黄病毒NS‘的研究课件.ppt

* The research of NS1’ 18 h after infection with WT or A30P KUNV viruses at an MOI of 5 Vero cells were transfected with DNA constructs coding for WT or A30P-mutated NS1-NS2A gene The putative pseudoknot structure in the NS2A gene is indeed required for the production of NS1’ and that the disruption of this RNA structure abolishes production of NS1’
. To con?rm the role of the slippery heptanucleotide and pseudoknot-forming sequences in the generation of NS1’ during KUNV infection, two viral mutants were constructed: A30A’(silent alanine) and FSSM (frameshift silent motif). A30A’ : contains two silent mutations, GCC AAG to GCU AAA, incodon positions 30/31 of the NS2A coding sequence FSSM : contains two silent mutations in the slippery heptanucle- otide, from U CCU UUU to U CCA UUC 4G4 (NS1/NS1’ speci?c) or FS-ab (NS1’ speci?c) These results demonstrate that, in the context of KUNV viral infection, production of NS1’ requires the presence of both a slippery heptanucleotide and a 3
adjacent pseudoknot-forming sequence. The presence or absence of NS1’ did not seem to play a role in virus replication in the examined cell lines; all mutant viruses accumulated to similar levels in BHK cells, and all but A30P virus replicated with similar ef?ciencies in C6/36 cells Absence of NS1’ correlates with reduced neuroinvasiveness in mice. Thus, viruses containing silent mutations in the frame- shift motif or pseudoknot structure showed reduced neuroinvasiveness, with the effect being intermediate between the WT and A30P viruses. Sequencing of viral progeny isolated from the brain of infected animals showed the retention of the introduced mutations (data not shown), demonstrating that mortality observed in mice inoculated with any of the frame- shift mutants was not due to reversions to the WT sequence. Overall, these results demonstrate that NS1’ plays some role in the neuroinvasiveness of KUNV. Cellular localization of plasmid-expressed NS1’ and NS1. Immuno?uoresc