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Endogenous viral elements (EVEs) have been for the most part described in animals and to a less extent in plants. The endogenization was proposed to contribute toward evolution of living organisms via horizontal gene transfer of novel genetic material and resultant genetic diversity. During the last two decades, several full-length and fragmented EVEs of pararetroviral and non-retroviral nature have been identified in different plant genomes, both monocots and eudicots. Prior to this work, no EVEs have been reported in alfalfa (Medicago sativa L.), the most cultivated forage legume in the world. In this study, taking advantage of the most recent developments in the field of alfalfa research, we have assessed alfalfa genome on the presence of viral-related sequences. Our analysis revealed segmented EVEs resembling two dsDNA reverse-transcribing virus species: Soybean chlorotic mottle virus (family Caulimoviridae, genus Soymovirus) and Figwort mosaic virus (family Caulimoviridae, genus Caulimovirus). The EVEs appear to be stable constituents of the host genome and in that capacity could potentially acquire functional roles in alfalfa’s development and response to environmental stresses.

Petunia plants are used for urban landscaping in many parts of the world, including South Korea. In this study, we aimed to investigate the occurrence of petunia vein clearing virus (PVCV) infection in petunia plants in Seoul, South Korea. PVCV was detected from 23 of 79 petunia samples collected from Seoul. We obtained the complete genome sequences of the Korean isolates in this study (called PVCV-Kr, Kr2, and Kr3), which were compared with the genome sequence of the USA isolate of the virus (PVCV-USA). The genomic DNA of the three PVCV isolates was found to comprise 7210-7267 nucleotides (nts), which is 4-15 nts longer than the PVCV-USA genome. The genomes of the Kr and Kr2 isolates encode a large polyprotein of 252 kDa (2180 amino acids (aa)). The genome of the Kr3 isolate encodes a large polyprotein of 255 kDa (2203 aa). The polyprotein has six protein domains: a movement protein (MP; 72 aa), a coiled-coil domain (CC; 33 aa), an RNA-binding domain (RB; 18 aa), a protease (PR; 21 aa), a reverse transcriptase (RT; 196 aa), and an RNase H (RH; 121 aa). The large polyprotein and six domains of the three isolates showed 93.9-100.0% sequence homology with those of PVCV-USA. Furthermore, the polymerase polyprotein gene (PR, RT, and RH) of the four PVCV isolates containing the USA isolate grouped with those of Rice tungro bacilliform virus and Soybean chlorotic mottle virus, which belong to the same family (Caulimoviridae). Our findings suggested that the Korean isolates represent a new isolate of PVCV. To our knowledge, this is the first report of PVCV detection in South Korea.

Soybean chlorotic mottle virus (SbCMV) was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR. The complete nucleotide sequence of NC113 was determined to be 8 210 nucleotides, and shared the highest similarity (91.7%) with sequences of SbCMV that was only reported in Japan. It encodes nine putative open reading frames (ORFs Ia, Ib and II–VIII), and contains a large intergenic region located at nucleotide 5 976–6 512 between ORFs VI and VII. Sequence analysis and phylogenetic tree indicated that NC113 is an isolate of SbCMV, and is more related to the soymoviruses Blueberry red ringspot virus (BRRSV), Peanut chlorotic streak virus (PCSV) and Cestrum yellow leaf curling virus (CmYLCV) than to other representative members in the Caulimoviridae family. Field survey of 472 legume plants from Jiangxi and Zhejiang provinces showed SbCMV was only detected from soybean in Nanchang City with a low incidence rate. This is the first report of Soybean chlorotic mottle virus identified in China.

The activity of a predicted promoter, PMC8, from Milk vetch dwarf virus was evaluated by comparing it with the cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues.

The double-stranded DNA genome of Blueberry red ringspot virus (BRRV), a member of the family Caulimoviridae, was cloned and sequenced. The genome organization and relationships of the 8303 nt sequence revealed BRRV to be a tentative member of the genus that has been provisionally named "Soybean chlorotic mottle-like viruses", rather than a member of the genus Caulimovirus, in which it had been placed previously. Insertion of the putative 35S promoter homolog of BRRV into promoterless constructs carrying the UidA (beta-glucuronidase) gene resulted in high-level transient expression from cranberry and stable expression from transgenic tobacco. Sequences of 5'-RACE clones derived from transcripts from transgenic tobacco were consistent with the map position of the promoter.

Soybean chlorotic mottle virus(SbCMV) is the type species of the genus ‘Soybean chlorotic mottle-like viruses’, within the familyCaulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1·3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNAMetprimer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case ofCauliflower mosaic virus.

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene.

