Pestivirus RNAs and the transfected in vitro synthesized recombining RNA molecules used in this study carry a 5’ triphosphate (5’-PPP) group. Furthermore, viral RNA in the cytoplasm of infected cells is protected from degradation by the formation of strong secondary structures (Brown et al. 1992). The results of the present showed by direct identification of the RNA degradation products in vivo that Xrn1-knockdown in cell culture resulted in a slower degradation of viral RNA (Fig. 15 and 16), although Xrn1 degrades specifically mRNAs with a 5’-terminal monophosphoryl group (5’-P) (Coller and Parker 2004, Nagarajan et al. 2013). Degradation of viral uncapped RNAs by Xrn1 has also been reported for other RNA viruses. HCV RNA is mainly degraded from the 5’ end by Xrn1 (Li et al. 2013, 2015). Interestingly, also Xrn2-knockdown resulted in increased amounts of accumulated HCV RNA, although functions of Xrn2 were believed to be restricted to the nucleus (Sedano and Sarnow
Discussion 2014, Li et al. 2015). Furthermore, binding of the liver-specific miR-122 to the HCV RNA uniquely protects it from degradation by Xrn1 (Li et al. 2013). For pestiviruses such a stabilization of the viral RNA by miRNA binding is yet not known. Studies focusing on tombusvirus RNA recombination showed that depletion of Xrn1p or knockdown of Xrn4 led to an increased amount of partially cleaved viral RNAs in yeast as well as in plant cells, respectively (Cheng et al. 2006, 2007, Jaag and Nagy 2009). These RNA fragments appear to arise by endoribonucleolytic cleavage and are then not further degraded by Xrn1p or Xrn4 (Jaag et al. 2011).
Although Xrn1 is shown to be involved in pestivirus RNA degradation, it remains unclear in which cellular compartment the viral RNA is degraded. It was shown that during HCV infection Xrn1 redistributes to lipid droplets, which are cytoplasmic foci required for infectious virus production (Miyanari et al. 2007, Ariumi et al. 2011, Pager et al. 2013). Thus, it was assumed that degradation of HCV RNA by Xrn1 occurs during the movement of the viral RNA from membranous webs to the sites of assembly on the surface of lipid droplets (Miyanari et al. 2007, Li et al. 2013). Live cell imaging of pestivirus infected cells demonstrated that BVDV NS5A localizes to lipid droplets (Isken et al. 2014). However, the role of P-bodies and the cytoplasmic distribution of Xrn1 during pestivirus infection have not been investigated so far. It can be speculated that infection with pestiviruses also triggers the distribution of Xrn1 to lipid droplets and that the degradation of the viral RNA by Xrn1 might occur in close proximity to them.
For the degradation by Xrn1 the viral RNA must undergo a certain cleavage process producing a 5’-P. In general for capped cellular mRNA, the 5’-P is obtained during the decapping process in the cytoplasm (Coller and Parker 2004, Nagarajan et al.
2013). However, uncapped mRNAs were shown to inhibit the decapping enzyme in vitro (LaGrandeur and Parker 1998). Initiation of RNA degradation by Xrn1 can also involve endoribonucleases producing a 5’-P (Coller and Parker 2004, Li et al. 2010, Nagarajan et al. 2013). Thus, the 5’-P of the viral RNA might be provided by other yet not identified ribonucleases. Sequence analysis revealed that the 5’ termini of partially degraded viral genome fragments were mostly located within single-stranded regions of the 3’NTR and clustered in SLII (Fig. 16B). The observation that the degradation sites were not distributed equally can be explained by the formation of specific secondary structures of the partially degraded viral RNAs, which may
Discussion
the production of subgenomic RNA species, which are a result of an incomplete degradation of the viral RNA, has only been observed during infection with flaviviruses (Pijlman et al. 2008). Interestingly, viral RNA recombination occurred preferentially in single-stranded regions (Austermann-Busch and Becher 2012). Also the analysis of 5’-terminal sequences of HCV RNAs, which were partially degraded by Xrn1, showed that the degradation sites were all located within single-stranded regions (Li et al. 2013). The crystal structure of Xrn1 revealed, that single-stranded regions are required to reach the active site of Xrn1 (Jinek et al. 2011). Therefore endoribonucleases, which cleave single-stranded RNAs, like IRE1, G3BP1 or PP11 (Li et al. 2010), are potential candidates involved in cytoplasmic viral RNA degradation. However, no significant impact on the RNA recombination frequency was observed when providing a 5’-P instead of a 5’-PPP at the 3’ recombination partner (Fig. 14). This might be due to a rapid cleavage of the viral RNA leading to an early availability of RNA fragments with 5’-P after transfection. Furthermore, the viral RNA with 5’-PPP might be directly degraded by Xrn1 without a previous processing as a weak in vitro activity of Xrn1 for substrates with 5’-PPP has been reported (Stevens and Poole 1995). Also a yet not identified cellular phosphatase or pyrophosphohydrolase might be involved in the cellular viral RNA degradation machinery (Li et al. 2013, Sedano and Sarnow 2014). This would also explain the observation that transfected synthetic BVDV genome fragments with intact 5’-terminal sequences were able to be ligated without a previous polyphosphatase treatment (Results 4.2.3).
Analysis of the viral RNA production in infected cells revealed that the amount of accumulated RNA only slightly decreased in Xrn1-knockdown cells (Fig. 18A). Also the efficient production of infectious viruses was not significantly influenced by Xrn1-knockdown (Fig. 18B). Previous studies focused on the influence of Xrn1 on viral replication for several members of the family Flaviviridae. For West Nile virus it was shown that Xrn1 is recruited to the sites of virus replication and that the depletion of P-body components led to decreased amounts of viral RNA (Chahar et al. 2013). For the closely related HCV increased (Jones et al. 2010, Ruggieri et al.
2012, Li et al. 2013, 2015), constant (Scheller et al. 2009), and decreased (Ariumi et al. 2011, Pager et al. 2013) viral RNA amounts after Xrn1 silencing were observed.
Furthermore, it was shown that the cytoplasmic distribution of Xrn1 in response to viral infection represents a dynamic process (Chahar et al. 2013). Accordingly,
Discussion effects of Xrn1 on the amount of viral RNA are less clear and might depend on the stage of infection.