title: A three-nucleotide mutation altering the Maize streak virus Rep pRBR-interaction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR-Rep binding.
abstract: Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA-pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA-pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover.
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa.
authors.forename: Dionne N
authors.initials: DN
authors.lastname: Shepherd
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa.
authors.forename: Darren P
authors.initials: DP
authors.lastname: Martin
authors.affiliation: John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
authors.forename: David R
authors.initials: DR
authors.lastname: McGivern
authors.affiliation: John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
authors.forename: Margaret I
authors.initials: MI
authors.lastname: Boulton
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Thomson
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D026762:Maize streak virus / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D016160:Retinoblastoma Protein / Q000378:metabolism*; D003313:Zea mays / Q000821:virology
chemical_list: D004268:DNA-Binding Proteins; D016160:Retinoblastoma Protein; D004247:DNA