Capítulo III: Propuesta artística y creativa
III.1 Desarrollo conceptual de la propuesta
IFN production can have protective effects against bunyavirus infection, which can be illustrated by the high susceptibility to RVFV infection in IFNAR knock out mice, compared to wild-type mice (Bouloy et al., 2001; Cyr et al., 2015; Yadani et al., 1999). During bunyavirus replication, products of each of the three viral RNA segments have the potential to activate IFN induction by the stimulation of RIG-I. As a consequence, bunyaviruses require efficient mechanisms to antagonise the IFN response. It is well- known that the NSs protein of bunyaviruses acts as a major virulence factor, contributing to pathogenesis by assisting the virus to evade the host innate immune system, first demonstrated for BUNV NSs (Bridgen et al., 2001; Weber et al., 2002). The NSs proteins of phleboviruses have also evolved a plethora of countermeasures to block the IFN response.
Within the Sandfly fever group of the Phlebovirus genus, the NSs protein of RVFV has been extensively studied. Unlike most phleboviruses, the NSs protein of RVFV localizes to the nuclei of infected cells, forming distinct filamentous structures (Brennan et al., 2014; Cyr et al., 2015; Struthers and Swanepoel, 1982; Yadani et al., 1999). Part of its IFN antagonistic activity is attributed to its nuclear localization, which enables RVFV to inhibit IFN-β mRNA synthesis. Initially, RVFV NSs was shown to inhibit general host cell mRNA transcription by inhibiting the assembly of the basal transcription factor IIH (TFIIH), as described in section 1.1.4 (Billecocq et al., 2004; Cyr et al., 2015; Kalveram et
al., 2011; Le May et al., 2004). However, RVFV NSs was shown to employ a different
mechanism for the suppression of IFN-β mRNA synthesis specifically. This mechanism involved an interaction between RVFV NSs and SAP30 (Sin3A associated protein 30), a protein which forms part of a repressor complex that regulates IFN-β gene expression (Le May et al., 2008). The interaction of SAP30 with RVFV NSs and its recruitment to the IFN-β promoter results in chromatin structure reorganization, which physically inhibits the recruitment of the CREB-binding protein (CBP) to the IFN-β promoter, therefore supressing histone acetylation and consequently transcriptional activation. RVFV NSs was also shown to support virus replication in infected cells by promoting the post- transcriptional proteasome-dependent down-regulation of the double-stranded RNA- dependent protein kinase (PKR) (Ikegami et al., 2009b; 2009a). Following IFN induction, PKR levels are up-regulated. A dsRNA-binding domain in PKR binds dsRNA generated during virus replication, which triggers its activation (F. Zhang, 2001). Once active, PKR can phosphorylate the eukaryotic initiation factor eIF2α (Srivastava et al., 1998). eIF2α is
a subunit of the IF2 initiation complex that is responsible for recruiting initiator MettRNA to the 40S ribosomal subunit in a guanosine triphosphate (GTP)-dependent manner.
Phosphorylation of eIF2α by PKR inhibits the exchange of GDP for GTP, thereby
resulting the inhibition of translation initiation due to the inability to recruit the initiator
MettRNA to the 43S pre-initiation complex (de Haro et al., 1996). Thus, the proteasomal
degradation of PKR by RVFV NSs supresses the phosphorylation of eIF2α and in this way prevents the inhibition of translation initiation induced by the IFN response (Habjan et al., 2009b; Ikegami et al., 2009b). Recently, it was shown that the degradation of PKR is mediated by the interaction of RVFV NSs with the F-box proteins FBXW11 and/or FBCW1, through a ‘degron’ sequence in RVFV NSs, which, curiously overlaps with the ΩXaV motif at its C terminus (Kainulainen et al., 2016; Mudhasani et al., 2016). It was suggested that perhaps the down-regulation of PKR turns the unfavourable environment induced by suppression of host cell mRNA transcription (by RVFV NSs) in infected host cells to one that supports viral replication, by preventing the inhibition of viral translation (Ikegami et al., 2009a). In this way, the dual functions of RVFV NSs could function concomitantly to inhibit host innate immune responses and to enable efficient RVFV replication (Ikegami et al., 2009b).
Early studies showed that TOSV induced IFN despite the ability of its NSs protein to antagonise the IFN response, which the authors indicate may be a result of a weak IFN antagonistic activity of TOSV NSs (Gori-Savellini et al., 2010). The ability to induce the proteasomal-mediated degradation of PKR by RVFV NSs is a property that is conserved in TOSV NSs (Kalveram and Ikegami, 2013). However, in comparison to RVFV, cellular transcription levels are unaffected following TOSV infection (Kalveram and Ikegami, 2013). Additionally, the type I IFN response was shown to be blocked by TOSV NSs by a direct interaction with RIG-I, and its targeting for proteasomal degradation, thereby preventing RIG-I-mediated IFN induction (Gori-Savellini et al., 2013). In a study where recombinant RVFV encoding PTV or SFSV NSs proteins instead of RVFV NSs were generated, the viruses encoding PTV and SFSV NSs proteins did not affect PKR levels (Kalveram and Ikegami, 2013; Lihoradova et al., 2013). However, the virus encoding PTV NSs was able to supress host cell mRNA transcription similarly to RVFV, whereas that encoding SFSV NSs had no inhibitory effect of host cell mRNA synthesis (Kalveram and Ikegami, 2013; Lihoradova et al., 2013).
