify viral proteins involved in infection and pathogenesis, we characterized five open reading frames (ORFs) of the ISKNV genome, ORF12, ORF65, ORF66, ORF99 and ORF111, which encode RING finger proteins (RFPs). We assessed the ubiquitin ligase (E3) activity of these recombinant RFPs fused to maltose-binding protein (MBP) using an in vitro ubiquitination assay and demonstrated that ORF12, ORF65, ORF66 and ORF111 possess the E3 activity in the presence of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin and zinc ion. E3 activity of ISKNV RFPs strictly depends on the UbcH5 E2 subfamily (ORF12 and ORF65 depend on UbcH5a/c, ORF66 and ORF111 depend on UbcH5a/b/c). Furthermore, point mutation in the RING domain completely abrogated ORF66 E3 activity, indicating the RING motif was essential for RFP of ISKNV. In addition, zinc ion was required as an enhancer for ISKNV RFP to exert its E3 function. Investigation of RFPs of ISKNV helps to understand their functions in the infection process and in the virus-host interaction.
Xie
Junfeng
J
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, People's Republic of China.
Zhu
Jingyi
J
Yang
Hongyan
H
Weng
Shaoping
S
Chen
Cheng
C
Chen
Hao
H
Xie
Qi
Q
Li
Mengfeng
M
He
Jianguo
J
D000818:Animals; D004266:DNA Virus Infections / Q000821:virology; D004926:Escherichia coli / Q000378:metabolism; D005393:Fish Diseases / Q000821:virology; D007497:Iridoviridae / Q000201:enzymology*; D016366:Open Reading Frames / Q000502:physiology; D010473:Perciformes / Q000821:virology; D011994:Recombinant Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D014764:Viral Proteins; D044767:Ubiquitin-Protein Ligases