Detection and Characterization of Viral cDNAs Amplified from Cells Expressing the JC Virus Early Coding Sequences.

Open Access
Author:
Barnes, Erin Stockwell
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Richard John Frisque, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • JC Virus
  • cDNA characterization
Abstract:
The early region of human polyomavirus JC virus (JCV) produces five proteins, large T antigen (TAg), small t antigen (tAg), and T´135, T´136, and T´165, encoded by transcripts alternatively spliced from one pre-mRNA. These multifunctional regulatory proteins mediate viral DNA replication and contribute to the oncogenic potential of the virus. Initially proposed to be proteolytic cleavage products of JCV TAg, the three T´ proteins have been shown to be authentic viral phosphoproteins that participate in JCV’s viral activities. T´ transcripts are generated by the removal of two introns from unspliced JCV early pre-mRNA; the first intron also being removed in the TAg transcript. Since the splice variants share their amino termini with TAg, all three T´ proteins contain the Rb binding motif LxCxE and the Hsc70 binding J domain, and thus exhibit some notable TAg functions such as stimulation of cell proliferation. The T´ cDNAs were first observed on polyacrylamide gels in RT-PCR experiments, in which RNA was extracted from JCV-infected cells, reverse transcribed into cDNA, and amplified by PCR. Five bands representing cDNAs encoding the JCV early T proteins were visualized on a polyacrylamide gel, two slower migrating “doublet” bands, and three faster migrating T´ bands 17-23 kD in size. Based upon the primers used, the TAg and tAg cDNAs are expected to be the same size and therefore form a single band positioned above the three T´ bands. The appearance of two bands has yet to be explained in an experimental setting, so to unravel this mystery, I have attempted to clone and identify the origin of these sequences within the cDNA doublet bands. Through a number of experimental approaches, our results have shown the repeated detection of the T´136 splice pattern within the cDNAs isolated from the doublet band. We believe that a small amount of T´136 cDNA remains “trapped” in the larger TAg/tAg cDNAs, and is preferentially selected for detection by our experimental methods. Further investigation is needed to conclude that TAg/tAg is the predominanat species in one of the doublet bands. Additional analysis will also be required to determine the origin of the non-TAg/tAg cDNA doublet band.