D. J. Robinson

Evidence that DNA-A of a geminivirus associated with severe cassava mosaic disease in Uganda has arisen by interspecific recombination.

Geminivirus isolates associated with the epidemic of severe cassava mosaic disease in Uganda were studied and compared with virus isolates from the part of Uganda outside the epidemic area, and with African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). Isolates of a novel type [the Uganda variant (UgV)] were detected in severely affected plants from the epidemic area, whereas those from plants outside the epidemic area were typical of ACMV. The complete nucleotide sequences of DNA-A of UgV (2799 nt) and of a Tanzanian isolate of EACMV (2801 nt) were determined and are extremely similar, except for the coat protein (CP) gene. The CP gene of UgV has three distinct regions: the 5 219 nt are 99% identical to EACMV (only 79% to ACMV); the following 459 nt are 99% identical to ACMV (75% to EACMV); and the 3 93 nt are 98% identical to EACMV (76% to ACMV). UgV DNA-A therefore is considered to have arisen by interspecific recombination of EACMV and ACMV. Despite the hybrid nature of their CP, UgV isolates were indistinguishable from ACMV in tests with 20 monoclonal antibodies (MAbs), including seven which reacted with ACMV but not EACMV. The discontinuous epitopes detected by these seven MAbs must involve amino acids which lie in the central part of the CP (residues 74-226) and which differ in ACMV and EACMV. UgV isolates were detected in severely mosaic-affected plants from all 11 widely separated locations sampled. The probable role of recombination in geminivirus evolution in the short to medium term is discussed.

Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra.

Complete DNA-A sequences of nine Pakistani geminivirus isolates from leaf curl-affected cotton (CLCuV-PK) or from okra, and the partial sequences of several additional isolates were determined. Sequences of isolates from cotton were of four types. Isolates from leaf curl-affected okra had virtually the same sequences as those from cotton. Isolates from yellow vein mosaic-affected okra were of two types (OYVMV types 201 and 301), both distinct from but closely related to the virus isolates from cotton. Of these six types, two types of CLCuV-PK are the most closely related but another (CLCuV-PK type 72b) is the most distinct. Of the encoded proteins, coat protein (CP) is the most strongly conserved (92-100% amino acid sequence identity), and AC4 protein the most variable (41-87%). The 5 and 3 halves of the intergenic region of some isolates had different affinities and occurred in seven combinations, suggesting that recombination had occurred and that the origin of replication was a favoured recombination site. Similarly, the first 1520 nt of CLCuV-PK type 804a DNA resembled those of OYVMV type 301 DNA but the remaining 1224 nt were very different. The AC1 (Rep) gene and 5 part of the intergenic region of CLCuV-PK type 72b closely resembled those of OYVMV type 301, whereas the rest of the sequence did not. The cotton leaf curl epidemic in Pakistan is caused by several distinct variants, with recombination events involving OYVMV and other unspecified geminiviruses having probably been involved in their evolution.

Nucleotide Sequence of Potato Leafroll Luteovirus RNA

A sequence of 5987 nucleotides is reported for the RNA of potato leafroll luteovirus (PLRV). The sequence contains six large open reading frames, and non-coding regions of 174 nucleotides at the 5 end, 141 nucleotides at the 3 end and 197 nucleotides between two large blocks of coding sequences. The 5 coding region encodes two polypeptides of 28,000 (28K) and 70K which overlap in different reading frames and circumstantial evidence suggests that the third open reading frame in the 5 block is translated by frameshift readthrough near the end of the 70K polypeptide to give a 118K polypeptide. The C-terminal part of the 118K protein contains the consensus sequence for RNA-dependent RNA polymerases. In vitro translation of PLRV RNA resulted in the synthesis mainly of 28K and 70K polypeptides and the largest product made was about 125K; these sizes are similar to those predicted for the translation products of the 5 block of coding sequence. The 3 block of coding sequence codes for three polypeptides: a 23K coat protein, a 17K polypeptide which is encoded in a different frame, and a 53K polypeptide which immediately follows the coat protein coding sequence, and is in the same reading frame. Circumstantial evidence suggests that the 53K polypeptide is translated by readthrough of the amber termination codon of the coat protein gene. The amino acid sequences encoded by the 3 block of coding sequence show many similarities with analogous polypeptides translated from the nucleotide sequences of RNA of barley yellow dwarf virus, PAV strain (BYDV) and, in particular, beet western yellows virus (BWYV). The 118K polypeptide has some similarities with the putative polymerase of southern bean mosaic virus and much more extensive similarities with the corresponding BWYV polypeptide but almost none with that of BYDV. In contrast, the amino acid sequence of the 28K polypeptide is not like that of proteins of the other luteoviruses or of viruses in other groups. The nucleotide sequences reported will appear in the EMBL, GenBank and DDBJ databases under the accession number X14600.

Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan

Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.

The complete nucleotide sequence of tobacco rattle virus RNA-1.

The complete nucleotide sequence of tobacco rattle virus (TRV) strain SYM RNA-1 was determined from a series of overlapping cDNA clones. cDNA prepared by primer extension was used to determine the exact 5 terminus. The RNA sequence was 6791 nucleotides in length and contained four open reading frames (ORFs). The ORF nearest the 5 terminus coded for a polypeptide of predicted mol. wt. 134,000 (134K) and terminated at an opal (UGA) stop codon. Readthrough of this stop codon would result in the production of a protein of 194K. The gene for a 29K polypeptide started one base beyond the 194K stop codon and, in turn, was followed by the gene for a 16K protein at the 3 end of RNA-1. Amino acid comparisons of the 194K protein with the putative replicase of tobacco mosaic virus showed three regions of strong homology, suggesting that the 134K and 194K proteins were similarly involved in virus replication. The 5 terminal sequences of both genome RNA species of TRV strains ORY, N5 and PRN together with that of SYM RNA-2 were also determined. Alignments of these sequences showed that there was a 22 base repeated sequence close to the 5 terminus in all these RNA species. It was also shown that the 5 terminus of RNA-1 was homologous with the same region in RNA-2.

Two Anomalous Tobravirus Isolates: Evidence For RNA Recombination in Nature

SUMMARY 16 and N5 are naturally occurring tobravirus isolates that produce symptoms in herbaceous plants similar to those induced by strains of tobacco rattle virus (TRV). In immunosorbent electron microscopy tests, however, they reacted with antisera to particles of pea early-browning virus (PEBV), not TRV. Furthermore, these tests indicated that 16 was related to the British serotype of PEBV and N5 to the Dutch. Pseudo-recombinant isolates were produced by reassortment of the genome parts of 16 or N5 with those of TRV, in any combination, but not in most combinations with those of PEBV. However, I6 RNA-2 was replicated in plants inoculated also with RNA-1 from an isolate of the British serotype of PEBV, but the PEBV RNA-1 was imperfectly packaged by 16 coat protein, and the virus particles seemed to have only limited stability. Nucleic acid hybridization experiments showed that the RNA-1 sequences of both I6 and N5 were similar to those of TRV strains. I6 RNA-2 contained sequences resembling those of the British serotype of PEBV, but with some TRV-like sequences at the 3 and 5 ends, whereas N5 RNA-2 contained more extensive TRV-like 3 and 5 ends flanking sequences that were related, but perhaps not closely, to those of the Dutch serotype of PEBV. Thus, the RNA-2 species of 16 and N5 were recombinant molecules that contained sequences typical of both TRV and PEBV, and which probably had separate but similar evolutionary origins. As a result of their hybrid nature, I6 and N5 were part of the gene pool and had the pathogenicity of TRV, while possessing the serological properties of PEBV.

Evidence that Potato Leafroll Virus RNA is Positive-stranded, is Linked to a Small Protein and Does Not Contain Polyadenylate

SummarynRNA extracted from particles of potato leafroll virus (PLRV) infected tobacco mesophyll protoplasts. Treating the RNA with proteinase K did not abolish its infectivity. In messenger-dependent rabbit reticulocyte lysate, PLRV RNA induced the synthesis of specific polypeptides: a major product of mol. wt. 71000 but no product the size of coat protein. PLRV RNA is therefore positive-stranded. A genome-linked protein (apparent mol. wt. 7000) was detected in preparations of PLRV RNA but no polyadenylate sequence was found. These features may prove to be characteristic of luteoviruses.

