Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi.
A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.
Yu
Qian
Q
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China.
Lin
Tiehao
T
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China.
Feng
Guozhong
G
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China.
Yang
Kai
K
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China.
Pang
Yi
Y
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China.
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D018411:Spodoptera / Q000235:genetics / Q000502:physiology* / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D051034:Inhibitor of Apoptosis Proteins; C117171:P49 protein, Spodoptera littoralis nucleopolyhedrovirus; D014764:Viral Proteins