My laboratory is interested in the molecular biology of geminiviruses, including viral pathogenesis and the regulation of viral gene expression and DNA replication. Current work is focused on understanding host defenses directed against geminiviruses, including posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) caused by methylation of viral chromatin, and how the geminivirus proteins AL2 and L2 counter these RNA silencing-based defenses.We are also studying a basal, metabolic defense conditioned by SnRK1 (SNF1) kinase, which is also countered by AL2 and L2. Finally, we are involved in a relatively new collaboration that is allowing us to explore the possibility that a mammalian virus, Herpes Simplex Virus Type 1, is also subject to and suppresses RNA silencing-based defenses.
Members of the Geminiviridae replicate circular ssDNA genomes in the nucleus through dsDNA intermediates that associate with cellular histones to form minichromosomes.These relatively simple viruses do not encode polymerases, and instead recruit host machinery to express viral mRNAs and replicate viral DNA. These characteristics make geminiviruses valuable model systems for studying the mechanisms and epigenetic regulation of these processes.
Most of our recent work involves the AL2 protein (also called AC2) encoded by members of theBegomovirus genus (e.g. Tomato golden mosaic virus and Cabbage leaf curl virus), and the related L2 (also called C2) protein found in the genus Curtovirus (e.g. Beet curly top virus). Both AL2 and L2 are pathogenicity factors, and AL2 in addition is a transcription factor that activates expression of viral genes needed late in infection.
Viral chromatin methylation as a defense against geminiviruses, and suppression of methylation and TGS as a viral counterdefense
siRNA-directed DNA and histone methylation is a fundamental epigenetic process that in the model plant Arabidopsis thalianais accomplished via the Pol IV/Pol V/DCL3/RDR2/AGO4pathway. Chromatin methylation is usually repressive, and leads to transcriptional gene silencing (TGS). Methylation is important for the regulation of gene expression during normal development, and also is critical and for the control of endogenous invasive DNAs such as resident transposons. It also directs the establishment of heterochromatin.
Our work has shown that plants use methylation of viral chromatin to limit geminivirus replication and transcription, and that geminiviruses counter this defense with proteins such as AL2 and L2, which inhibit methylation and suppress TGS. One mechanism used by AL2 and L2 to inhibit methylation involves their ability to inactivate adenosine kinase (ADK), which is required for efficient production of a necessary methyltransferase co-factor. Current studies are focused on using geminiviruses and their suppressor proteins to better understand viral pathogenesis and siRNA-directed methylation pathways.
PTGS suppression by AL2 and L2
Geminivirus transcripts are vulnerable to post-transcriptional gene silencing (PTGS). We have found that AL2 and L2 are versatile proteins that suppress PTGS as well as TGS. Further, although they have somewhat different capabilities, AL2 and L2 act by multiple mechanisms to block several aspects of PTGS, including intra- and intercellular silencing. Continued analysis of AL2 and L2 suppression mechanisms will allow us to gain a better appreciation of the multiple branches of silencing pathways and their possible points of intersection.
AL2 and L2 suppress the cellular stress response by inactivating SnRK1 (SNF1-related kinase)
Our work indicates that the cellular stress response (CSR) conditioned by SnRK1 is a component of basal antiviral defenses, and that AL2 and L2 inhibit the CSR by inactivating SnRK1.
Interestingly, SnRK1 is activated by increased cellular AMP levels which can result from increased ADK activity, and these kinases are further linked by the fact AL2 and L2 interact with and inhibit both of them in vivo. Thus ADK may be a "high value" target, since by inhibiting its activity, geminiviruses can simultaneously interfere with silencing and the CSR. We are investigating the possibility that ADK and SnRK1 are part of a signaling pathway that is activated by pathogen attack.
Buchmann, R.C., Asad, S., Wolf, J.N., Mohannath, G., and Bisaro, D.M. (2009) Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation. Journal of Virology 83: 5005-5013.
Wu, Z., Zhu, Y., Bisaro, D.M., and Parris, D.S. (2009) Herpes simplex virus type 1 suppresses RNA-induced gene silencing in mammalian cells. Journal of Virology 83: 6652-6663.
Golenberg, E.M., Sather, D.N., Hancock, L.C., Buckley, K.J., Villafranco, N.M., and Bisaro, D.M. (2009) Development of a gene silencing DNA vector derived from a broad host range geminivirus. BMC Plant Methods 5:9
Raja, P., Sanville, B.C., Buchmann, R.C., and Bisaro, D.M. (2008) Viral genome methylation as an epigenetic defense against geminiviruses. Journal of Virology 82: 8997-9007.
Yang, X., Baliji, S. Woody, S., Buchmann, R.C., Wang, H., Lindbo, J., Sunter, G., and Bisaro, D.M. (2007) Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction. Journal of Virology 81: 11972-11981.
Bisaro, D.M. (2006) Silencing suppression by geminivirus proteins.Virology344: 158-168.
Wang, H., Buckley, K., Yang, X.Y., Buchmann, C., and Bisaro, D.M. (2005) Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins.Journal of Virology79: 7410-7418.
Hao, L., Wang, H., Sunter, G., and Bisaro, D.M. (2003) Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase.Plant Cell15: 1034-1048.
Wang, H., Hao. L., Sunter, G., and Bisaro, D.M. (2003) Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins. Plant Cell 15: 3020-3032.
Sunter, G. and Bisaro, D.M. (2003) Identification of a minimal sequence required for activation of the tomato golden mosaic virus coat protein promoter in protoplasts.Virology305: 452-462.