Mutagenesis analysis of the zinc-finger antiviral protein.
BACKGROUND
The zinc-finger antiviral protein (ZAP) specifically inhibits the replication of certain viruses, including murine leukemia virus (MLV), by preventing the accumulation of viral mRNA in the cytoplasm. ZAP directly binds to the viral mRNA through the zinc-finger motifs and recruits the RNA exosome to degrade the target RNA. RNA helicase p72 is required for the optimal function of ZAP. In an attempt to understand the structure-function relationship of ZAP, we performed alanine scanning analysis.


RESULTS
A series of ZAP mutants was generated, in which three consecutive amino acids were replaced with three alanines. The mutants were analyzed for their antiviral activities against pseudotyped MLV vector. Out of the nineteen mutants analyzed, seven displayed significantly lower antiviral activities. Two mutations were in the very N-terminal domain, and five mutations were within or around the first and second zinc-finger motifs. These mutants were further analyzed for their abilities to bind to the target RNA, the exosome, and the RNA helicase p72. Mutants Nm3 and Nm63 lost the ability to bind to RNA. Mutants Nm 63 and Nm93 displayed compromised interaction with p72, while the binding of Nm133 to p72 was very modest. The interactions of all the mutants with the exosome were comparable to wild type ZAP.


CONCLUSIONS
The integrity of the very N-terminal domain and the first and second zinc-finger motifs appear to be required for ZAP's antiviral activity. Analyses of the mutants for their abilities to interact with the target RNA and RNA helicase p72 confirmed our previous results. The mutants that bind normally to the target RNA, the exosome, and the RNA helicase p72 may be useful tools for further understanding the mechanism underlying ZAP's antiviral activity.
Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Xinlu
X
Wang
Fengxiang
F
Lv
Guangxia
G
Gao
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D004252:DNA Mutational Analysis; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins; D051381:Rats
D002352:Carrier Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C465959:Zc3hav1 protein, rat; D000409:Alanine