The replication initiator protein of a geminivirus interacts with host monoubiquitination machinery and stimulates transcription of the viral genome

Abstract

Geminiviruses constitute a group of plant viruses, with a ssDNA genome, whose replication in the nucleus of an infected cell requires the function of geminivirus-encoded replication initiator protein (Rep). Our results suggest that monoubiquitinated histone 2B (H2B-ub) promotes tri-methylation of histone 3 at lysine 4 (H3-K4me3) on the promoter of Chilli leaf curl virus (ChiLCV). We isolated homologues of two major components of the monoubiquitination machinery: UBIQUITIN-CONJUGATING ENZYME2 ( NbUBC2) and HISTONE MONOUBIQUITINATION1 ( NbHUB1) from N. benthamiana. ChiLCV failed to cause disease in NbUBC2-, and NbHUB1-silenced plants, at the same time, H2B-ub and H3-K4me3 modifications were decreased, and the occupancy of RNA polymerase II on the viral promoter was reduced as well. In further investigations, Rep protein of ChiLCV was found to re-localize NbUBC2 from the cytoplasm to the nucleoplasm, like NbHUB1, the cognate partner of NbUBC2. Rep was observed to interact and co-localize with NbHUB1 and NbUBC2 in the nuclei of the infected cells. In summary, the current study reveals that the ChiLCV Rep protein binds the viral genome and interacts with NbUBC2 and NbHUB1 for the monoubiquitination of histone 2B that subsequently promotes trimethylation of histone 3 at lysine 4 on ChiLCV mini-chromosomes and enhances transcription of the viral genes.

Author summary

Histone post translation modification plays vital role in regulation of gene expression in eukaryotes. In plants, Ubiquitin-Conjugating Enzyme 2 and Histone Monoubiquitination1 monoubiquitinates histone 2B that promotes trimethylation of histone H3 at lysine 4 and both modifications eventually activates host transcription. The chromatinized genome (viral DNA associated with host histone proteins) of geminiviruses serves as a potential template for histone modification enzymes. Our study reveals the role of Chilli leaf curl virus ( Geminiviridae) encoded Replication initiator protein (Rep) in stimulation of viral transcription via histone modifications on the viral minichromosome. Furthermore, the Rep protein redirects NbUBC2 from cytoplasm to the nucleus to form monoubiquitination machinery in association with NbHUB1 and recruits the machinery to accelerate the H2B-ub deposition on the viral promoter. The silencing of NbUBC2 and NbHUB1 did not alter Rep localization in the nucleus but affect ChiLCV pathogenesis. Taken together, our results showed for the first time that geminivirus Rep protein recruits the NbUBC2 and NbHUB1 on the viral promoter for monoubiquitination of H2B and stimulates viral gene expression.