Elaboración de la obra

Co-immunoprecipitation (Co-IP) was carried out either on transfected or infected cell monolayers. For transfections, 4 x105 HEK293T cells were seeded in 6-well plates and transfected with 1 µg pCMVXNSs-V5 (where X refers to UUKV, HRTV or SFTSV), or mock-transfected. In some cases, the plasmids were transfected along with with 1 µg pCMVRIG-I-N-FLAG, pCMVMAVS-FLAG, pCMVTBK-1-FLAG, pCMVIKKε-FLAG, or pCMVIRF-3(5D). For Co-IP of NSs proteins in virus-infected cells, HEK293T or A549 cells were infected with UUKV or HRTV at an MOI of 20 or 10 FFU/cell, respectively. At the indicated time point, cells were lysed in RIPA lysis buffer or UUKV NSs IP lysis buffer (specifically for UUKV NSs IP), and incubated by rotation at 4°C for 30 min. For the IP of HRTV NSs, cells were lysed on ice for 30 min, the cell lysates transferred to a tube and removed from containment level 3 conditions, before incubation by rotation for another 30 min at 4°C. The cell lysates were clarified by centrifugation at 12,000 rpm for 20 min at 4°C, and transferred to a fresh tube. Note at this stage the whole cell lysate (WCL) fraction was also taken.

For co-IP of FLAG-tagged proteins, anti-FLAG M2 magnetic beads (Sigma) were used. For co-IP of UUKV or HRTV NSs proteins, clarified cell lysates were incubated overnight at 4°C with rabbit polyclonal anti- UUKV or HRTV NSs antibodies, followed by the addition of Protein A magnetic Dynabeads® (Thermofisher). For co-IP of V5-tagged proteins, clarified cell lysates were incubated overnight at 4°C with mouse anti-V5 antibody, followed by the addition of Protein G magnetic Dynabeads® (Thermofisher). All beads were used according to the manufacturer’s instructions. Following incubation of the cell lysates with the beads for 1.5 h at 4°C, the beads were washed five times with RIPA lysis buffer (or UUKV NSs IP lysis bufer) without detergent, and two times with PBS before elution of antibody complexes. Elution of proteins was carried out by the addition of

reducing protein disruption buffer and boiling at 95°C for 10 min. Eluates were analysed by western blotting analysis as described in section 4.1.2.

4.4 Statistical analysis

All data were analysed using Prism 5 software (GraphPad) and presented as mean ±

standard deviation (SD) or standard error of the mean (SEM). Statistical significance for the comparison of means between groups was determined by one-way analysis of variance (ANOVA), followed by post-hoc tests. p-values ≤ 0.05 were considered significant (****, p ≤ 0.0001; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05).

Chapters

5-8

RESULTS

5 Establishment of a reverse genetics system for

Uukuniemi virus

5.1 Introduction

Reverse genetics is an approach used for studying the effect of targeted gene modifications on phenotype. Reverse genetics tools in virus research range from minigenome systems to virus-like particles (VLPs) and virus rescue systems. While minigenome systems and VLPs allow studying specific aspects of a virus’ life cycle, virus rescue systems enable the investigation of the life cycle in its entirety. Minigenome systems consist of an analogue of the virus genome, in which the open reading frame (ORF) of a virus segment is replaced by a reporter gene. Supplementing the genome analogue with trans-acting viral nucleocapsid (N) and RNA-dependent RNA polymerase (L) proteins results in reporter gene activity, which can be measured to investigate the promoter strength of untranslated regions (UTRs), functionality of viral UTRs and N and L proteins, or the effect that other viral or cellular factors may have on minigenome activity. Therefore, minigenome systems allow modelling and simulating virus transcription and replication processes. So far, minigenome systems have been established for a number of bunyaviruses, such as BUNV (Kuismanen et al., 1984; 1982; Murphy et al., 1973), SBV (Elliott and Blakqori, 2012), OROV (Acrani et al., 2015), LACV (Blakqori et al., 2003), RVFV (Ikegami et al., 2005a), SFTSV (Brennan et al., 2015), and CCHFV (Devignot et al., 2015).

