Cloned Infectious cDNAs of Lettuce Chlorosis Virus (LCV) and Characterization of a cis-Acting Element in the 3' Non-Coding Region of LCV RNA 2 Involved in Minus-Strand RNA Synthesis
- Author(s): Mongkolsiriwattana, Chawin
- Advisor(s): Ng, James C.K.
- et al.
For positive-sense single-stranded(ss) RNA virus, determinants that mediate minus-strand RNA synthesis have been often found to reside in 3'-terminal region of the viral genome. Although significant progress has been made in understanding these determinants in several model positive-sense ssRNA viruses, the knowledge of this particular aspect is significantly behind for many economically important viruses including those that belong to the genus Crinivirus (family Closteroviridae). Lettuce chlorosis virus (LCV) is a relatively new member of crinivirus with a bipartite single-stranded positive-sense RNA genome. This dissertation identified and examined determinants that mediate minus-strand RNA synthesis of LCV. Since no molecular tool of LCV was available for examining determinants involved in minus-strand RNA synthesis, Chapter 1 described the construction of infectious cDNA clones of full-length LCV genomic RNAs (1 and 2). Transcripts synthesized from infectious cDNA clones of LCV RNAs were shown to exhibit wild type LCV virion RNAs property in inoculated tobacco protoplasts: similar accumulation kinetics of RNAs 1 and 2, the synthesis of the major coat protein, and formation of virion-like particles. In addition, several novel LCV RNA 1-derived RNAs were detected, including first reported crinivirus defective RNAs derived from RNA 1. Next, we investigated determinants that mediate minus-strand LCV RNA 2 synthesis in Chapter 2 of this dissertation. First, we found that the exchange of conserved region of 3' non-coding regions (NCRs) of LCV RNAs showed no detrimental effects on minus-strand RNA synthesis, suggesting determinants involved in minus-strand RNA synthesis are similarly present in both 3' NCRs of LCV RNAs 1 and 2. Second, deletion analysis of 3' NCR of LCV RNA 2 showed that minimum sequences required for minus-strand RNA 2 synthesis lie between 25 to 98 nucleotides from the 3' terminus. Third, computer modeling and newly developed SHAPE (Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension) analysis revealed a presence of 3'-terminal stem-loop (SL) structure, and mutational analysis showed that overall structure, but not loop, top stem and internal loop, of SL is required for minus-strand RNA synthesis. In addition, SHAPE analysis of LCV RNA 2 defective RNA showed that SL is a part of a larger Y-shaped structure.