The 5′ untranslated region of Bean pod mottle virus RNA2 tolerates unusually large deletions or insertions
ABSTRACT Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA virus of Secoviridae. We recently reported that a 137 nucleotide (nt) stretch (#263-399) of the 466 nt 5' untranslated region (5' UTR) of BPMV RNA2 can be deleted without compromising BPMV propagation in host plants [Lin et al., J. Gen. Virol. (2013) 94, 1415-1420]. Here we demonstrate that nonviral insertions of up to 625 nt is tolerated by the same region. Furthermore, one insertion mutant underwent recombination in infected plants, leading to the truncation of nt #250-361, thus extending the dispensable sequence to 150 nt (nt #250-399). We are unaware of any other (+) RNA virus that tolerates insertion/deletion of these sizes (625 nt/150 nt) within its 5' UTR. Importantly, tolerance of large insertions within the RNA2 5' UTR offers a novel, more convenient site for incorporating host gene fragments, making BPMV a more versatile vector of virus-induced gene silencing.
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ABSTRACT: Chimeric genomes of poliovirus (PV) have been constructed in which the cognate internal ribosomal entry site (IRES) element was replaced by genetic elements of hepatitis C virus (HCV). Replacement of PV IRES with nt 9-332 of the genotype Ib HCV genome, a sequence comprising all but the first eight residues of the 5' nontranslated region (5'NTR) of HCV, resulted in a lethal phenotype. Addition of 366 nt of the HCV core-encoding sequence downstream of the HCV 5'NTR yielded a viable PV/HCV chimera, which expressed a stable, small-plaque phenotype. This chimeric genome encoded a truncated HCV core protein that was fused to the N terminus of the PV polyprotein via an engineered cleavage site for PV proteinase 3CPpro. Manipulation of the HCV core-encoding sequence of this viable chimera by deletion and frameshift yielded results suggesting that the 5'-proximal sequences of the HCV open reading frame were essential for viability of the chimera and that the N-terminal basic region of the HCV core protein is required for efficient replication of the chimeric virus. These data suggest that the bona fide HCV IRES includes genetic information mapping to the 5'NTR and sequences of the HCV open reading frame. PV chimeras replicating under translational control of genetic elements of HCV can serve to study HCV IRES function in vivo and to search for anti-HCV chemotherapeutic agents.Proceedings of the National Academy of Sciences 03/1996; 93(4):1412-7. DOI:10.1073/pnas.93.4.1412 · 9.67 Impact Factor
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ABSTRACT: The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.Journal of Virology 12/1997; 71(11):8868-74. · 4.44 Impact Factor
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ABSTRACT: Mutational analysis has been used to investigate cis-acting sequences in the 3'- and 5'-untranslated regions of red clover necrotic mosaic virus RNA2 required for replication in the presence of wild-type RNA1. Deletion of a sequence near the 3' end of RNA2, which is potentially capable of forming a stable stem-loop structure, abolished the ability of RNA2 to replicate in Nicotiana clevelandii protoplasts, as judged by the failure to detect production of either the positive or negative RNA strand. A base-paired structure in the stem was shown to be essential for replication, but a mutant RNA2 in which every base of the stem had been altered was able to replicate in N. clevelandii protoplasts, to produce lesions on leaves of Vigna unguiculata plants, and to infect N. clevelandii plants systemically. However, changing three bases in the loop of the potential stem-loop structure abolished the replication of RNA2. Upstream sequences in the 3'-untranslated region of RNA2 were also shown to be important for RNA2 replication. Deletions in the 5'-untranslated region of RNA2 showed that sequences across the whole of this region were needed for production of the positive strand but not for production of the negative strand. Some of the cis-acting sequences required for replication of RNA2 have almost identical counterparts in RNA1, but others appear to be unique to RNA2.Virology 02/1999; 253(1):115-24. DOI:10.1006/viro.1998.9495 · 3.32 Impact Factor