Structural and functional analysis of the 3′ untranslated region of bamboo mosaic potexvirus genomic RNA

The secondary structure of a 170 nt transcript derived from a cDNA clone containing the 3' untranslated region of bamboo mosaic potexvirus (BaMV) with 32 adenine residues of the poly(A) tail, was investigated in solution by using enzymatic and chemical probes. Three consecutive stem-loops forming a cloverleaf-like structure (domain ABC) and a major stem-loop (domain D) containing a bulge and an internal loop were identified as connected to a previously identified pseudoknot domain (domain E) comprising at least 13 adenylate residues of the 3' poly(A) tail. The highly conserved hexamer nucleotides (ACc/uUAA) among potexviruses are located in loop D and the putative polyadenylation signal (AAUAAA) is located in the internal loop of domain D. Based on the data of the structural probing, a three-dimensional structure was modeled. Mutants with domain ABC deleted showed no detectable signal in protoplasts, while changes in domain D, except for the bulge deletion, showed interference of BaMV RNA accumulation in protoplasts. Mutants with disrupted stem D formation impaired BaMV accumulation. However, the mutant with compensatory mutations restored stem formation which could only improve the viral accumulation to 58% that of the wild-type structure. (C) 1999 Academic Press.