Interaction between Geminivirus Replication Protein and the SUMO-Conjugating Enzyme Is Required for Viral Infection

Abstract

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transientreplication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.Geminiviruses constitute a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (77, 82). They infect a broad range of plants and cause devastating crop diseases (57, 63). The family Geminiviridae is classified into four genera, Begomovirus, Curtovirus, Topocuvirus, and Mastrevirus, based on their genome organizations, host ranges, and insect vectors (25,26). The largest genus corresponds to the begomoviruses, which can have bipartite genomes (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV).Geminiviruses replicate through double-stranded DNA (dsDNA) intermediates (34). Begomoviruses encode two proteins involved in viral replication. AL1 (also called AC1, C1, and Rep) is essential for replication (23), while AL3 (also called AC3, C3, and REn) enhances viral DNA accumulation (86). AL1 is a multifunctional protein that mediates the virusspecific recognition of its cognate origin (28), is required for the initiation and termination of viral DNA synthesis (28,49,70), and acts as a DNA helicase (18,19). A variety of protein interactions have been demonstrated for TGMV AL1 and other geminivirus replication proteins, including the formation of homomultimers (72) and interactions with AL3/REn (83, 84) a...

SCE1, the SUMO-Conjugating Enzyme in Plants That Interacts with NIb, the RNA-Dependent RNA Polymerase of Turnip Mosaic Virus, Is Required for Viral Infection

“…To detect the SUMOylated form of NIb, an NIb-His fusion protein was transiently expressed in plants. However, possibly due to the reversible nature of the SUMOylation process or to only a very small amount of viral protein being SUMOylated, as suggested previously (20), the SUMOylated form of NIb was not detected by immunoblotting (data not shown). An E. coli strain system that was reconstituted with the SUMO conjugation pathway (30) was employed for SUMOylation assay.…”

“…3C). This phenomenon was frequently ob- served for proteins containing multiple SUMOylation sites, possibly owing to the fact that each SUMOylation site may be conjugated by a different number of SUMO molecules (20). Indeed, analysis of the entire NIb by use of the SUMOylation prediction program revealed additional potential SUMOylation motifs (data not shown).…”

“…So far, the only published data related to plant viruses have come from studies of Tomato golden mosaic virus (TGMV), a geminivirus. In this case, the interaction between SCE1 and the viral replicase protein AL1 is essential for the infectivity of this DNA virus (19,20).…”

Quantitative Proteomics Reveals Factors Regulating RNA Biology as Dynamic Targets of Stress-induced SUMOylation in Arabidopsis

“…Similar connections have been observed in animals, including roles in anaerobic tolerance (34,35) and genotoxic stress (36). Numerous plant and animal pathogens also have been shown to synthesize factors that promote infection by interfering with host SUMOylation (37,38).…”

Ubiquitin and Plant Viruses, Let’s Play Together!

“…Further analysis revealed that the interaction between Rep and SUMO E1 is required for viral DNA replication and viral infectivity. Overexpression of Rep did not alter the general SUMOylation pattern of plant proteins, but as few additional conjugated proteins were detected, it was proposed that modulation of SUMOylation may be limited to specific host proteins, which remain to be defined (Sánchez-Durán et al, 2011).…”