Serological Relationships and Genome Homologies among Geminiviruses

SummarynIn immunosorbent electron microscopy (ISEM) tests, strong relationships were detected between five whitefly-transmitted geminiviruses: African cassava mosaic (ACMV), bean golden mosaic, euphorbia mosaic, squash leaf curl and tomato golden mosaic. Among five leafhopper-transmitted geminiviruses, beet curly top and tobacco yellow dwarf viruses were distantly related but no relationship was detected between either chloris striate mosaic, maize streak or wheat dwarf viruses and any of the other four. No relationship was detected between any whitefly-transmitted and any leafhopper-transmitted virus. A similar pattern of relationships was found by spot hybridization experiments in which extracts from infected leaves were tested with probes for ACMV DNA-1 or DNA-2. Imperfect nucleotide sequence homologies were found between ACMV DNA-1, which contains the particle protein gene, and the DNA of five other whitefly-transmitted viruses: bean golden mosaic, tomato golden mosaic, tobacco leaf curl, tomato leaf curl and tomato yellow leaf curl, the last three of which are not sap-transmissible. Thus, relationships were established between sap-transmissible and sap non-transmissible geminiviruses. No homologies were detected with a full-length probe for ACMV DNA-2. Extracts from plants infected with three leafhopper- transmitted viruses (beet curly top, maize streak and wheat dwarf) did not react with probes for ACMV DNA-1 or DNA-2. Because each of the leafhopper-transmitted geminiviruses has a different vector species whereas the whitefly-transmitted geminiviruses all have the same vector, Bemisia tabaci, the genome homologies and antigenic relationships detected among members of the group could be explained if their coat proteins have a key role in transmission by vectors.

A satellite RNA of groundnut rosette virus that is largely responsible for symptoms of groundnut rosette disease

SummarynGroundnut (Arachis hypogaea) plants with rosette disease contain a manually transmissible virus, groundnut rosette virus (GRV), which depends on a luteovirus, groundnut rosette assistor virus (GRAV), for transmission by the aphid Aphis craccivora. No virus-like particles have been reported for GRV but infected plants yield infective ssRNA. Infected leaves also contain dsRNA with prominent electrophoretic species of 4.6 kbp (dsRNA-1) and 1.3 kbp (dsRNA-2), a very abundant species of 900 bp (dsRNA-3), and numerous minor species of intermediate mobility. In studies with GRV(C), an isolate from groundnut plants with a chlorotic form of rosette, cDNA to dsRNA-1 reacted with dsRNA-1 and dsRNA-2 but not with dsRNA-3 or any of the minor dsRNA species. In contrast, cDNA to dsRNA-3 reacted with dsRNA-3 and several of the minor dsRNA species but reacted only weakly or not at all with dsRNA-1 or dsRNA-2. An isolate lacking dsRNA-3 (isolate G96) was derived from GRV(C) by passage through Gomphrena globosa. When dsRNA-3 recovered from agarose gels was melted and inoculated to Nicotiana benthamiana plants, it was not infective on its own but multiplied in plants that were also infected with G96. Similar results were obtained with sucrose density gradient fractions containing RNA molecules of the size expected for ssRNA-3. These results show that dsRNA-3 represents a satellite RNA. Addition of dsRNA-3 to the G96 culture resulted in a slight amelioration of symptoms in N. benthamiana and N. clevelandii. However, only cultures containing RNA-3 induced rosette symptoms in groundnut, though the symptoms were intensified by further addition of GRAV. The results show that the satellite RNA is largely responsible for rosette disease symptoms in groundnut.

An Umbraviral Protein, Involved in Long-Distance RNA Movement, Binds Viral RNA and Forms Unique, Protective Ribonucleoprotein Complexes

ABSTRACT Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [ΤMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with ΤMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.