Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helpervirus genome except for two short motifs present in separate putative stem-loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.
The complete sequence of the RNA genome of tobacco necrosis virus strain D (TNV-D) consisting of 3759 nucleotides has been determined. The positive strand contains five open reading frames (ORFs). The 5'proximal ORF encodes a 22K protein terminating with an amber codon which may be read through to produce a 82K protein (p82). Two small centrally located ORFs each encode two out-of-frame 7K proteins (p7a and p7b). The 3'-proximal ORF encodes the 29K coat protein (CP), the N terminus of which has been sequenced directly. The genomic organization of TNVD is very similar to that of TNV-A but differs in the placement of the p7a ORF, which does not overlap the p82 ORF in TNV-D, and in the absence of an ORF downstream of the CP gene in TNV-D. The p82 ORF shows extensive sequence similarity with the putative polymerases of the carmovirus group. This ORF is also as closely related to the corresponding ORF of TNV-A as it is to the corresponding ORF of the tombusvirus cucumber necrosis virus. The amino acid sequence of the TNV-D CP gene is similar to both the TNV-A and southern bean mosaic virus CP genes. Of the two p7 ORFs, p7a exhibits amino acid sequence similarity with corresponding proteins from TNV-A, melon necrotic spot virus, carnation mottle virus, turnip crinkle virus and maize chlorotic mottle virus, whereas the p7b ORF appears to be unique to TNV-A and TNV-D. Only the 3'-terminal three nucleotides of TNV-D genomic RNA are identical to the 3'-terminal nucleotides of the TNV satellite virus
Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or Cl) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.
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