Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.     

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.     

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.     

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single-stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem-loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling-circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral-strand DNA synthesis independently from its termination and also to identify the sequence within which rolling-circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem-loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem-loop sequence. Here we show that viral-strand DNA synthesis efficiently initiates at both sequences.     

The nucleotide sequence of cloned wheat dwarf virus DNA

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.     

Replication of wheat dwarf virus DNA in protoplasts and analysis of coat protein mutants in protoplasts and plants.

The replication of wheat dwarf virus (WDV) in protoplasts derived from a Triticum monococcum suspension cell system was investigated. The production of circular viral double-stranded DNA (dsDNA) forms consistent with the replication of the viral genome was observed. In comparison to whole plants, the production of viral single-stranded DNA (ssDNA) was reduced, possibly due to only low levels of viral coat protein being produced in the protoplasts. Mutations introduced into the viral coat protein open reading frame (ORF) did not affect the ability of the viral DNA to replicate, and a deletion of ca. 400 bp was tolerated. However, these mutations abolished the infectivity of the viral genome when agroinoculated onto wheat plants, providing evidence that, contrary to the case for the bipartite geminiviruses, the coat protein is essential for infection by WDV.     

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.     

Preliminary Functional Studies on AC2, a Novel Trans-acting Factor from Cotton Leaf Curl Virus

Studies on tomato golden mosaic virus and African cassava mosaic virus suggested that virion sense promoter was trans -activated in transient expression by A C2 encoded by geminivirus. The AC2 gene fragment of cott on leaf curl virus (CLCuV) was obtained from total DNA of CLCuV infected tobacco leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into vector. Transient expres sion vectors were constructed by fusing the AC2 gene fra gment with CaMV 35S prom oter and nopaline terminator. These constructs were delivered into tobacco [ WT(Nicotiana tabacum L.) and cotton ( Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. Results indicated that activity of virion sense promoter was activated by AC2 and increased remarkably. However, the activity of trans-activated virion sense promoter was still lower than that of complementary sense promoter. Expression pattern of transactivated virion sense promoter was similar to that of complementary sense promoter with the high activity in both mesophyll and vascular of leaf vein. In this paper, the expression behavior of AC2 in Agrobacterium -mediated transgenic plants was also discussed.      

Expression of a bacterial gene in plants mediated by infectious geminivirus DNA

A viable coat protein deletion mutant of cassava latent virus (CLV) DNA 1 has been isolated, suggesting that this geminivirus might be exploited as a gene replacement vector. An extensive deletion of 727 nucleotides within the coat protein gene renders DNA 1 non-infectious. Chimeric clones have been constructed in which the deleted coat protein open reading frame has been replaced by the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene. Infectivity is restored to DNA 1 when the CAT gene is inserted in either orientation, producing symptoms typical of CLV infection. The results demonstrate that the coat protein plays no essential role in virus spread throughout the host. Levels of CAT expression of 80 U/mg soluble protein occur in systemically infected Nicotiana benthamiana leaves when the CAT gene is fused in-frame to the amino terminus of the coat protein, providing a sensitive assay for viral DNA replication.     

Determination of the origin cleavage and joining domain of geminivirus Rep proteins.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'rolling circle replication initiator protein', the M(r) 41 kDa viral Rep protein, encoded by the C1 or AC1 genes. Using wheat dwarf virus (WDV) and tomato yellow leaf curl virus (TYLCV) as examples, we show that not only the full-size Rep proteins, but also a putative 30 kDa translation product of WDV open reading frame C1-N as well as an artificially shortened 24 kDa Rep of TYLCV, cleave and join single-stranded origin DNA in vitro. Thus the pivotal origin recognition and processing activities of geminivirus Rep proteins must be mediated by the amino-terminal domain of Rep.     

Tomato leaf curl geminivirus associated with cantaloupe yellow leaf disease in Thailand

Geminivirus associated with yellow leaf disease of cantaloupe plants was detected using polymerase chain reaction (PCR) with geminivirus-specific degenerate primers which anneal within the AC1 ORF (replication initiator protein gene) and the AV1 ORF (coat protein gene). A DNA fragment of 1.2 kbp was amplified, cloned and sequenced. The 32-base stem loop region was found in the amplified fragment. This included the conserved nonanucleotide sequence TAATATTAC present in all geminiviruses. The nucleotide sequence of the intergenic region (IR) was compared with 28 whitefly-transmitted geminiviruses. The geminivirus associated with yellow leaf disease of cantaloupe plants showed 96.2% sequence identity with DNA A of tomato leaf curl geminivirus from India (ToLCV-In2). These data suggest that cantaloupe yellow leaf disease was caused by ToLCV.