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In a yeast two-hybrid interaction screen of an MA104 cDNA library, that employed NSP1 of a bovine rotavirus (strain B641) as bait, the interferon regulatory factor 3 (IRF3) was identified as the primary cellular interaction partner of NSP1 (Graff et al., 2002). The same research group also found that NSP1 synthesized in infected MA104 cells interacted with host cellular IRF3 in a GST pull-down assay suggesting that the interaction was not unique to the yeast two-hybrid system (Graff et al., 2002). Similarly, NSP1 of another bovine rotavirus (strain UKtc) has also been shown to interact with IRF3 in a yeast two-hybrid assay (Goodbourn et al., unpublished results) and in addition, this interaction caused IRF3 degradation in a proteasome dependent manner (Barro and Patton, 2005). Despite these results with bovine rotavirus strains, in MA104 cells infected with a porcine rotavirus (strain OSU), the cellular IRF3 levels remained stable, little interaction between OSU NSP1 and IRF3 was observed in a GST pull-down assay. From these results it was

concluded that the binding between OSU NSP1 and IRF3 was less than 10% of that seen with the bovine B641 NSP1 (Graff et al., 2007).

The porcine NSP1 was however shown to interact and promote degradation of a second component involved in the innate immune response, namely the β-transducin repeat containing protein (β-TrCP), which functions as part of an E3 ligase

Skp1/Cull/F-box complex (SCFβ-TrCP) involved in the ubiquitination of inhibitor kappa B (IκBα) (Graff et al., 2009). By contrast UKtcNSP1 has been shown to have little effect on β-TrCP (Arnold and Patton, 2011). Therefore, based on the fact that UKtcNSP1 and OSUNSP1 were able to interact and degrade different cellular

proteins and they share only 59.3% and 66% conservation at the amino acid and the nucleotide sequence levels respectively (Xu et al., 1994), it was decided to use these two NSP1s for generating chimeric NSP1 hybrid constructs to map the important regions in NSP1 responsible for their specific interactions with the different cellular proteins. Figure 3.1 illustrated the amino acid sequence comparison between a few NSP1 proteins from different rotavirus strains including the selected bovine UKtc, porcine OSU strains used in this study and some other strains analysed in others’ studies (Figure 3.1).

The strategy employed was to select or create by site directed mutagenesis restriction enzyme (RE) sites spread at intervals across both UKtc and OSU NSP1 sequences to facilitate the swapping of cDNA fragments between the two parental molecules to generate a series of reciprocal hybrid gene constructs. A total of seven possible restriction enzyme sites spread across the two sequences were initially identified although a Nru I site (at UKtcNSP1 nucleotide position 669) was eliminated as it is the only RE site of those identified that was sensitive to dam methylation. Six RE sites finally chosen to be generated in the two parental NSP1 sequences are shown (Figure 3.2A). These positions were selected on the basis that the generation of the RE sites should not cause any change in the amino acid sequence in order to

eliminate the possibility that their introduction would change the structure of the NSP1 proteins. The predicted secondary structures of both proteins were also taken in consideration when selecting these potential RE sites (Figure 3.2B) to ensure that the higher order structure of the protein was maintained as much as possible in the hybrids being generated.

Figure 3.1 Sequence comparision between NSP1s of different rotavirus strains.

Sequence alignment was generated using DNASTAR’s Lasegene Core Suite

software. The three NSP1s from the bovine strains (B641, NCDV and UKtc) showed over 90% of sequence identity among themselves. However the NSP1 of the porcine strain OSU showed only around 60% of sequence identity with bovine NSP1, but around 85% with the NSP1 from the human strain Wa. The NSP1 from the simian stratin SA11 showed less than 40% sequence identity with the porcine OSU NSP1, the human Wa NSP1 and all the bovine NSP1s.

Figure 3.2 Schematic diagrams illustrating the selected restriction enzyme (RE) sites to be generated in both UKtc and OSU NSP1 sequences.

(A). Schematic diagram of six selected RE sites including Stu I, Pml I, Pst I, Hind III, Acl I and Hpa I to be generated in both UKtc and OSU NSP1 sequences (Hpa I already exists in OSUNSP1). The amino acid positions of the corresponding sites in UKtcNSP1 are indicated. The nucleotides highlighted in red indicate those in both NSP1 sequences mutated to generate the RE site without changing the amino acid sequence of the protein.

(B). Predicted secondary structures of the two NSP1 proteins (adapted from Xu et al., 1994) with the selected RE sites highlighted indicating that the overall structure of the NSP1 proteins was maintained mostly in the hybrids constructed while

introducing the six RE sites. E indicates positions predicted to have β-sheet structure, H represents positions predicted as being α-helix and L denotes positions predicted to be in loops. Positions at which no prediction with an acceptable level of confidence could be made are indicated by ‘-’ and gaps introduced to optimize the alignment by a ‘.’.