Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.