Silencing of the baculovirus Op-iap3 gene by RNA interference reveals that it is required for prevention of apoptosis during Orgyia pseudotsugata M nucleopolyhedrovirus infection of Ld652Y cells

Abstract

The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during OpMNPV infection, we used RNA interference (RNAi) to silence Op-iap3 expression during OpMNPV infection of Ld652Y cells. Infected cells treated with Op-iap3 double-stranded RNA (dsRNA) did not accumulate detectable Op-iap3 mRNA, confirming that the Op-iap3 gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in OpMNPV-infected cells treated with Op-iap3 dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-iap3 dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during OpMNPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function.

FIG. 1.

AcMNPV infection does not inhibit RNAi. Sf21 cells were infected with AcMNPV, and 1 h later the cells were mock transfected (No RNA) or transfected with 80 μg of the indicated dsRNAs. Total RNA was isolated at the indicated times, and RT-PCR was performed to detect the presence of Sf-actin (A) or Sf-iap mRNA (B). HPR, hours post-dsRNA addition.

FIG. 2.

Silencing of Op-iap3 during infection with vOpIAPR-11, an AcMNPV recombinant that expresses Op-iap3 and lacks p35, induces apoptosis in Sf21 cells. (A) Sf21 cells were left untreated (i) or infected with vOpIAPR-11 (ii to iv) and transfected with 160 μg of control CAT dsRNA (ii and iv) or Op-iap3 dsRNA (iii). Photographs were taken at 18 (i to iii) or 74 (iv) h after dsRNA addition. Magnifications, ×400 (i to iii) and ×1,000 (iv). (B) Ethidium bromide-stained agarose gel showing DNA fragmentation in vOpIAPR-11-infected cells transfected with Op-iap3 dsRNA. Lane 1, untreated Sf21 cells; lane 2, vOpIAPR-11-infected Sf21 cells transfected with CAT dsRNA; lane 3, uninfected Sf21 cells transfected with Op-iap3 dsRNA; lane 4, uninfected Sf21 cells transfected with CAT dsRNA; lane 5, Sf21 cells treated with actinomycin D; lane 6, vOpIAPR-11-infected Sf21 cells transfected with Op-iap3 dsRNA.

FIG. 3.

Op-iap3 dsRNA transfection during vOpIAPR-11 infection of Sf21 cells eliminates Op-IAP3 protein expression. The levels of Op-IAP3 protein were determined by immunoblotting with antiserum made against Op-IAP3 at the indicated times following mock transfection of vOpIAPR-11-infected cells (A) or transfection of vOpIAPR-11-infected cells with Op-iap3 dsRNA (B). PC, positive control lysate from Sf21 cells transfected with a vector overexpressing Op-IAP3; U, lysate from untreated Sf21 cells; HPR, hours post-dsRNA addition.

FIG. 4.

Time course of Op-IAP3 expression during OpMNPV infection of Ld652Y cells. Ld652Y cells were infected with OpMNPV and harvested at the indicated hours p.i. (HPI), and the levels of Op-IAP3 protein were determined by immunoblotting with antiserum against Op-IAP3. (A) PC, positive control lysate from Sf21 cells transfected with a plasmid overexpressing Op-IAP3; U, lysate from untransfected Sf21 cells. (B) U, lysate from uninfected Ld652Y cells. Molecular mass markers are indicated to the left of each panel in kilodaltons.

FIG. 5.

Silencing of Op-iap3 during OpMNPV infection of Ld652Y cells induces apoptosis. (A) Ld652Y cells were either untreated (i), transfected with control CAT dsRNA during OpMNPV infection (ii), transfected with Op-iap3 dsRNA during OpMNPV infection (iii), or transfected with Op-iap3 dsRNA in the absence of virus infection (iv). Photographs were taken at 48 h after the transfection of dsRNA. Magnification, ×400. (B) DNA fragmentation was observed in Ld652Y cells treated with actinomycin D (lane 1) or Op-iap3 dsRNA during OpMNPV infection (lane 2) but not in OpMNPV-infected cells treated with CAT dsRNA (lane 3) or in untreated cells (lane 4).

FIG. 6.

Transfection of Op-iap3 dsRNA silences Op-iap3 expression during OpMNPV infection. (A) The presence of Op-iap3 (top panel) and hrf-1 (bottom panel) mRNA was determined by RT-PCR at the indicated times in OpMNPV-infected Ld652Y cells following mock transfection (No RNA) or transfection with CAT or Op-iap3 dsRNA. U, uninfected Ld652Y cells; HPR, hours post-dsRNA addition. (B) The levels of Op-IAP3 protein were determined by immunoblotting in OpMNPV-infected Ld652Y cells at the indicated times following mock transfection (upper panel) or transfection with Op-iap3 dsRNA (lower panel). PC, positive control lysate from Sf21 cells transfected with a vector overexpressing Op-IAP3; U, uninfected Ld652Y cell lysate used as a negative control; HPR, hours post-dsRNA addition.

FIG. 7.

Association of Op-IAP3 with purified OpMNPV budded virions. Sucrose gradient-purified BV was analyzed by immunoblotting with polyclonal antisera against Op-IAP3 or OpMNPV gp37 or a monoclonal antibody against OpMNPV IE-1. IC, infected cell lysates harvested at 48 h p.i. An equal amount of total protein was loaded in each lane.