Geminivirus replication origins have a modular organization.

Abstract

Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for replication, and their A components encode trans-acting factors required for this process. We showed that virus-specific interactions between the cis-and trans-acting functions are required for TGMV and BGMV replication in tobacco protoplasts. We also demonstrated that, similar to the essential TGMV AL1 replication protein, BGMV AL1 binds specifically to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificities of the B components were exchanged by site-directed mutagenesis, but the resulting mutants were not replicated by either A component. These results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Geminivirus genomes also contain a stem-loop sequence that is required for origin function. A BGMV B mutant with the TGMV stem-loop sequence was replicated by BGMV A, indicating that BGMV AL1 does not discriminate between the two sequences. A BGMV B double mutant, with the TGMV AL1 binding site and stemloop sequences, was not replicated by either A component, indicating that an additional element in the TGMV origin is required for productive interaction with TGMV AL1. These results suggested that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is required for replication but does not contribute to virus-specific recognition of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.

Regulated Nuclear Trafficking of rpL10A Mediated by NIK1 Represents a Defense Strategy of Plant Cells against Virus

“…Tobacco leaves were biolistically inoculated with partial tandem repeats of Tomato golden mosaic virus (TGMV) DNA-A and DNA-B [26]. Likewise, NIK1-overexpressing tomato plants at the six-leaf stage were inoculated with tandem repeats of ToYSV DNA-A and DNA-B [29] by biolistic delivery [44].…”

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants

“…Cells were transfected with 15 μg of wild‐type or mutant TGMV A replicon DNA. Total DNA was extracted 3 days post‐transfection, digested with Xho I and Dpn I, and analyzed for viral DNA accumulation by DNA gel blot hybridization using a TGMV A specific probe (Fontes et al ., 1994b). Viral DNA was quantified by phosphoimager analysis.…”

Replication of Plant Viruses

“…The replication origin for mastreviruses consists of a large cis-acting region where the Rep protein forms multiple complexes (Castellano et al, 1999) and that for the begomoviruses contains one binding site for Rep (Lazarowitz et al, 1992;Fontes et al, 1992Fontes et al, , 1994bOrozco and Hanley-Bowdoin, 1998). The replication origin for mastreviruses consists of a large cis-acting region where the Rep protein forms multiple complexes (Castellano et al, 1999) and that for the begomoviruses contains one binding site for Rep (Lazarowitz et al, 1992;Fontes et al, 1992Fontes et al, , 1994bOrozco and Hanley-Bowdoin, 1998).…”