- Volume 77, Issue 9, 1996

Complementary DNA clones representing the entire genome of groundnut rosette umbravirus (GRV) were obtained and sequenced. GRV RNA comprises 4019 nucleotides and contains four large open reading frames (ORFs). The second ORF from the 5′ end includes sequences that encode motifs characteristic of viral RNA-dependent RNA polymerases and is probably expressed by a -1 frameshift mechanism as a fusion protein with the product of the 5′-most ORF. The other two ORFs are almost completely overlapping in different reading frames, and are probably expressed from subgenomic RNA. One of the putative products has significant sequence similarity with viral movement proteins. None of the putative proteins encoded by GRV RNA seems to be a structural protein. In genome organization and in the amino acid sequences of its potential products, the RNA of GRV is similar tot hat of carrot mottle mimic umbravirus, and to the umbravirus-like RNA-2 of pea enation mosaic virus.

The M RNA of peanut bud necrosis virus (PBNV; synonym groundnut bud necrosis virus) is 4801 nucleotides in length. It comprised two ORFs in an ambisense organization and terminal inverted repeats. The 3′ large ORF (3363 nucleotides in the virus-complementary strand) encoded a protein with a predicted size of 127.2 kDa which was identified as the glycoprotein precursor (GP) of the G1 and G2 glycoproteins. A comparison of the deduced amino acid sequence of GP revealed 37% identity and 58–59% similarity with that of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), and 21–23% identity and 44–47% similarity with those of other members of the genus Bunyavirus. The 5′ small ORF (924 nucleotides in the virus-sense strand) encoded a 34.2 kDa protein which was identified as the non-structural (NSm) protein based on 41–43% identity and 60–63% similarity with that of TSWV and INSV. Defective RNA molecules derived from the genomic M RNA were detected during continuous passage of the virus by sap inoculations.

Previously we made a series of deletion mutants in the 5′ non-coding region of tomato mosaic tobamo-virus (ToMV) RNA and checked their ability to replicate in tobacco protoplasts. Long deletions in this region caused the virus to lose the ability to replicate. Several mutants with deletions of about 10 nucleotides (short deletion mutants; SDM) retained the ability to replicate. In this study, we inoculated SDMs onto systemic host plants and observed their symptoms. One mutant (19/32) caused severe mosaic symptoms on some tobacco plants (Nicotiana tabacum cv. Samsun) but no symptoms on others. Virus accumulation in 19/32-inoculated plants paralleled the severity of symptoms. Four CAA repeat sequences were deleted in 19/32. Progeny 19/32 from plants showing severe systemic mosaic symptoms had acquired additional nucleotides in this region. We conclude that the CAA repeat sequence is related to the fitness of the virus population to replicate in whole plants rather than to translation of ToMV replicase genes.

The complete, 19226 nt sequence of the RNA genome from VT, a seedling yellows strain of citrus tristeza virus (CTV), was determined and found to have a genome organization identical with that of the previously determined CTV-T36 isolate, except that ORF 1 of CTV-VT was 70 nt shorter due to two widely separated 18 nt deletions. Sequence comparison of CTV-VT and CTV-T36 revealed approximately 89% identity throughout the ten 3′ ORFs, but only 60–70% identity throughout ORF 1. The 5′ nontranslated regions were only 60% identical whereas the 3′ nontranslated regions were 97% identical. The transition between regions of similarity and deviation was gradual, suggesting that the sequence similarities and differences compared to CTV-T36 were unlikely to have arisen from a recent recombination event between a close T36 relative and a distantly related CTV isolate. This is the first attempt to compare in detail the variation between the genomes of two strains of a member of the closterovirus group. The observed deviation between the large RNA genomes of the two CTV strains is greater than that among different viruses of most other groups, raising the question of how to define the taxonomy of these viruses.

To investigate the specificity of comoviral 24 kDa (‘24K’) proteinases, a full-length cDNA copy of red clover mottle virus (RCMV) RNA 1 has been cloned downstream of a T7 promoter. Translation in rabbit reticulocyte lysates of in vitro transcripts from this clone resulted in the synthesis of a 200K protein which was processed in a manner similar to that of the equivalent protein from cowpea mosaic virus (CPMV). Full-length cDNA clones of the RNA 1 molecules of RCMV and CPMV were used to create hybrid RNA 1 molecules. RNA transcribed in vitro from these hybrids was translated in vitro and the ability of the 24K proteinase from one comovirus to cleave the 32K/170K processing site from the other assessed. The results of the experiments show that the 24K proteinases are virus-specific in cis.