Whilst within the Phlebovirus genus most studies have focused on understanding the mechanism employed by mosquito-borne virus NSs proteins to antagonize the IFN response, the role of tick-borne phlebovirus NSs proteins in innate immunity was neglected until the emergence of the novel highly pathogenic SFTSV. Upon the discovery of SFTSV, research efforts focused on understanding its underlying molecular mechanisms of virulence and pathogenicity. Consequently, SFTSV NSs was quickly characterized and was found to form cytoplasmic viroplasm-like structures, also known as inclusion bodies (IBs). The structures were described in infected cells, but also when NSs is expressed in the absence of other viral proteins (Ning et al., 2014; Santiago et al., 2014). The IBs were found to colocalise with lipid droplets and viral dsRNA (X. Wu et al., 2014). Inhibitors of fatty acid synthesis hindered the formation of IBs and virus replication, thus suggesting that NSs-formed IBs play a role in virus replication (X. Wu et al., 2014). However, the confirmation of such conclusion would require the generation of a recombinant SFTSV lacking NSs. Another study found that IBs colocalised with the early endosomal marker Rab5, but not with Golgi apparatus or endoplasmatic reticulum markers, suggesting the endosomal system is utilised for the formation of IBs (Santiago et al., 2014). Finally, it was suggested that SFTSV NSs-formed IBs are secreted into the extracellular space, and that the extracellular vesicles containing infectious virions could mediate the receptor- independent transmission of SFTSV, through endocytosis into uninfected neighbouring cells, where they could sustain virus replication (Silvas et al., 2015). As well as the roles suggested by NSs-induced IBs in virus replication and receptor-independent endocytosis, the IBs were described as a novel, elegant strategy that SFTSV employs to antagonise the IFN response. In fact, SFTSV NSs was characterized as a potent antagonist of IFN induction through the spatial isolation and sequestration of TRIM25 and RIG-I (Santiago
et al., 2014), TBK1 (Ning et al., 2014; X. Wu et al., 2014), and IRF-3 (Ning et al., 2014;
X. Wu et al., 2014) into the IBs. The interaction between IKKε or IRF-3 and SFTSV NSs was reported to be indirect, facilitated by the interaction with TBK1 (X. Wu et al., 2014). However, one study also showed a direct interaction with IKKε (Ning et al., 2014). Similarly, while the direct interaction between SFTSV NSs and RIG-I was reported in one study (Santiago et al., 2014), such an interaction was not observed by others (Ning et al., 2014). As well as inhibiting IFN induction, a potent inhibitory activity of SFTSV NSs on IFN signalling was described, and was attributed to the interaction between SFTSV NSs and STAT1 or STAT2. This interaction results in the spatial isolation of STAT1 and STAT2, therefore inhibiting the JAK/STAT signalling pathway (Chaudhary et al., 2015; Ning et al., 2015). Interestingly, a study using liver epithelial cells showed that while
SFTSV NSs strongly inhibited IFN-β promoter activation, NFκB activation was enhanced by SFTSV NSs (Q. Sun et al., 2015). The differential effect of SFTSV NSs on IFN-β and NFκB promoter activities may explain elevated levels of proinflammatory cytokines and chemokines regulated by NFκB signalling in liver tissues of mice infected with SFTSV (Q. Sun et al., 2015).
Despite our understanding of the role of SFTSV NSs in impairing the IFN response, little is known about the mechanisms utilised by other tick-borne phleboviruses. Recent reviews have highlighted the need of comparative studies to elucidate the countermeasures that phleboviruses have evolved to hinder the IFN response (Ly and Ikegami, 2016; Wuerth and Weber, 2016), as viruses belonging to this genus are continuously emerging and appear to have evolved divergent mechanisms to supress host cell responses to virus infection.
Chapter
2
AIMS
2 Aims
It well-known that the main virulence factor evolved by bunyaviruses to counteract the host innate immune system is their non-structural protein NSs. However, it is also evident that we have limited information about the role of tick-borne phlebovirus NSs proteins in modulating the innate immune system.
The overall aim of this project was to carry out a molecular characterization of the NSs protein of the tick-borne phleboviruses UUKV and HRTV, utilizing reverse genetics as a tool to assist with these investigations.
Specifically, I aimed to:
(i) Develop minigenome and virus-like particle assays for UUKV, as a first step towards developing a reverse genetics system for this virus.
(ii) Rescue UUKV and exploit the ability to manipulate the UUKV genome to generate recombinant viruses lacking its NSs protein.
(iii) Compare the ability of UUKV and recombinant UUKV lacking NSs to hinder interferon production.
(iv) Compare the ability of UUKV and HRTV NSs proteins to block interferon production and interferon signalling, using the well-characterized SFTSV NSs as a comparison.
(v) Investigate the molecular mechanisms of antagonism employed by UUKV and HRTV NSs proteins at the level of interferon induction and interferon signalling.
(vi) Sequence the genome of the HRTV stock available in our laboratory and compare it to the published sequence.
(vii) Develop a reverse genetics system for HRTV.
(viii) Use the available reverse genetics systems of tick-borne phleboviruses as a molecular tool to assess the possibility of reassortment between tick-borne phleboviruses.