David Bisaro

Focus

Molecular biology of geminiviruses; including viral pathogenesis and regulation of viral gene expression.

Research interests

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 more general metabolic defense conditioned by 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 RNA silencing-based defenses.

Geminiviruses

Geminiviruses replicate circular ssDNA genomes in the nucleus through dsDNA intermediates that associate with cellular histones to form minichromosomes. Viral genomes have a limited coding capacity varying from four to seven genes, depending on the virus, and do not encode polymerases. Instead host machinery is recruited to express viral mRNAs and replicate the viral DNA. These characteristics make geminiviruses valuable model systems for studying the mechanisms and epigenetic regulation of these processes.

All geminiviruses package their ssDNA genomes in paired isometric particles from which the family derives its name. Most of our studies involve Tomato golden mosaic virus (TGMV), Cabbage leaf curl virus (CaLCuV), and Beet curly top virus (BCTV). BCTV is the type member of the genus Curtovirus. Curtoviruses have monopartite genomes of ~3.0 kb and have broad host ranges within the dicots. TGMV and CaLCuV are members of the Begomovirus genus. Begomoviruses also infect dicots but have rather narrow host ranges. Some have monopartite genomes but many, including CaLCuV and TGMV, are bipartite. The two genomic DNAs are similar in size (~2.6 kb) and share a nearly identical common region (CR) of 200-250 bp. The CR is part of a larger intergenic region (IR) that contains divergent promoters flanking the origin of replication, which includes a conserved hairpin found in all geminiviruses.

Most of our recent work involves the AL2 protein (also called AC2) encoded by begomoviruses, and the related L2 protein encoded by curtoviruses. 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 as a viral counterdefense

siRNA-directed DNA and histone methylation is a fundamental epigenetic process that in the model plant Arabidopsis thaliana is accomplished via the DCL3/RDR2/AGO4 pathway. Methylation is important for the regulation of gene expression during normal development. In addition, because most methylation events are inhibitory to gene expression, methylation is critical to the establishment of heterochromatin and for the control of endogenous invasive DNAs such as resident transposons. We now have evidence that methylation is used to inhibit exogenous invasive DNAs (i.e. DNA viruses) as well.

Our working hypothesis is that plants use methylation of viral chromatin to limit geminivirus replication and transcription, and that geminiviruses counter this defense with proteins that inhibit methylation. It is based on several key findings from this laboratory: 1) Methylation-deficient Arabidopsis mutants are exquisitely sensitive to geminivirus infection and show greatly enhanced disease symptoms. 2) Geminivirus DNA and associated histone proteins (notably histone H3 at lysine 9; H3K9) are methylated in infected plants. 3) Viral DNA methylation is significantly reduced in methylation-deficient mutant plants that display enhanced susceptibility. 4) The geminivirus AL2 and L2 proteins can inhibit methylation by inactivating a cellular enzyme (adenosine kinase; ADK) required for efficient production of the methyl group donor S-adenosyl methionine. Current studies are focused on using geminiviruses as models to better understand siRNA-directed methylation pathways.

PTGS suppression by AL2 and L2

Geminivirus transcripts are vulnerable to cytoplasmic PTGS. While most viral silencing suppressors are more or less specialized in their activities, AL2 and L2 are versatile proteins that can suppress both TGS (see above) and PTGS. Further, although they have somewhat different capabilities, we have found that AL2 and L2 act by multiple mechanisms to block several aspects of PTGS. Specifically, they can inhibit local silencing, block systemic silencing spread, and reverse established PTGS, presumably by interfering with maintenance pathways. Continued analysis of AL2 and L2 suppression mechanisms will allow us to gain a better appreciation of the multiple branches of RNA silencing pathways and possible points of intersection.

AL2 and L2 suppress the cellular stress response by inactivating SNF1 kinase

Transgenic tobacco plants that express AL2 or L2 show a novel enhanced susceptibility (ES) phenotype following inoculation with TGMV, BCTV or Tobacco mosaic virus (an RNA virus). ES is characterized by a reduction in latent period and a large decrease in the inoculum concentration needed to infect the transgenic plants. However, ES does not result in increased disease symptoms and viral nucleic acids do not accumulate to significantly greater levels. We subsequently demonstrated that ES is due to AL2- and L2-mediated inactivation of SNF1 kinase, a global regulator of metabolism that conditions the cellular stress response (CSR). Following nutritional and environmental stresses that deplete ATP (and increase AMP levels), SNF1 turns off energy consuming biosynthetic pathways and activates energy generating systems. Thus we identified the CSR as a component of innate, non-specific, antiviral defenses and its inhibition by AL2 and L2 as a counterdefense. How active SNF1 responses alter infectivity is not known.

Interestingly, SNF1 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 in vivo. In addition, we have shown that ADK levels are normally increased by DNA and RNA virus infection. We are investigating the possibility that ADK and SNF1 are part of a signaling pathway that is activated by viral pathogen attack.

Selected Publications

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. In press.

Bisaro, D.M. (2006) Silencing suppression by geminivirus proteins. Virology 344: 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 Virology 79: 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 Cell 15: 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. Virology 305: 452-462.

 

BISARO LAB MEMBERS

Postdoc: Dr. Kenneth Buckley

Graduate Students: Cody Buchmann, Priya Raja, Gireesha Mohannath

Undergraduates: Bradley Sanville, Natalie Villafranco, Leslie King

Visiting Scientist: Dr. Shaheen Asad

Publications

• Yang, X., Baliji, S., Buchmann, R.C., Wang, H., Lindbo, J.A., Sunter, G., and Bisaro, D.M. (2007) Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction. Journal of Virology. in press.
• Bisaro, D.M. (2006) Silencing suppression by geminivirus proteins . Virology 344: 158-168.
• Wang, H., Buckley, K.J., Yang, X., Buchmann, R.C., and Bisaro, D.M. (2005) Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins. Journal of Virology 79: 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 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. Virology 305: 452-462.
• Wang, H., Hao, L., Shung, C.-Y., Sunter, G. and Bisaro, D.M. (2003) Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins. Plant Cell 15: 3030-3032.