Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

E.P Fontes,P.A Eagle,P.S Sipe,V.A Luckow,L Hanley-Bowdoin

doi:10.1016/s0021-9258(17)37216-2

E.P Fontes, P.A Eagle + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(17)37216-2

Copy DOI

Abstract

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Highlights

The geminivirus, tomato golden mosaicvirus (TGMV), encodes one protein, AL1, that isabsolutely required for viral DNA replication
We have investigated the nature ansdignificance of fillorigin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays
The ALJ Binding SiteIs Located between TGMV Positions 54 and 84-An earlier study established that tThGe MV AL1 prowere subcloned into pNSB69 digested with NdeI, repaired using Kle- tein expressed ineithertransgenic Nicotiana benthamiana and digested withBgZII

Summary

11 Towhom correspondence shouldbe addressed

Dept. of Biochemistry, Box7622, North Carolina State University, Raleigh, NC 276957622. The function of AL1 in replication is (34).Thefinalconcentration of radiolabeled probe DNA was 1 m not known,the protein shows limited sequence identity to site- (-60,000 cpm), and competitorDNA concentrations rangedfrom 2.5 to specific endonucleases that mediate initiation of rolling circle. Binding to specific DNA sequences distinct from the cleavage Bound DNAin reactions with competitor was normalized to bDoNuAnd sites in their replication origins (32, 33). The 52-bp fragment overlaps the TGMV origin of replication and includes the transcription start site in reactions without competito(1r 00%).The indexof competition (IC6,,) is the concentration of competitor DNA that caused a 50% reduction in the binding of radiolabeled probe to ALl. Probe and competitor DNAs were prepared by restriction enzyme digestion followed by fractionation on agarose gels and purification via and TATAboxof the AL1 promoter. Competitor DNAs were isolated from pNSB73 (122 and 15 bp)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION