Two Domains of the AL1 Protein Mediate Geminivirus Origin Recognition

Abstract

The geminiviruses tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) have bipartite genomes. Their A and B DNA components contain cis-acting sequences that function as origins of replication, while their A components encode the trans-acting replication proteins--AL1 and AL3. Earlier experiments demonstrated that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication and that the AL1 proteins of the two viruses specifically bind their respective origins. In the current study, characterization of AL1 and AL3 proteins produced from plant expression cassettes in transient replication assays revealed that interaction between AL1 and the origin is responsible for virus-specific replication. The AL3 protein does not contribute to specificity but can be preferred by its cognate AL1 protein when replication is impaired. Analysis of chimeric proteins showed that two regions of AL1 act as specificity determinants during replication. The first domain is located between amino acids 1 and 116 and recognizes the AL1 origin binding site. The second region, which is between amino acids 121 and 209, is not dependent on the known AL1 DNA binding site. Analysis of wild type and chimeric proteins in transient transcription assays showed that AL1 also represses its own promoter in a virus-specific manner. Transcriptional specificity is conferred primarily by AL1 amino acids 1-93 with amino acids 121-209 making a smaller contribution. Together, these results demonstrated that the virus-specific interactions of AL1 during replication and transcription are complex, involving at least two discreet domains of the protein.

A NAC Domain Protein Interacts with Tomato leaf curl virus Replication Accessory Protein and Enhances Viral Replication

“…The other viral protein required for high levels of viral DNA accumulation, REn, is involved in several protein-protein interactions. It binds to Rep and may increase the affinity of this protein for the viral origin of replication (Fontes et al, 1994;Settlage et al, 1996;Gladfelter et al, 1997), an activity proposed to enhance viral replication (Hanley-Bowdoin et al, 1999). REn also interacts with the host Rb and PCNA proteins, suggesting that its role in replication is multifaceted (Settlage et al, 2001;Castillo et al, 2003).…”

Replication promiscuity of DNA-β satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-β satellite localizes sequences involved in replication

“…Replication of DNA-A and DNA-B components relies on an interaction between the DNA-A-encoded replicationassociated protein (Rep) and reiterated motifs (termed iterons) located within the origin of replication (Argüello-Astorga et al, 1994;Fontes et al, 1992Fontes et al, , 1994Laufs et al, 1995;Gladfelter et al, 1997), which usually limits Rep trans-replication to its own DNA-A and DNA-B components. In contrast, the ability of intact and defective DNA-b satellites to be trans-replicated by distinct begomoviruses (Dry et al, 1997;Saunders et al, 2002;Mansoor et al, 2003) indicates that productive origin recognition is more relaxed for the satellite.…”

Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins

“…For ssDNA binding assays and DNA cleavage assays, individual oligonucleotides were heated 5 min at 100°C and immediately placed on ice. For dsDNA and ssDNA binding assays, wild-type and mutant AL1 1-180 proteins (2.5 g) were resuspended in binding buffer (25 was modeled based on the nuclear magnetic resonance (NMR) structure for amino acids 4 to 121 of TYLCSV Rep (PID 1L2M) (8). SWISS-MODEL was used to create the original pdb file based on alignment of TGMV AL1 to TYLCSV Rep (3,36,55).…”