Geminivirus Replication Research Articles

Mutational Analysis of a Putative NTP-Binding Domain in the Replication-Associated Protein (AC1) of Bean Golden Mosaic Geminivirus

Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA geminivirus with a bipartite genome. AC1 is the only ORF required for geminiviral replication. A putative NTP-binding motif, EGX 4GKTX 32DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263). We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies. Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from K228 to H or D262 to R within the putative NTP-binding site. A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain. Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from I190 to R. Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication. The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed.

Geminivirus replication: Genetic and biochemical characterization of rep protein function, a review

Replication of the single-stranded DNA genome of plant geminiviruses follows a rollin circle mechanism. It strictly depends on a ‘replication initiator protein’ (Rep) which is the sole viral protein essential for replication. Rep protein catalyzes multiple reactions duringthe reproductive cycle of the virus. Here we summarize the recent advances of in vivo and in vitro analyses of the various Rep protein activities in a model for replication initiation and termination. In addition, the position of the geminivirus Rep protein within a general context of bacterial and mammalian replication initiator proteins is discussed.

Geminivirus replication: analysis of Rep protein functions

Geminivirus replication: analysis of Rep protein functions

A DNA sequence required for geminivirus replication also mediates transcriptional regulation.

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, requires a single virus-encoded protein for DNA replication. We show that the TGMV replication protein, AL1, also acts during transcription to specifically repress the activity of its promoter. An earlier study established that AL1 binds to a 13-bp sequence (5'-GGTAGTAAGGTAG) that is essential for activity of the TGMV replication origin. Analysis of AL1 binding site mutants in transient expression assays demonstrated that the same site, which is located between the transcription start site and TATA box in the AL1 promoter, also mediates transcriptional repression. These experiments revealed that the repeated motifs in the AL1 binding site contribute differentially to repression, as has been observed previously for AL1-DNA binding and viral replication. Introduction of the AL1 binding site into the 35S promoter of the cauliflower mosaic virus was sufficient to confer AL1-mediated repression to the heterologous promoter. Analysis of a truncated AL1 promoter and of mutant AL1 proteins showed that repression does not require a replication-competent template or a replication-competent AL1 protein. Transient expression studies using two different Nicotiana cell lines revealed that, although the two lines replicate plasmids containing the TGMV origin similarly, they support very different levels of AL1-mediated repression. These results suggest that geminivirus transcriptional repression and replication may be independent processes.

Geminivirus Replication Origins Have a Group-Specific Organization of Iterative Elements: A Model for Replication

A phylogenetic and structural analysis of the Intergenic region of 22 dicot-infecting and 8 monocot-infecting geminiviruses was carried out. The analysis allowed the identification of iterative sequence motifs 8-12 nucleotides in length, whose organization (number, orientation, and spacing) is highly conserved within each of the three major lineages of dicotgeminiviruses, according to the phylogeny derived from the amino acid sequences of the replication-associated protein (AL1). The iterated elements differ in sequence even between closely related viruses, and are found in the Vicinity of the putative TATA box of the AL1 gene in all dicot-infecting geminiviruses. Analogous elements were identified also in monocot-infecting geminiviruses, but the arrangement was different, since one of the iterative sequences is part of the conserved hairpin structure essential for replication of all the members of this viral family. We propose here that the iterated sequences are the specific binding sites of the geminiviral replication-associated proteins and show that the hypothesis is in agreement with the experimental data available to date. Additionally, a model of geminivirus replication that involves the participation of host transcription factors in the process is presented.

A DNA Sequence Required for Geminivirus Replication Also Mediates Transcriptional Regulation

A DNA Sequence Required for Geminivirus Replication Also Mediates Transcriptional Regulation

Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Geminivirus replication origins have a modular organization.

Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for replication, and their A components encode trans-acting factors are required for this process. We showed that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication in tobacco protoplasts. We also demonstrated that, similar to the essential TGMV AL1 replication protein, BGMV AL1 binds specifically to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificities of the B components were exchanged by site-directed mutagenesis, but the resulting mutants were not replicated by either A component. These results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Geminivirus genomes also contain a stem-loop sequence that is required for origin function. A BGMV B mutant with the TGMV stem-loop sequence was replicated by BGMV A, indicating that BGMV AL1 does not discriminate between the two sequences. A BGMV B double mutant, with the TGMV AL1 binding site and stem-loop sequences, was not replicated by either A component, indicating that an additional element in the TGMV origin is required for productive interaction with TGMV AL1. These results suggested that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is required for replication but does not contribute to virus-specific recognition of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.

Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins.

It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family. Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA. These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication. It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick. The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain. By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately. The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed.

A Geminivirus Replication Protein Is a Sequence-Specific DNA Binding Protein

A Geminivirus Replication Protein Is a Sequence-Specific DNA Binding Protein

A geminivirus replication protein is a sequence-specific DNA binding protein.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.