MultipleCisElements Contribute to Geminivirus Origin Function

Orozco, Beverly M.; Gladfelter, Heather J.; Settlage, Sharon B.; Eagle, Patricia A.; Gentry, Renee N.; Hanley‐Bowdoin, Linda

doi:10.1006/viro.1997.9013

Cited by 72 publications

(40 citation statements)

References 48 publications

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“…Another origin mutant, i.e., OriMЈ, with an A3T mutation 32 nucleotides upstream of the 5Ј end of the 9-mer sequence, was constructed (YCpO Ϫ -OriMЈ-2A) in which neither the DNA binding nor the specific cutting site of the recombinant IMYMV Rep protein was affected (data not shown). A similar mutation in tomato golden mosaic virus DNA did not alter the replication origin activity of tomato golden mosaic virus (17). As expected, the replicative efficiency of YCpO Ϫ -OriMЈ-2A was the same as that of plasmid YCpO Ϫ -2A (Fig.…”

Section: Two Tandem Copies Of the Viral Genome Can Replicate In Yeastsupporting

confidence: 77%

The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells

Raghavan

Malik

Choudhury

et al. 2004

J Virol

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Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.The complexities of host features might hinder the addressing of specific issues related to host-virus interactions, including biosynthesis of viral nucleic acids. The factors required for DNA replication of the plant DNA viruses are difficult to discover in a systematic manner by employing the current approaches of plant biology. The variability associated with DNA transfections of plant protoplasts, the problems related to plant transformation, and other issues limit the progress of studies of DNA replication of viruses such as geminiviruses, which are potent phytopathogens. Moreover, the intermediates of replicated products are difficult to isolate from infected plant tissues. The lack of genetic mutants and of sequence information for the host genomes also compounds the problem. In order to circumvent these difficulties, we looked for a model eukaryotic host that supports DNA replication of the geminiviruses.Geminiviruses represent a family of viruses which are characterized by twin icosahedral particles, each of which encapsidates a small (ϳ2.7 kb) single-stranded circular DNA. Though no crop is immune to the geminiviruses, each strain of geminivirus can infect crop plants within a narrow window of host specificity (19). Indian mung bean yellow mosaic virus (IMYMV), a member of the Geminiviridae family, possesses bipartite genomes, namely DNA-A and DNA-B, and is a menace in legume cultivation (18). The 2,745-bp DNA-A component encodes the information for viral DNA replication, transcription, and encapsidation, whereas the 2,616-bp DNA-B encodes two movement proteins responsible for virus translocation.Studies with viruses related to IMYMV have suggested that the viral DNA frequently, but not exclusively, replicates in a rolling circle mode (RCR) (10, 16, 21) The replication origins of all geminiviruses include an invariant 9-mer sequence (TAATATT2AC) which is site-specifically nicked (at the position of the arrow) by a virus-encoded replication initiator protein (Rep; also called AC1 or AL1) to initiate RCR (28). For efficient replication, Rep is assisted by a repl...

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Section: Two Tandem Copies Of the Viral Genome Can Replicate In Yeastsupporting

confidence: 77%

The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells

Raghavan

Malik

Choudhury

et al. 2004

J Virol

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“…The CR contains modular cis-acting elements of the origin of replication (ori) (18,29,33). Five open reading frames (ORFs) are conserved among the DNA A of the New World begomoviruses.…”

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confidence: 99%

Emergence of a New Cucurbit-Infecting Begomovirus Species Capable of Forming Viable Reassortants with Related Viruses in the Squash leaf curl virus Cluster

Brown¹,

Idris²,

Alteri³

et al. 2002

Phytopathology®

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Cucurbit leaf curl virus (CuLCV), a whitefly-transmitted geminivirus previously partially characterized from the southwestern United States and northern Mexico, was identified as a distinct bipartite begomovirus species. This virus has near sequence identity with the previously partially characterized Cucurbit leaf crumple virus from California. Experimental and natural host range studies indicated that CuLCV has a relatively broad host range within the family Cucurbitaceae and also infects bean and tobacco. The genome of an Arizona isolate, designated CuLCV-AZ, was cloned and completely sequenced. Cloned CuLCV-AZ DNA A and B components were infectious by biolistic inoculation to pumpkin and progeny virus was transmissible by the whitefly vector, Bemisia tabaci, thereby completing Koch's postulates. CuLCV-AZ DNA A shared highest nucleotide sequence identity with Squash leaf curl virus-R (SLCV-R), SLCV-E, and Bean calico mosaic virus (BCaMV) at 84, 83, and 80%, respectively. The CuLCV DNA B component shared highest nucleotide sequence identity with BCaMV, SLCV-R, and SLCV-E at 71, 70, and 68%, respectively. The cis-acting begomovirus replication specificity element, GGTGTCCTGGTG, in the CuLCV-AZ origin of replication is identical to that of SLCV-R, SLCV-E, and BCaMV, suggesting that reassortants among components of CuLCV-AZ and these begomoviruses may be possible. Reassortment experiments in pumpkin demonstrated that both reassortants of CuLCV-AZ and SLCV-E A and B components were viable. However, for CuLCV-AZ and SLCV-R, only one reassortant (SLCV-R DNA A/CuLCV-AZ DNA B) was viable on pumpkin, even though the cognate component pairs of both viruses infect pumpkin. These results demonstrate that reassortment among sympatric begomovirus species infecting cucurbits are possible, and that, if generated in nature, could result in begomoviruses bearing distinct biological properties.

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“…The 5Ј-intergenic region separating the transcription units is nearly identical between the two DNA components and includes the plus strand origin of replication (7,8). The promoter for complementary sense transcription overlaps the replication origin (5,9) and shares some of the cis-elements involved in origin function (10).…”

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confidence: 99%

“…A hairpin structure with a 9-base pair loop sequence that is conserved among all geminiviruses is essential for replication and contains the cleavage site for initiating plus strand DNA synthesis (4,13,14). A conserved sequence between the AL1 binding site and the hairpin, the AG-motif, is also required for replication (8). The third element, the CA motif, is located outside of the minimal origin but its deletion reduced replication 20-fold (8).…”

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confidence: 99%

The Multifunctional Character of a Geminivirus Replication Protein Is Reflected by Its Complex Oligomerization Properties

Orozco

Kong

Batts

et al. 2000

Journal of Biological Chemistry

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Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157-159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157-159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein.Geminiviruses are a large family of plant viruses with circular, single-stranded DNA genomes that replicate in the nuclei of infected cells (reviewed in Ref. 1). The single-stranded genome is converted to a double-stranded DNA that serves as the template for rolling circle replication (2-4) and transcription (5, 6). Geminiviruses do not encode their own polymerases and, instead, rely on host enzymes for viral DNA and RNA synthesis. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms.The geminivirus, tomato golden mosaic virus (TGMV), 1 has a bipartite genome that encodes seven open reading frames that are divergently transcribed. The 5Ј-intergenic region separating the transcription units is nearly identical between the two DNA components and includes the plus strand origin of replication (7, 8). The promoter for complementary sense transcription overlaps the replication origin (5, 9) and shares some of the cis-elements involved in origin function (10). A directly repeated sequence, GGTAG, is required for origin recognition (11) and transcriptional repression of the complementary se...

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MultipleCisElements Contribute to Geminivirus Origin Function

Cited by 72 publications

References 48 publications

The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells

The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells

Emergence of a New Cucurbit-Infecting Begomovirus Species Capable of Forming Viable Reassortants with Related Viruses in the Squash leaf curl virus Cluster

The Multifunctional Character of a Geminivirus Replication Protein Is Reflected by Its Complex Oligomerization Properties

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