Spindle-E Acts Antivirally Against Alphaviruses in Mosquito Cells

Abstract

Mosquitoes transmit several human- and animal-pathogenic alphaviruses (Togaviridae family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in Aedes aegypti-derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in Drosophila melanogaster, resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in Ae. aegypti cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes.

PIWI pathway against viruses in insects

“…Infection of mosquito cells by SINV produces vpiRNAs that derive from hotspots corresponding mostly to the subgenomic region of the viral genome (Brackney et al, ; Miesen et al, ; Vodovar et al, ). Preferential targeting of the subgenomic region was also observed for SFV, which is not affected by Dcr2 or Spn‐E knockdown (Schnettler, Donald, et al, ; Varjak et al, ; Varjak, Maringer, et al, ). The bias for the coding strand and the 5′ end of the subgenomic RNA indicates preferential targeting, depending on features such as viral sequence or structure (Schnettler, Donald, et al, ).…”

“…aegypti Aag2 cell line (Dietrich, Shi, et al, ). When infected with the (+) RNA virus Semliki Forest virus (SFV), silencing of piwi4 enhanced not only viral replication but also virion production, while it had no effect on vpiRNA production (Schnettler, Donald, et al, ; Varjak et al, ; Varjak, Donald, et al, ; Varjak, Maringer, et al, ). Thus, Piwi4 was suggested to have the role of an effector protein that targets the virus, but is not necessary for the production of SFV‐specific vpiRNAs (Schnettler, Donald, et al, ).…”

“…Results on the effect of Ago3 on viral replication are contradictory for SFV (Varjak, Donald, et al, ) (Schnettler, Donald, et al, ) but positive for RVFV (Dietrich, Jansen, et al, ). However, with regard to the production of SFV vpiRNAs an involvement of Ago3 and Piwi5 was demonstrated (Varjak et al, ; Varjak, Maringer, et al, ); moreover, concomitant silencing of all 7 piwi and ago3 genes abolished completely the production of SFV‐vpiRNAs (Schnettler, Donald, et al, ). Piwi5, Ago3 and Piwi6 were shown to have a role in the biogenesis of virus‐specific piRNAs in Ae.…”

“…Piwi5, Ago3 and Piwi6 were shown to have a role in the biogenesis of virus‐specific piRNAs in Ae. aegypti in studies where infections with SINV, DENV2 and CHIKV were analyzed (Miesen et al, ; Miesen et al, ; Varjak et al, ). In the case of SINV, Piwi5 and Ago3 (and less importantly also Piwi6) proved to be major Ping‐Pong biogenesis factors for vpiRNAs, as they were shown to immunoprecipitate with the vpiRNAs possessing the corresponding “piRNA signature,” i.e., uridine at position 1 (1U) and adenine at position 10 (10A) (Miesen et al, ).…”

“…Similarly, viral replication of BUNV was decreased when piwi6 and ago3 were silenced, while only piwi5 knockdown had the same effect for the midge‐borne SBV (Dietrich, Shi, et al, ). Lately, a possible antiviral role for Spn‐E has appeared as silencing of spn‐E caused a decrease (50%) of SFV‐specific vpiRNAs, in contrast to the absence of any effect observed on viral replication levels for BUNV and ZIKV (Varjak et al, ).In the case of Non‐Retroviral Integrated RNA Virus Sequences (NIRVS, see further below), experimental data show that the piRNA pathway is important as well. NIRVS are viral fossils existing as genomic integrations in Aedes sp.…”