Investigating the role of rotavirus GS10(NSP4) and GS6(VP6) in reassortment

Abstract

Rotaviruses are dsRNA viruses and are the leading causative agent of severe gastroenteritis. All current licenced rotavirus vaccines contain live-attenuated strains and are capable of reassorting their genome segments with those of co-circulating field strains. This requires elucidation of the determinants of reassortment to produce vaccine seed strains that incorporate novel safety features. Rotavirus GS6(VP6) and GS10(NSP4) are possible candidates. Analyses of circulating field strains have shown that GS6(VP6) and GS10(NSP4) co-reassort and that VP6 subgroup and NSP4 genotype is often associated. Reverse genetics can be used to interrogate the functions of specific amino acids in protein-protein interactions. A NWU SA11-N5 pSMART reverse genetics system was developed previously, but it has not been reproducible due to plasmid instability. To start to elucidate the mechanism of GS6(VP6)/GS10(NSP4) reassortment, the stability of the SA11-N5 pSMART plasmid set needed to be improved. Previous single plasmid NGS and Sanger sequencing data of the SA11-N5 pSMART plasmid set was reanalysed and showed that the plasmids containing GS7(NSP3) and GS9(VP7) did not contain ribozymes, and could be traced back to the original glycerol stocks. It was decided to reorientate the transcription cassettes to determine whether this improved plasmid stability by avoiding collisions between the transcription and replication machinery. All 11 plasmids were reorientated successfully. Sanger sequencing and NGS showed that they were still stable even when the NGS was repeated a year later from plasmids that were extracted from archived glycerol stocks. Rescue was attempted using the original protocol without success. Rescue was then attempted using seven different transfection reagents, five different transfection and infection cell lines, and an expression plasmid approach. Rescue could not be achieved in any attempt. Eventuallly, PCR testing of our transfection and infection cell lines showed that our MA104 and ST cells were heavily contaminated with mycoplasma. The cell lines were exchanged with uncontaminated ones, but rescue could not be attempted extensively. This resulted in the use of the SA11-L2 pT7 system for the remainder of this project. Reverse genetics showed that NSP4 amino acids 169 and 174 may be important in determining its receptor interaction with VP6. Random NGS-generated sequences of 36 RVA VP6 and 38 NSP4s from Genbank were analysed. For DS-1-like strains NSP4 amino acids 169K/S and 174A/S were observed. For Wa-like strains, 169I/S and 174S were observed. For VP6 DS-1-like strains (SGI, I2), strains had 172A, 305A, and 306A at their subgroup determining epitopes. Whereas, the Wa-like strains (SGII. I1) had 172M, 306N, and 306A.

VP6s from a Wa-like Moz0060a and Moz308 strain, an SA11 NSP4 with the Moz0060a binding domain, and two SA11 NSP4s, SA11 NSP4 R169I and SA11 NSP4 A174S were tested. Only rSA11 Moz308 VP6, rSA11 NSP4 0060a DLP-BD, rSA11 NSP4 R169I were rescued. rSA11 Moz0060a VP6, rSA11 Moz0060a VP6 DLP-BD, and rSA11 NSP4 A174S could not be rescued. Growth kinetics showed rSA11 Moz308 VP6 replicated to a titre nearly 2 logs lower than rSA11. A single amino acid difference in the VP6/VP2 contact may destabilise the DLP. Alhough VP4 and VP7 also contact VP6, no significant amino acids changes were seen in the contacts been SA11 and the Moz308 strain. rSA11 NSP4 R169I replicated to a titre similar to rSA11. Non - rescue of rSA11 NSP4 A174S may show that this amino acid is essential to the interaction and that a non-polar/polar 169/174 pair is required in an SA11 background. This result was confirmed by the growth curve of rSA11 NSP4 0060a DLP-BD, a virus that contains a non-polar/ polar 169/174 NSP4 DLP-BD pair. Taken together this provides new insights into a rotavirus protein-protein interaction that requires further investigation. Also, additional work is required to determine the reasons for non-rescue of the SA11-N5 pSMART reverse genetics system.