Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence,genome organization and homology to other tospoviruses

Summary The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22–34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82–86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis virus (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To.     

Class II defective avian sarcoma viruses: Comparative analysis of genome structure

The genomes of class II avian sarcoma viruses PRCII, PRCII-p, PRCIV, and Fujinami sarcoma virus (FSV), were studied by oligonucleotide fingerprinting, heteroduplex mapping, and nucleic acid hybridization. All of these viruses are genetically defective and have a small RNA genome between 4.5 and 6.1 kilobases (kb) in length. They contain helper-related sequences at both the 5′- and 3′-ends, but most of the retroviral sequences in the middle of the genome are deleted. In place of this deleted information, a contiguous stretch of transformation-specific sequences, termed fps, is found. These putative oncogenic sequences are about 1.2 kb in PRCII, and those in PRCII-p and PRCIV are roughly 2.9 kb. From the analysis of oligonucleotides, it appears that the fps sequences of PRCII represent a subset of those of PRCII-p. Most of the additional sequences present in PRCII-p but absent from PRCII are at the 5′-half of fps. The helper-related sequences in PRCII and PRCII-p are almost indistinguishable, except that PRCII-p contains slightly more retroviral information at the 3′-end of the genome. Therefore, it is possible that PRCII has been derived by deletion from PRCII-p. By contrast, PRCII-p and PRCIV were found to contain identical fps sequences, but their helper-related sequences have diverged substantially. These two sarcoma viruses either represent two independent isolates or, if derived from a single isolate, they have undergone extensive mutation and recombination with diverse avian retroviruses. FSV was found to differ to a greater extent from other class II sarcoma viruses in both helper-related and fps sequences. The difference in fps sequences is localized in the 5′-half of that region. Considering the variation in fps among all members of class II avian sarcoma viruses, it appears that the 3′-half of that genetic region is more conserved than the 5′-half.     

A tymovirus with an atypical 3′-UTR illuminates the possibilities for 3′-UTR evolution

We report the complete genome sequence of Dulcamara mottle virus (DuMV), confirming its membership within the Tymovirus genus, which was previously based on physical and pathology evidence. The 5′-untranslated region (UTR) and coding region of DuMV RNA have the typical characteristics of tymoviral RNAs. In contrast, the 3′-UTR is the longest and most unusual yet reported for a tymovirus, possessing an internal poly(A) tract, lacking a 3′-tRNA-like structure (TLS) and terminating at the 3′-end with –UUC instead of the typical –CC(A). An expressible cDNA clone was constructed and shown to be capable of producing infectious DuMV genomic RNAs with –UUC 3′-termini. A chimeric Turnip yellow mosaic virus (TYMV) genome bearing the DuMV 3′-UTR in place of the normal TLS was constructed in order to investigate the ability of the TYMV replication proteins to amplify RNAs with –UUC instead of –CC(A) 3′-termini. The chimeric genome was shown to be capable of replication and systemic spread in plants, although amplification was very limited. These experiments suggest the way in which DuMV may have evolved from a typical tymovirus, and illuminate the ways in which viral 3′-UTRs in general can evolve.     

An internal element of the measles virus antigenome promoter modulates replication efficiency

The cis-acting sequence elements that direct measles virus (MV) genome synthesis reside in the 109 base non-coding region at the 5′ trailer (3′ antigenomic) end of MV genome that makes up the antigenomic promoter (AGP). The MV-AGP nucleotides 79–96, corresponding to nucleotide hexamers 14, 15 and 16 (the C′ element), show sequence similarity with the equivalent region of many paramyxoviruses and are analogous to the three nucleotide hexamers that form the second replication control element in the Sendai virus AGP. In this study, results of two independent procedures demonstrate that the MV C′ element also is a replication control sequence. Results of in vivo nucleotide selection experiments show that selection pressure for retaining the wild type nucleotides at the first position of each of the three hexamers, and for the fifth position of the 14th hexamer was relatively high. However, with continued replication, preference for the conservation of wild type nucleotides across the entire C′ element was clearly evident. Results of mutational analysis of individual nucleotides in one or more hexamers in a measles-helper-virus driven reporter gene rescue system agreed with these results. Substitutions at the first position of the 14th, the 15th or the 16th hexamers reduced minireplicon activity dramatically. In contrast, changes at the other five positions of any one hexamer had little or no effect on minireplicon activity, even when all the five bases were changed at the same time. However, when minireplicons were analyzed which contained point mutations at equivalent positions in all three hexamers, it was evident that the nucleotides, particularly those at the 5th position, were also important components of the C′ element. This pattern of sequence requirement in the C′ element based on mutational analysis could be described as a distinct motif, 5′-(GNNNAN)₂GNNNCN-3′, that is important for MV replication.     

Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA

Two overlapping cDNA clones corresponding to the 5' region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an ambisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5' terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3' termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression.     

The complete genome sequence of polygonum ringspot virus

The complete genome sequence of polygonum ringspot virus (PolRSV), genus Tospovirus, family Bunyaviridae, was determined. This is the first report of the complete genome sequence for a European tospovirus isolate. The large RNA of PolRSV was 8893 nucleotides (nt) in size and contained a single open reading frame of 8628 nucleotides in the viral-complementary sense, coding for a predicted RNA-dependent RNA polymerase of 330.9 kDa. Two untranslated regions of 230 and 32 nucleotides were present at the 5′ and 3′ termini, respectively, which showed conserved terminal sequences, as commonly observed for tospovirus genomic RNAs. The medium and small (S) RNAs were 4710 and 2485 nucleotides in size, respectively, and showed 99 % homology to the corresponding genomic segment of a previously partially characterized PolRSV isolate, Plg3. Protein sequences for G_N/G_C, N and NSs were identical in length in the two PolRSV isolates, while an amino acid insertion was observed for the NSm protein of the newly characterized isolate. The noncoding intergenic region of the S RNA was very short (183 nt) and was not predicted to form a hairpin structure, confirming that this unique characteristic within tospoviruses, previously observed for Plg3, is not isolate specific.     

Chromosome 7 abnormalities in acute megakaryoblastic leukemia associated with Down syndrome

Deletions of the 5′ ABL region adjacent to the t(9;22)(q34;q11) have recently been reported in 8–32.7% of patients with chronic myeloid leukemia (CML). The deletions were visualized with fluorescence in situ hybridization using, in the majority of the cases, the Vysis LSI BCR/ABL ES (extra signal) probe. In our series, 10 of 99 CML patients (10.1%) were characterized by a 5′ ABL deletion. We show that 3′ BCR losses are observed in nearly all the cases with 5′ ABL deletions. Moreover, the different genetic events (Philadelphia chromosome formation; 5′ ABL and 3′ BCR deletions) occur simultaneously in a one-step process without any evidence for genetic instability in the target bone marrow cells.