Structural and Functional Analysis of the cis -Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

Abstract

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3 terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/⌬5, with a deletion of the 3-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/⌬16 and Ba-77/⌬31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3 end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.There are at least two major steps involved in the replication of positive-sense viral RNA: the first is the synthesis of the complementary strand of genomic RNA, which is followed by the generation of progeny RNAs using the newly synthesized minus-strand RNA templates (37). For these reactions to proceed faithfully, the 3Ј ends of both plus-and minus-strand RNAs must contain important elements, the sequences and/or structures needed to direct the replicase to initiate the RNA synthesis (7, 18). The 3Ј-terminal secondary structures of Alfalfa mosaic virus RNAs (12,13,33), Turnip crinkle virus RNAs (39), and Tomato bush stunt virus defective interfering RNA (8) and the 3Ј tRNA-like structures (TLS) of Brome mosaic virus (BMV), Tobacco mosaic virus, and Turnip yellow mosaic virus (TYMV) RNAs have been identified to be important for minus-strand RNA synthesis (5,6,17,26,36).Bamboo mosaic virus (BaMV) is a single-stranded positivesense RNA virus. Its 6,366-nucleotide (nt) genome [excluding the poly(A) tail] contains a 5Ј m 7 GpppG structure and a 3Ј poly(A) tail (24). Five open reading frames (ORF) encoding polypeptides of 155, 28, 13, 6, and 25 kDa could be identified in the genome. Besides the genomic RNA which encodes the 155-kDa protein comprising a capping enzyme domain (21, 22), a 5Ј triphosphatase and helicase-like domain (9,11,16), and an RNA-dependent RNA polymerase (RdRp) activi...

Structural homology between bamboo mosaic virus and its satellite RNAs in the 5'untranslated region

“…We note that, in the region which forms critical structures in both positive-and negative-strand RNA, double mutations that were designed for maintaining structures in one strand may seriously disrupt structures present in the other strand. For instance, the unexpected decrease in the accumulation of our mutant M2 may also be attributed to alterations in the proposed negative-strand hairpins (Lin et al, 2005). Likewise, the surprisingly low replication observed by Lin et al (2005) of the double mutant (BaLSR), which was designed to restore the stem of a hairpin in negative-strand RNA, could be the result of disrupting stem-I and stem-III in positive-strand RNA (Fig.…”

“…2c; Lin et al, 2005), it is of interest to address how these substitutions interfered with the secondary structure of our model for positive-strand RNA. We note that, in the region which forms critical structures in both positive-and negative-strand RNA, double mutations that were designed for maintaining structures in one strand may seriously disrupt structures present in the other strand.…”

“…For BaMV, a previous study of the corresponding region focused on cis-acting elements in negative-strand RNA (Lin et al, 2005). For PVX, the sequence of AL, IL-I and the C : C mismatch were shown to be critical for gRNA accumulation (Miller et al, 1998(Miller et al, , 1999, although a C : A mismatch was temporarily functional (Miller et al, 1999).…”

“…These structural elements have been implicated in the initiation of minus-strand synthesis by the viral RNAdependent RNA polymerase (Huang et al, 2001;Cheng et al, 2002). However, structural elements in the 59UTR of BaMV gRNA that potentially play roles in replication and/or translation have not been investigated in detail, although two stem-loop structures in the corresponding region of the negative-strand RNA have been suggested to be involved in positive-strand RNA synthesis (Lin et al, 2005).Using Mfold (Zuker, 2003), a large stem-loop structure was predicted in the 59 117 nt of BaMV positive-strand RNA (Fig. 1a).…”

Cell-to-cell movement of three genera (+) ss RNA plant viruses

“…In line with the given assumptions, MP can either directly or indirectly change properties of the wall surrounding PD, which can lead to PD broadening. Possibly it is connected with transport to ER with the use of cytoskeleton or ER vesicles which contains MP-vRNA complex moving across PD (Kawakami et al 2004;Boevink and Oparka 2005;Lin et al 2005;Wright et al 2007). When MP is not present, PD ER is stationary (Heinlein and Epel 2004).…”