Background

Under virus infection, the activation of host immune system usually starts with the recognition of viral pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) (Medzhitov and Janeway, 2002). Viral PAMPs, associated with virus invasion, include virus surface glycoproteins, viral DNA and RNAs (Mogensen and Paludan, 2005). In case of RNA virus infection, virus replication generated RNAs are essential PAMPs portion which is able to be recognized by the host intracellular PRRs (Alexopoulou et al., 2001). Two possible candidates for the role of such viral-generated PAMP RNAs have been recently reported. First, classical viral PAMP RNAs are dsRNA replication forms that are recognized by RIG-I and MDA5 and non-capped positive-strand RNAs (polyA+) generated by alphaviruses (Sokoloski et al., 2015) that may be recognized by RIG-I (Akhrymuk et al., 2015). Second, non-classical viral PAMP RNAs are short non-polyadenylated dsRNAs with 5’ triphosphate. They are IFN-inducing RNAs generated by viral replicase using cellular RNA templates (Nikonov et al., 2013). In Semliki Forest virus (SFV) infected cells these RNAs represent the majority of PAMP RNAs (Nikonov et al., 2013).

Given the essential role of viral PAMP RNAs to activate host immune system, a robust protocol to analyze different types of virus-generated PAMP RNAs is useful for virus-host interaction study. Quantitative real time PCR could be used to quantify classical viral dsRNAs by targeting negative strands. However, it is hard to differentiate between capped and non-capped positive strands. Furthermore, the approach is neither able to tell if the targeted PAMP RNA is functional nor able to access the PAMP efficiency and cannot detect the non-classical viral PAMP RNAs. To analyze the functional PAMP RNAs that are able to trigger host immune response, we established this protocol to analyze both classical and non-classical viral PAMP RNAs. To assess the total viral PAMP RNAs, total RNA from the infected cells were extracted for the assessment. Since all types of classical alphaviral PAMP RNAs have unpaired polyA tails and they are presumably the biggest proportion in the viral PAMP RNAs, they may overshadow other types of PAMP RNAs. Therefore, in a parallel experiment, we separated and examined the polyA- fraction from the total RNAs (Nikonov et al., 2013). To assess the non-classical viral PAMP RNAs, viral replicase constructs were used for analysis because the system did not have replication competent viral RNA templates and therefore no classical viral PAMP RNAs were produced. Cop5 cells were used as IFNα/β activation host for all PAMP RNAs analyses. The quantity of the IFNα/β produced by the transfected PAMP RNAs can be determined by ELISA assay.

This protocol is a reliable method to analyze alphavirus-generated PAMP RNAs during the virus replication and, with exception of separation of polyA- fraction, applied to viruses that lack unpaired polyA tails. It is not only to measure the level of the PAMP RNAs but also is able to assess the PAMP RNAs efficiency. Therefore, this protocol is suitable for the study involving the interaction between RNA virus invasion and the host immune response.