Functional implications of residues of the B′ motif of geminivirus replication initiator protein in its helicase activity

Abstract

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site-and strand-specific endonuclease, ligase, ATPase, and helicase. B' motif and b-hairpin loop of the geminivirus Rep are conserved and important for Rep-mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and b-hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild-type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a b-hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate in planta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and b-hairpin loop of the C-terminal region of the Rep protein.

Betasatellite-encoded βC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein

“…The motor function of Rep protein is driven by ATP hydrolysis activity of its C-terminal region. Recent studies have shown Lys-272 (Bʹ motif residue), Lys-286 and Asp-289 [27,43,44]. In the current study, the Rep protein region associated with the ATP hydrolysis and DNA unwinding function of the protein was found to be the region interacting with the βC1 protein.…”

“…The motor function of Rep protein is driven by ATP hydrolysis activity of its C-terminal region. Recent studies have shown Lys-272 (Bʹ motif residue), Lys-286 and Asp-289 (hairpin loop residues), Lys-227 (Walker-A residue), and Asp-261 and Asp-262 (Walker-B residues) to be the crucial amino acid residues for Rep protein function [9–11, 27]. The residues involved in ATP hydrolysis, DNA binding and DNA helicase function of Rep protein are conserved across the super family 3 (SF3) helicase members [27].…”

“…The residues involved in ATP hydrolysis, DNA binding and DNA helicase function of Rep protein are conserved across the super family 3 (SF3) helicase members [27]. Geminivirus-Rep proteins encoded by tomato leaf curl New Delhi virus, tomato yellow leaf curl virus, bean golden mosaic virus, African cassava mosaic virus, beet curly top virus, turnip curly top virus, tomato pseudo curly top virus, maize streak virus and wheat dwarf virus have been reported to have the conserved SF3 signatures [27, 40–42]. Interestingly, various SF3 helicase proteins encoded by viruses belonging to several other virus families – with examples of these proteins including E1 protein encoded by papillomaviruses, large T antigen encoded by polyomaviruses, Rep78 encoded by dependoparvovirus and NS protein encoded by protoparvoviruses – have been found to include the conserved Walker A, Walker B and B′ motif residues [27, 43, 44].…”

“…To delineate the βC1protein-meditated inhibition of ATP hydrolysis activity of Rep protein, we attempted to identify the amino acid residues of Rep protein that are vital for its interaction with βC1. The residues of Rep protein essential for its function, in particular Walker-A residue K227, Walker-B residue D262, Bʹ motif residue K272, and hairpin loop residues K286 and D289 [24,27], were selected for the interaction studies. For this, a Y2H assay was carried out to test whether βC1 can interact with any of the mutated RepC proteins, namely RepC-D227A, RepC-D262A, RepC-K272A, RepC-K286A and RepC-D289A.…”

Rapid multilocus adaptation of clonal cabbage leaf curl virus populations toArabidopsis thaliana

“…The coordinates of the oligomerization domain and RETINOBLASTOMA protein interaction surface are inferred based on alignment to the Rep protein sequence from tomato golden mosaic virus (Orozco and Hanley-Bowdoin 1998, Kong et al 2000, Reyes 2012). Walker motifs were inferred based on Ruhel et al (2021). Positions of amino acids L145 and E176 are shown with a purple line and a blue line.…”