Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells

Abstract

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either proapoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

Rapid selection against arbovirus-induced apoptosis during infection of a mosquito vector

“…To assess whether increasing or decreasing apoptosis during virus replication affected the ability of SINV to infect the midgut, mosquitoes that had been given a blood meal containing MRE/Rpr or control virus were dissected and midguts were examined for levels of infection at 3, 5, and 7 dpi by immunofluorescence, using anti-E1 antibody. Each midgut was assigned an infection score based on the surface area that was infected and the intensity of the antibody staining, as previously described (42,43). Prevalence of infection (the proportion of mosquitoes that were positive for SINV antigen in midgut) was significantly lower among mosquitoes infected with MRE/Rpr than control virus MRE/Mx-as at 3 dpi ( Fig.…”

“…1A). This recombinant SINV, called MRE/Rpr, has been previously shown to induce apoptosis in cultured mosquito cells (42). Two control constructs, MRE/ Rpr-as and MRE/Mx-as, containing the sequence of the A. aegypti michelob_x gene, were used that contained noncoding (antisense orientation) sequences of similar size as reaper to control for the effects of genome size on virus replication.…”

“…To examine the effects of inducing apoptosis on arbovirus-vector interactions, we used an infectious clone based on the SINV strain 5′dsMRE16ic (41) that was engineered to express the proapoptotic Drosophila gene reaper via a duplicated subgenomic promoter (42) (Fig. 1A).…”

“…Recombinant 5′dsMRE16ic-based SINV clones containing sense and antisense sequences for Drosophila reaper (MRE/Rpr) and an additional antisense control containing the antisense sequence of A. aegypti michelob_x (MRE/Rpr-as or MRE/Mx-as) were previously described (42). These viruses have insert sizes of 198 nt for MRE/Rpr and MRE/Rpr-as and 339 nt for MRE/ Mx-as.…”

“…This could be a result of at least two factors: first, Reaper expression from the viral subgenomic promoter occurs relatively late in the virus replication cycle, and thus may only cause a delay in cell-to-cell spread of virus. Some virus would likely be produced before MRE/Rpr-infected cells died by apoptosis, as was seen when cultured cells were infected (42). However, after the infected cells die, they would no longer be able to serve as virus factories, as they would in a normal persistent infection.…”