J. Virol., Jan 1996, 148-158, Vol 70, No. 1
Copyright © 1996, American Society for Microbiology

A DNA structure is required for geminivirus replication origin function

BM Orozco and L Hanley-Bowdoin
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.

This article has been cited by other articles:

• Ha, C., Coombs, S., Revill, P., Harding, R., Vu, M., Dale, J. (2008). Molecular characterization of begomoviruses and DNA satellites from Vietnam: additional evidence that the New World geminiviruses were present in the Old World prior to continental separation. J. Gen. Virol. 89: 312-326 [Abstract] [Full Text]  
• Choudhury, N. R., Malik, P. S., Singh, D. K., Islam, M. N., Kaliappan, K., Mukherjee, S. K. (2006). The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase. Nucleic Acids Res 34: 6362-6377 [Abstract] [Full Text]  
• Shen, W., Hanley-Bowdoin, L. (2006). Geminivirus Infection Up-Regulates the Expression of Two Arabidopsis Protein Kinases Related to Yeast SNF1- and Mammalian AMPK-Activating Kinases. Plant Physiol. 142: 1642-1655 [Abstract] [Full Text]  
• Morilla, G., Castillo, A. G., Preiss, W., Jeske, H., Bejarano, E. R. (2006). A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication.. J. Virol. 80: 3624-3633 [Abstract] [Full Text]  
• Cheung, A. K. (2004). Palindrome Regeneration by Template Strand-Switching Mechanism at the Origin of DNA Replication of Porcine Circovirus via the Rolling-Circle Melting-Pot Replication Model. J. Virol. 78: 9016-9029 [Abstract] [Full Text]  
• Cheung, A. K. (2004). Detection of Template Strand Switching during Initiation and Termination of DNA Replication of Porcine Circovirus. J. Virol. 78: 4268-4277 [Abstract] [Full Text]  
• Castillo, A. G., Kong, L. J., Hanley-Bowdoin, L., Bejarano, E. R. (2004). Interaction between a Geminivirus Replication Protein and the Plant Sumoylation System. J. Virol. 78: 2758-2769 [Abstract] [Full Text]  
• Raghavan, V., Malik, P. S., Choudhury, N. R., Mukherjee, S. K. (2004). The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells. J. Virol. 78: 2405-2413 [Abstract] [Full Text]  
• Schnippenkoetter, W. H., Martin, D. P., Willment, J. A., Rybicki, E. P. (2001). Forced recombination between distinct strains of Maize streak virus. J. Gen. Virol. 82: 3081-3090 [Abstract] [Full Text]  
• Navas-Castillo, J., Sánchez-Campos, S., Noris, E., Louro, D., Accotto, G. P., Moriones, E. (2000). Natural recombination between Tomato yellow leaf curl virus-Is and Tomato leaf curl virus. J. Gen. Virol. 81: 2797-2801 [Abstract] [Full Text]  
• Brister, J. R., Muzyczka, N. (2000). Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking. J. Virol. 74: 7762-7771 [Abstract] [Full Text]  
• Brister, J. R., Muzyczka, N. (1999). Rep-Mediated Nicking of the Adeno-Associated Virus Origin Requires Two Biochemical Activities, DNA Helicase Activity and Transesterification. J. Virol. 73: 9325-9336 [Abstract] [Full Text]