The genome of sugarcane bacilliform virus (ScBV), a badnavirus, consists of a circular dsDNA. The complete sequence of a cloned infective ScBV genome is reported here. The genome is 7568 bp in size and possesses a number of features suggesting that ScBV is a pararetrovirus. A tRNA(Met)-binding site that may serve as a primer for minus-strand synthesis is present. The plus-strand of the ScBV genome contains three open reading frames (ORFs) which are capable of encoding proteins with calculated M(r) values of 22K, 13K and 215K. The 215K protein has regions with similarity to the RNA-binding domains, aspartic proteases and replicases of retro-elements. In addition, the 215K protein also has a region with restricted similarity to the intercellular transport proteins of plant viruses. Comparisons with the other sequenced badnaviruses, Commelina yellow mottle (CoYMV) and rice tungro bacilliform (RTBV) viruses, indicate that the arrangement of the ORFs in these viruses is conserved. Located next to the putative RNA-binding domain is a cysteine-rich region that is unique to the badnaviruses. When the molecular relationships of a portion of the reverse transcriptases of plant pararetroviruses were determined, two badnaviruses, CoYMV and ScBV, form one distinct cluster, whereas three caulimoviruses, cauliflower mosaic virus, carnation etched ring virus and figwort mosaic virus, form a second cluster. The badnavirus RTBV and the caulimovirus soybean chlorotic mottle virus occupy intermediate positions between the clusters. When introduced by Agrobacterium-mediated inoculation, a construct containing 1.1 copies of the cloned ScBV genome is infectious to both rice and banana.

The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII–PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154–201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII–PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection.

Cestrum yellow leaf curling virus (CmYLCV) has been characterized as the aetiological agent of the Cestrum parqui mosaic disease. The virus genome was cloned and the clone was proven to be infectious to C. parqui. The presence of typical viroplasms in virus-infected plant tissue and the information obtained from the complete genomic sequence confirmed CmYLCV as a member of the Caulimoviridae family. All characteristic domains conserved in plant pararetroviruses were found in CmYLCV. Its genome is 8253 bp long and contains seven open reading frames (ORFs). Phylogenetic analysis of the relationships with other members of the Caulimoviridae revealed that CmYLCV is closely related to the Soybean chlorotic mottle virus (SbCMV)-like genus and particularly to SbCMV. However, in contrast to the other members of this genus, the primer-binding site is located in the intercistronic region following ORF Ib rather than within this ORF, and an ORF corresponding to ORF VII is missing.

Summary The DNA of soybean chlorotic mottle virus (SoyCMV) has been isolated. When analysed by gel electrophoresis, the native DNA formed multiple bands resembling DNA from other caulimoviruses. The viral DNA was cloned in bacterial vectors. Cloned DNA was used to map restriction sites on the SoyCMV DNA. Comparison of cloned DNAs of SoyCMV and cauliflower mosaic virus (CaMV) by Southern transfer hybridization revealed the absence of significant sequence homology. Electrophoresis in denaturing gels showed that SoyCMV DNA had three single-strand discontinuities (‘gaps’), one in one strand and two in the other, and the positions of these gaps were mapped on the viral DNA. Potential primer sites for reverse transcription were detected in the cloned DNA by sequence analysis of regions around the gap sites. The results obtained definitely establish SoyCMV as a member of the caulimovirus group and suggest a replication mechanism for this virus similar to that described for CaMV.

The nucleic acid of soybean chlorotic mottle virus (SoyCMV) was isolated and analysed. CsCl equilibrium density gradient centrifugation and thermal denaturation kinetics revealed that the nucleic acid is a double-stranded DNA. Electrophoresis in agarose gels separated SoyCMV DNA into multiple bands, similar to those of cauliflower mosaic virus (CaMV) DNA, which indicated that this DNA consisted of both linear and circular molecules. The existence of these different topological forms was confirmed by electron microscopy. Using six different restriction endonucleases a physical map was constructed which showed the SoyCMV genome to be a circular DNA molecule of approximately 8, 200 base pairs long. The results obtained support the previous assignment of SoyCMV to the caulimovirus group.

The complete nucleotide sequence of an infectious clone of soybean chlorotic mottle virus (SoyCMV) DNA was determined and compared with those of three other caulimoviruses, cauliflower mosaic virus (CaMV), carnation etched ring virus and figwort mosaic virus. The double-stranded DNA genome of SoyCMV (8,175 bp) contained nine open reading frames (ORFs) and one large intergenic region. The primer binding sites, gene organization and size of ORFs were similar to those of the other caulimoviruses, except for ORF I, which was split into ORF Ia and Ib. The amino acid sequences deduced from each ORF showed only short, highly homologous regions in several of the corresponding ORFs of the three other caulimoviruses. A promoter fragment of 378 bp in SoyCMV ORF III showed a strong expression activity, comparable to that of the CaMV 35S promoter, in tobacco mesophyll protoplasts as determined by a beta-glucuronidase assay using electrotransfection. The fragment contained CAAT and TATA boxes but no transcriptional enhancer signal as reported for the CaMV 35S promoter. Instead, it had sequences homologous to a part of the translational enhancer signal reported for the 5'-leader sequence of tobacco mosaic virus RNA.