FUNCTIONAL ANALYSIS OF THE UPSTREAM PSEUDOKNOT DOMAIN AND ITS SUBSTITUTION WITH A POLY(A) TRACT IN TOBACCO MOSAIC VIRUS (TMV)

Abstract

THE UPSTREAM PSEUDOKNOT DOMAIN (UPD) OF TOBACCO MOSAIC VIRUS (TMV) GENOME PLAYS AN IMPORTANT ROLE DURING VIRUS REPLICATION AND TRANSLATION. THE FUNCTIONS OF UPD WERE ANALYZED USING A SUBSTITUTED MUTANT TMV-43A IN WHICH AN INTRODUCED 43 NT POLY(A) TRACT REPLACED THE UPD IN TMV GENOME. TMV-43A DISPLAYED REDUCED REPLICATION EFFICIENCY AND INDUCED MOSAIC SYMPTOMS IN NICOTIANA BENTHAMIANA. DEEP SEQUENCING RESULTS SHOWED THAT SMALL RNA PROFILES DIFFERED BETWEEN TMV- AND TMV-43A-INFECTED PLANTS. SEVERAL VIRAL SMALL INTERFERING RNAS DERIVED FROM TMV-UPD WERE PREDICTED TO TARGET HOST GENES. RNA STRUCTURES WERE ANALYZED USING SELECTIVE 2'-HYDROXYL ACYLATION ANALYZED BY PRIMER EXTENSION (SHAPE), WHEN BOTH INTERNAL POLY(A) TRACT AND UPD WERE PRESENT IN THE VIRAL GENOME. RESULTS SHOWED THAT THE LONGER THE INTERNAL POLY(A) TRACT INTRODUCED TO A LOCATION UPSTREAM OF UPD, THE SLOWER VIRUS REPLICATION AND INFECTIVITY. IN ADDITION, A NOVEL STEM LOOP STRUCTURE THAT REGULATES VIRAL REPLICATION WAS DISCOVE