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 co ntaining the 3' untranslated region of bamboo mosaic potexvirus (BaMV) with 32 adenine residues of the poly(A) tail, was investigated in solution by u sing enzymatic and chemical probes. Three consecutive stem-loops forming a cloverleaf-like structure (domain ABC) and a major stem-loop (domain D) con taining a bulge and an internal loop were identified as connected to a prev iously identified pseudoknot domain (domain E) comprising at least 13 adeny late residues of the 3' poly(A) tail. The highly conserved hexamer nucleoti des (ACc/uUAA) among potexviruses are located in loop D and the putative po lyadenylation 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, sho wed interference of BaMV RNA accumulation in protoplasts. Mutants with disr upted stem D formation impaired BaMV accumulation. However, the mutant with compensatory mutations restored stem formation which could only improve th e viral accumulation to 58% that of the wild-type structure. (C) 1999 Acade mic Press.