In vitro transcription and translation of the human pathogenic parvovirus B19 nonstructural (NS1) gene : an honors thesis [(HONRS 499)]

The human parvovirus B19 contains inverted terminal repeats (ITRs) which form stable hairpin structures. Although ITRs have not been shown to be transcribed, recent studies indicate that they function as enhancers. To define the role of ITRs in transcription, we subcloned the B19 non-structural (NS1) gene, with or without the ITR sequence upstream of the NS1 gene, into a prokaryotic vector pSP65. Plasmid pWS602 contained the ITR sequence whereas this sequence was deleted in plasmid pWS603. Both plasmids were transcribed in vitro using the SP6 RNA transcription system. Equivalent amounts of in vitro synthesized mRNAs were translated in vitro using the rabbit reticulocyte lysate system. Whereas mRNA transcribed from pWS603 yielded two NS1 proteins of mol. wts. 78 kD and 68 kD respectively, no translation of NS1 proteins occurred with mRNA transcribed from pWS602. These studies document that the presence of a hairpin structure upstream of the NS1 gene has no effect on the efficiency of transcription but effectively inhibits translation of the NS1 gene products in vitro. These results are thus consistent with the observed lack of expression of ITRs which, in turn, would negatively affect the production of viral gene products in vivo.Honors CollegeThesis (B.?.