We examined whether the 130K and 180K proteins of tobacco mosaic virus (TMV), the putative virus-encoded replicase components, produced by a replication-competent TMV mutant could complement a replication-defective mutant in a single cell. The replication-competent mutant (LDCS29) had a deletion in the coat protein gene and the replication-defective mutant (LDR28) had a large deletion in the gene encoding the 130K and 180K proteins. Neither the replication of LDR28 nor the production of the coat protein from LDR28 or LDCS29 was detected when the mutants were inoculated separately into tobacco protoplasts. However, when the two mutants were co-inoculated, the production of the LDR28 genomic RNA and the subgenomic RNA for the coat protein and accumulation of the coat protein were observed. These results show that the virus-encoded replicase components of TMV complemented the replication-defective mutant in trans.
"Replicase components produced by a replication-competent mutant of TMV are able to complement replication-defective mutants with various deletions in trans (Knapp et al., 2001; Lewandowski & Dawson, 1998; Ogawa et al., 1991, 1992). However, efficient transcomplementation of a full-length replication-deficient genome is uncommon among tobamoviruses (Ogawa et al., 1991). Full-length defective RNA encoding only the 126-kDa protein could not be complemented in trans by a construct encoding the functional 126/183-kDa proteins (Lewandowski & Dawson, 1998; Ogawa et al., 1992), as it contains sequences that are inhibitory to replication (Lewandowski & Dawson, 1998). "
[Show abstract][Hide abstract] ABSTRACT: Sequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of T-->C at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.
Journal of General Virology 09/2004; 85(Pt 8):2447-57. DOI:10.1099/vir.0.80070-0 · 3.18 Impact Factor
"The replication of tobamovirus genomic RNAs appears to occur primarily by a cis-preferential mechanism based on the inability of mutant full-length viruses to be replicated in trans by a competent helper virus (Lewandowski and Dawson, 1998). However, it is known that tobamovirus replicase complexes can amplify RNAs in trans since a satellite virus of TMGMV occurs in nature (Valverde and Dodds, 1986) and, more recently, it was found that TMV and ToMV RNAs could be replicated in trans if specific internal sequences were removed (Ogawa et al., 1991, 1992; Raffo and Dawson, 1991; Lewandowski and Dawson, 1998). The 3Ј replication signals required for the cis-preferential replication of the tobamovirus genomic RNA have been identified (Takamatsu et al., 1990, 1991; Watanabe et al., 1996) and are contained primarily within the NTR. "
[Show abstract][Hide abstract] ABSTRACT: The viral replicase complex of positive-stranded RNA viruses interacts with cis-acting elements that are usually located at the termini of the viral RNAs. On comparison of the replication requirement of a tobacco mosaic virus (TMV)-based defective RNA (dRNA) and its helper virus, we found different requirements for replication of TMV RNAs in cis and in trans. The level of replication of full-length TMV RNA decreased substantially in the absence of pseudoknot (pk) 1 and/or 2, whereas identical deletions in dRNAs did not affect their replication. However, pk3 was required for replication of both full-length TMV RNAs and dRNAs. The requirements for homologous sequences were greater for dRNA replication than for replication of full-length TMV RNAs. Defective RNAs with heterologous 3′ nontranslated regions (NTRs) failed to be replicated or replicated minimally, whereas replication of similarly mutated full-length RNAs was much less affected. Increasing amounts of contiguous heterologous sequences in the dRNAs compensated for the impaired interactions between the replicase and 3′ NTR. The precision requirement appeared to involve the terminal 28 nucleotides, specifically the pseudoknot in the aminoacyl acceptor arm of the tRNA like structure, which was important in replication of both dRNAs and full-length TMV RNAs.
"for replication of TMV RNA in tobacco protoplasts was referred to earlier . TMV deri - vatives with most of the coding regions for the 126 - kDa / 183 - kDa proteins removed also replicated well in proto - plasts , and spread in plants , in the presence of a wild - type TMV helper virus ( Ra¡o & Dawson 1991 ) . Similar observations were reached by Ogawa et al . ( 1991 , 1992 ) , who also noted that removal of the 3 ' - terminal one - third of the read - through portion of the coding region for the 183 - kDa protein increased the accumulation of this subgenomic replicon , suggesting that this sequence may have a regulatory role in RNA replication ."
[Show abstract][Hide abstract] ABSTRACT: The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.
Philosophical Transactions of The Royal Society B Biological Sciences 04/1999; 354(1383):613-27. DOI:10.1098/rstb.1999.0413 · 7.06 Impact Factor
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