piRNAs are a type of small RNA in the RNAi mechanism. Current thinking limits them to an anti-transposable element role in the germline; however, a new paper in PLoS One by Vodovar et al adds to the growing body of evidence that this strict classification may be an oversimplification, and that piRNAs may have an antiviral role.
There are two things that distinguish piRNAs from other small RNAs: they are 25-30 nucleotides long, compared to siRNAs which are 21nt long; and they have a bias for uridine at their first position and adenosine at their tenth.
So to find if piRNAs function against viruses, the authors injected cells from the mosquitos Aedes albopictus and Aedes aegypti with Sindbis Virus (SINV), and extracted and sequenced the small RNAs produced in response.
They found small RNAs of 21 and 25-29 nucleotides in length (displayed below): these are the appropriate sizes for siRNAs and piRNAs respectively. (all figures adapted from Vodovar et al, 2012)
Then they looked at whether the sequences of these small RNAs corresponded to anywhere on the SINV genome, and found matches across the genome: it is therefore likely that these small RNAs were derived from the viral genome. This is displayed below: the higher the bar, the more small RNAs mapped to that location.
Thirdly, they sequenced the small RNAs and looked at how biased each position was (i.e. whether A, C, G or U occurred more than 25% of the time). They found a strong bias for U at their first position, and A at the tenth position, characteristic of piRNAs (displayed below, with the size of each letter corresponding to its frequency relative to the other three bases).
They confirmed this finding by analysing the data from an earlier study by Brackney et al on A. albopictus infected with La Crosse Virus (LACV), and found the same U and A biases in small RNAs in the same 25-29 nucleotide size range
Finally, they checked whether piRNAs in these mosquitos are still fulfilling their original function of defence against transposable elements. They did this by extracting small RNAs extracted from A. aegypti cells under normal (i.e. no virus present) conditions, and mapping these to the A. aegypti genome sequence, which contains sequences for the transposable elements in this species. They found the same results as during viral exposure: peaks at 21 and 25-29 nucleotides long; a first position U and tenth position A bias; and matches to the sequences of many different transposons.
This paper presents convincing results for piRNAs being both an antiviral and anti-transposable element mechanism in mosquitos. While they only present data from cell culture, a previous paper by Morazzani et al (2012) has found a similar phenomenon in whole mosquitos, thus increasing the confidence with which these conclusions can be extended to natural insect immune responses. Importantly, the authors find the same results using cells with a normal RNAi mechanism and cells deficient in Dcr-2, a protein integral to the siRNA pathway: it seems that this piRNA production is not merely a back-up for the siRNA response, but a response in its own right. Their discussion also highlights vertical (mother to offspring) viral transmission in arthropods: could the presence of vertically transmitted viruses in the germline have been the selective pressure that drove an antiviral function in the piRNA pathway? And does this limit the prospect of finding antiviral piRNAs in other animals, which don’t transmit viruses vertically?
Vodovar, N., Bronkhorst, A.W., van Cleef, K.W.R, Miesen, P., Blanc, H., van Rij, R.P. and Saleh, M-C (2012) Arbovirus-derived piRNAs exhibit a Ping-Pong signature in mosquito cells. PLoS One 7: 1-8
Morazzani, E.M., Wiley, M.R., Murreddu, M.G., Adelman, Z.N. and Myles, K.M. (2012) Production of virus-derived Ping-Pong-dependent piRNA-like small RNAs in the mosquito soma. PLoS Pathogens 8: 1-11