Studies on the 5' non-coding region of the genome of poliovirus.
thesisposted on 19.11.2015, 09:03 by Michael A. Sullivan
The last decade has seen widespread application of recombinant DNA technology to the study of picornaviruses. Comparative sequence analysis has revealed that the most highly conserved region amongst many members of this family of viruses is the 5' non-coding region. Using recombinant type 3 polioviruses it has been shown that a single point mutation located in this region dramatically reduces neurovirulence and inhibits the intracellular life-cycle of the virus. Mutation at this nucleotide contributes to the observed reversion to neurovirulence of the Sabin attenuated poliovirus type 3 vaccine strain currently used in vaccination programmes throughout the world. Knowledge concerning the function of the 5' non-coding region remains scant, and as a result, the mechanism whereby a single point mutation within this region results in alteration of the expressed phenotype of the virus remains unknown. Clearly, an understanding of the molecular mechanism(s) involved requires greater knowledge of the function of the 5' non-coding region. This thesis describes the design and construction of vectors that allow analysis of the role of the 5' non-coding region in the control of viral translation, replication, and encapsidation of viral RNA. In the plasmid pRSV-5'polio-CATm2 (N+), the 5' non-coding region of poliovirus was fused to the coding region of the bacterial chloramphenicol acetyltransferase reporter gene. The presence of the 5' non-coding region resulted in the inhibition of CAT expression when this plasmid was introduced into eukaryotic cells in culture. Deletion analysis of the 5' non-coding region in this vector identified two regions that were responsible for the marked inhibition of expression of the reporter gene. It would appear from the results of these experiments that the poliovirus/CAT chimaeric message is translatsed as a normal eukaryotic mRNA and is subject to the rules of the "scanning model". This observation suggests that the 5' non-coding region of poliovirus on its own does not possess features which enable a message containing it to be translated efficiently. It is concluded that a second factor, present in infected cells, is required for the efficient translation of poliovirus. A second plasmid was designed and constructed to investigate the role of the 5' non-coding region in replication and encapsidation of viral RNA. Preliminary data suggest that the product of this vector does undergo replication while its ability to be encapsidated has still to be tested.