Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells

The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.     

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.     

High-frequency reversion of geminivirus replication protein mutants during infection

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.     

A DNA structure is required for geminivirus replication origin function.

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.     

Genetic analysis of the tomato golden mosaic virus. II. The product of the AL1 coding sequence is required for replication.

Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.     

Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.

The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and squash leaf curl virus (SqLCV) have two single-stranded circular genomic DNAs, the A and B components, thought to be replicated from double-stranded circular DNA intermediates. Although it has been presumed that the origin sequences for viral replication are located in the highly conserved 200-nucleotide common region (CR) present in both genomic components and that the viral-encoded AL1 protein interacts with these sequences to effect replication, there has been no evidence that this is in fact so. We have investigated these questions, demonstrating selectivity and sequence specificity in this protein-DNA interaction. Simple component switching between the DNAs of TGMV and SqLCV and analysis of replication in leaf discs showed that whereas the A components of both TGMV and SqLCV promote their own replication and that of their cognate B component, neither replicates the noncognate B component. Furthermore, using an in vivo functional replication assay, we found that cloned viral CR sequences function as a replication origin and direct the replication of nonviral sequences in the presence of AL1, with both circular single-stranded and double-stranded DNA being synthesized. Finally, by the creation of chimeric viral CRs and specific subfragments of the viral CR, we demonstrated sequence-specific recognition of the replication origin by the AL1 protein, thereby localizing the origin to an approximately 90-nucleotide segment in the AL1 proximal side of the CR that includes the conserved geminiviral stem-loop structure and approximately 60 nucleotides of 5' upstream sequence. By deletional analysis, we further demonstrated that the conserved stem-loop structure is essential for replication. These studies identify the functional viral origin of replication within the CR, demonstrating that sequence-specific recognition of this origin by the AL1 protein is required for replication.     

A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted α-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.     

DNA sequences essential for replication of the B genome component of tomato golden mosaic virus.

The genome of the geminivirus tomato golden mosaic virus (TGMV) is divided between two DNA components, designated A and B, which differ in sequence except for a 230-nucleotide common region. The A genome component is known to encode viral functions necessary for viral DNA replication, while the B genome component specifies functions necessary for spread of the virus through the infected plant. To identify cis-acting sequences required for viral DNA replication, several mutants were constructed by the introduction of small insertions into TGMV B at selected sites within and just outside the common region. Other mutants had the common region inverted or deleted. All of the mutants were tested for their effects on infectivity and DNA replication in whole plants and leaf discs. Our results indicate that the common region in its correct orientation is required for infectivity and for replication of TGMV B. Furthermore, the conserved hairpin loop sequence located within the TGMV common region and found in all geminiviruses is necessary for DNA replication, and may be part of the viral replication origin.     

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.     

Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants

Tomato golden mosaic virus (TGMV), a member of the geminivirus group, has a genome consisting of two DNA molecules designated the A and B components. Both are required for infectivity in healthy plants, although the former has been shown to replicate independently in transgenic plants containing tandem direct repeats of the A genome component. In the studies presented here, petunia plants transgenic for either both components (A×B hybrids) or the A component alone were examined for the presence of virus particles and encapsidated, single stranded viral DNA. The results of DNase protection experiments and direct observation of extracts from transgenic plants by electron microscopy indicate that single stranded TGMV DNA is in both cases packaged into paired particles identical to those obtained from virus-infected plants. DNase-treated virions isolated from A×B hybrid petunia are infectious when inoculated onto healthy Nicotiana benthamiana. Likewise, virions obtained from transgenic A petunia are infectious for plants transgenic for the B component.Our observations of TGMV replication in transgenic plants indicate that TGMV A DNA encodes all viral functions necessary for the replication and encapsidation of viral DNA. The possible role of the B component in TGMV replication is discussed.     

Interaction between geminivirus replication protein and the SUMO-conjugating enzyme is required for viral infection

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transient-replication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.     

Interaction between a geminivirus replication protein and the plant sumoylation system

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells after accumulation of host replication machinery. Tomato golden mosaic virus (TGMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) encode a protein, RepAC1 (or Rep), that is essential for viral replication. Rep/RepAC1 is an oligomeric protein that binds to double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and is sufficient for host induction. It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1. To identify other cellular proteins that interact with Rep/RepAC1 protein, a Nicotiana benthamiana cDNA library was screened with a yeast two-hybrid assay. The host cell sumoylation enzyme, NbSCE1 (N. benthamiana SUMO-conjugating enzyme, homolog to Saccharomyces cerevisiae UBC9), was found to interact specifically with RepAC1. Mapping studies localized the interaction to the N-terminal half of RepAC1. Effects on geminivirus replication were observed in transgenic plants with altered levels of SUMO, the substrate for UBC9.     

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants

Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1-pRBR interactions. Plants infected with the E-N140 mutant, which is wild-type for pRBR binding, developed wild-type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1-pRBR interactions during geminivirus infection of plants.     

PCNA interacts with Indian mung bean yellow mosaic virus rep and downregulates Rep activity

Proliferative cell nuclear antigen (PCNA), a conserved plant protein as well as an important replication factor, is induced in response to geminivirus infection in the resting cells of the phloem tissues. The biochemical role of PCNA in rolling circle replication (RCR) of geminivirus DNA has not been explored in detail. The initiation of RCR of the bipartite genome of a geminivirus, Indian mung bean yellow mosaic virus (IMYMV), is mainly controlled by viral protein Rep (or AL1 or AC1). The role of host PCNA in RCR of IMYMV was revealed by studying the physical and functional interactions between recombinant PCNA and recombinant IMYMV Rep. Pea nuclear PCNA as well as recombinant pea PCNA showed binding to recombinant Rep in experiments involving both affinity chromatography and yeast two-hybrid approaches. The contacting amino acid residues of PCNA seemed to be present throughout a wide region of the trimeric protein, while those of Rep appeared to be localized only in the middle part of the protein. The site-specific nicking-closing activity and the ATPase function of IMYMV Rep were impaired by PCNA. These observations lead to interesting speculations about the control of viral RCR and dynamic profiles of protein-protein interactions at the RCR origin of the geminiviruses.     

Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.