Supplementing a minigenome system with viral glycoproteins allows the generation of transcription and replication-competent virus-like particles (VLPs), which have a lipid envelope embedded with the viral glycoproteins. VLPs can package the reporter minigenome and infect target cells to deliver ribonucleoprotein complexes (RNPs), which are able to undergo replication and transcription. In addition to enabling the study of transcription and replication, utilizing VLPs allows to investigate packaging and budding processes, as well as virus morphogenesis.

Uukuniemi virus (UUKV) is a tick-borne phlebovirus originally isolated in 1964 in Finland (Oker-Blom et al., 1964). While antibodies to UUKV (or UUKV-like viruses) have been isolated in humans and other vertebrates such as birds, rodents and cows, there is no evidence of disease in these species (Hubálek and Rudolf, 2012; Saikku and Brummer-Korvenkontio, 1973). Therefore, it serves as a safe laboratory model to study

tick-borne phleboviruses. Indeed, UUKV has served as a prototype virus of the

Bunyaviridae family for many years, contributing to advances in bunyavirus structure and

architectural studies (Freiberg et al., 2008; Overby et al., 2008), glycoprotein studies (Overby et al., 2007) and bunyavirus entry into mammalian cells (Lozach et al., 2010) (Mazelier et al., 2016).

For UUKV, an M segment-based minigenome system utilising chloramphenicol acetyltransferase (CAT) or GFP as reporter genes has already been reported (R. Flick and Pettersson, 2001). Moreover, the authors reported the generation of VLPs containing the reporter minigenomes following superinfection with UUKV. UUKV VLPs generated using a glycoprotein precursor expression plasmid instead of superinfection has also been reported (Överby et al., 2006). However, these systems rely on constructs driven by RNA polymerase I (Pol I), which is disadvantageous because while Pol I localises to the nucleus, bunyaviruses replicate exclusively in the cytoplasm (Plyusnin and Elliott, 2011). In fact, most bunyavirus reverse genetics systems have been developed using a bacteriophage T7 RNA polymerase (RNAP) (Brennan et al., 2015; Tilston-Lunel et al., 2016) (Bergeron et

al., 2015; Elliott et al., 2013; Lowen et al., 2004; Varela et al., 2013), which localises to

the cytoplasm (Elroy-Stein and B. Moss, 1990).

Finally, even though UUKV Pol I-driven minigenome and VLP systems have been developed, no virus rescue entirely from cDNA has been described. The availability of a rescue system allows the generation of recombinant infectious virus from cDNA clones, enabling the genetic engineering of the viral genome. In this way, the effect of genetic changes on the viral life cycle in its entirety can be explored. There is a need to develop a reverse genetics system for UUKV for the ultimate manipulation of the UUKV genome. Such a system would facilitate the use of UUKV as a safe comparative model to understand the molecular basis for virulence and pathogenicity of highly pathogenic tick- borne phleboviruses, such as Heartland virus (HRTV) and Severe Fever with Thrombocytopenia Syndrome virus (SFTSV).

5.2 Aims

The primary aim of the studies described in this chapter was to establish a T7 RNAP- driven reverse genetics system for UUKV. This included the development of a UUKV minigenome system for all segments using luciferase reporter genes, the generation of virus-like particles, and finally the recovery of UUKV entirely from cDNA clones.

Secondly, I aimed to manipulate the UUKV genome to generate recombinant viruses lacking the NSs protein, which could be used in the future to gain an understanding of the function that UUKV NSs may play in the virus life cycle. Finally, I aimed to generate a recombinant UUKV encoding eGFP in the NSs locus, such that it could be used as a quick tool to investigate permissibility of various mammalian cell lines to UUKV infection.

5.3 Results