Structural and Functional Analysis of the 3 0 Untranslated Region of Bamboo Mosaic Potexvirus Genomic RNA Chi-Ping Cheng and Ching-Hsiu Tsai* Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan The secondary structure of a 170 nt transcript derived from a cDNA clone containing the 3 0 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 identi®ed as connected to a previously identi®ed pseudoknot domain (domain E) comprising at least 13 adenylate residues of the 3 0 poly(A) tail. The highly conserved hexamer nucleotides (ACc/uUAA) among potexviruses are located in loop D and the putative polyadenylation sig- nal (AAUAAA) is located in the internal loop of domain D. Based on the data of the structural probing, a three-dimensional structure was mod- eled. 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. How- ever, the mutant with compensatory mutations restored stem formation which could only improve the viral accumulation to 58 % that of the wild-type structure. # 1999 Academic Press Keywords: bamboo mosaic potexvirus; RNA; 3 0 UTR; structural mapping; pseudoknot *Corresponding author Introduction Bamboo mosaic potexvirus (BaMV) has a ¯exu- ous rod-shaped morphology (Lin et al., 1977) and consists of a single-stranded, positive sense RNA genome with a 5 0 m7GpppG structure and a 3 0 poly(A) tail. The entire nucleotide sequence of iso- late O comprising 6366 nt (excluding the 3 0 poly(A) tail) has been determined (Lin et al., 1994). Five open reading frames (ORF1 to 5) typical of potex- viral genomes encode 155 kDa, 28 kDa, 13 kDa, 6 kDa, and 25 kDa polypeptides, respectively. Only the 155 kDa polypeptide can be synthesized directly from the virion RNA of an in vitro rabbit reticulocyte lysate (Lin et al., 1992). Two major sub- genomic RNAs of 2.0 and 1.0 kb in length are 3 0 co-terminal (Lin et al., 1992). ORF1 encodes a protein containing a methyl- transferase-like domain (Rozanov et al., 1992), an RNA helicase-like domain (Hodgman, 1988; Gorbalenya et al., 1989), and an RNA-dependent RNA polymerase domain (Argos, 1988; Koonin, 1991) in order from the N to the C terminus. ORF2 to 4 of white clover mosaic potexvirus were shown to be required for viral cell-to-cell movement (Beck et al., 1991; Lough et al., 1998); ORF5 encodes the viral coat protein. The 3 0 untranslated regions (UTR) of single- stranded positive sense RNA viruses have been identi®ed in several different species to form a ter- tiary structure which is reported to play an essen- tial role in viral RNA ampli®cation. The 3 0 ends of brome mosaic virus (BMV) and turnip yellow mosaic virus (TYMV) RNAs terminate in tRNA- like structures that can be speci®cally tyrosylated and valylated, respectively. Alterations in tRNA- like structures of both viral RNAs interfered with infectivity (Dreher et al., 1989, 1992; Tsai & Dreher, E-mail address of the corresponding author: chtsai1@dragon.nchu.edu.tw Abbreviations used: BaMV, bamboo mosaic virus; ORF, open reading frame; UTR, unstranslated region; BMC, brome mosaic virus; TYMV, turnip yellow mosaic virus; IL, internal loop; DEPC, diethylpyrocarbonate; RdRp, RNA-dependent RNA polymerase. Article No. jmbi.1999.2716 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 288, 555±565 0022-2836/99/190555±11 $30.00/0 # 1999 Academic Press