Strucutre-function studies of the venezuelan equine encephalitis virus 5'UTR promoter element and its role in attenuation of the virus

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RNA structural elements play critical roles in several viral processes. An \r\nattempt to elucidate the role of one such RNA structural element encoded by the 5’ \r\nUntranslated Region (5’UTR), in regulating virus replication and attenuation of \r\nVenezuelan equine encephalitis virus (VEEV) is described. VEEV is one of the \r\npathogenic members of the Alphavirus genus in the Togaviridae family. VEEV \r\ninfection causes debilitating illness complicated by neurological manifestations. The \r\nonly available vaccine for VEE infection, the attenuated strain VEEV TC-83 provides \r\nminimal protection against virulent strains, but the molecular basis for its attenuation \r\nremains poorly understood. Interestingly however, the attenuation of TC-83 was \r\nshown to strongly depend on two point mutations, one of which, the G3A mutation, \r\nwas found in the 5’UTR of the viral genome. \r\nResults from my biochemical and biophysical studies demonstrate that the \r\nG3A mutation strongly affects the structure of the VEEV 5’UTR. Further functional \r\nanalysis revealed that this change in 5’UTR RNA structure affects various processes \r\nin virus replication. The G3A mutation moderately enhanced translation of the \r\ndownstream polyprotein, and strongly increased replication of the viral genome, but \r\nled to a significant decrease in the synthesis of subgenomic RNA (sgRNA). Based on \r\nmy findings and those of others, I propose a model for attenuation of the vaccine \r\nstrain TC-83. The enhanced functionality of the TC-83 5’UTR in viral processes prompted further investigation into the structural requirements within the VEEV \r\n5’UTR for efficient virus replication. \r\nResults from these studies revealed that the sequence, secondary structure and \r\nstability of the stem-loop in this region are critical for virus replication. Mutations \r\naffecting any of the above resulted in pseudorevertants that either acquired \r\ncompensatory AU or AUG repeat sequences in the 5’UTR, or accumulated mutations \r\nin the VEEV non-structural proteins. Results from my mutational analyses thus \r\nprovide evidence that during the replication of the viral genome, the ends of the \r\ndsRNA replication intermediate stay single stranded and fold into individual stem- \r\nloops that are critical for virus replication, and the sequence and folding determines \r\nthe efficiency of the promoter in this region for genomic RNA synthesis.