Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion

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 TRC 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.

A Toxic Mutator and Selection Alternative to the Non-Mendelian RNA Cache Hypothesis for hothead Reversion

“…Different proteins display different responses to selection for mutant reversion. With some proteins, precise reversion of deleterious mutations is the predominant response when strong selection for restoration of protein activities is applied (Rossiter et al, 1990;Coombs et al, 1994;Gee et al, 1994;Wang et al, 2004). It is possible that the HTH protein may belong to this category and that its activity may be difficult to restore by secondary intragenic changes.…”

An infectious RNA with a hepta-adenosine stretch responsible for programmed −1 ribosomal frameshift derived from a full-length cDNA clone of Hibiscus latent Singapore virus

“…Among plant viruses, reversions of mutated sites to wild-type sequences are not uncommon (Arguello-Astorga et al, 2007;Shepherd et al, 2005;Wang et al, 2004). In HLSV, the 7A stretch was extended to 8A during its replication.…”

“…Therefore, HLSV with 8A becomes dominant in HLSV-7A inoculated leaves at 6 dpi. For tobamoviruses such as Tobacco mosaic virus and Odontoglossum ringspot virus, their replicase proteins produced by a replication-competent mutant are able to complement replication-defective mutants in trans but the efficiency is low (Ogawa et al, 1991;Wang et al, 2004). Thus, the dominance of HLSV with 8A is due to its faster replication through production of higher amount of replicase proteins.…”

Exploring the Multifunctional Roles of Odontoglossum Ringspot Virus P126 in Facilitating Cymbidium Mosaic Virus Cell-to-Cell Movement during Mixed Infection

“…This single-stranded, positive-sense RNA genome has four open reading frames (ORFs) that encode P126 replicase and its readthrough product P183, movement protein (MP) and capsid protein (CP). The mutation of Phe 50 to Ser 50 in P126/183 of an ORSV infectious cDNA clone abolished ORSV replication [26]. Moreover, the ORSV replicase is responsible for host range restriction in Nicotiana sylvestris [27].…”