Conservation of potential phosphorylation sites in the NS proteins of the New Jersey and Indiana serotypes of vesicular stomatitis virus.

A full length cDNA copy of the NS mRNA of the Missouri strain (Hazelhurst subtype, New Jersey serotype) of vesicular stomatitis virus (VSV) has been cloned and sequenced. The mRNA is 856 nucleotides long (excluding polyadenylic acid) and encodes a protein of 274 amino acids (mol. wt. 31 000). Comparison with the NS gene of the Ogden strain (Concan subtype, New Jersey serotype) showed 15% difference at the nucleotide level and 10% difference at the amino acid level; the majority of the changes were located in the 3' half of the mRNA. Comparison with the NS genes of two strains representing the Indiana serotype showed about 50% nucleotide and 33% amino acid sequence homology between the serotypes. In a four-way comparison of the proteins, two regions of higher homology were noted which may be of functional importance. Eighteen potential phosphorylation sites (Ser or Thr) were conserved between the four proteins; five of these sites correspond to the residues which have been suggested to be constitutively phosphorylated and may be essential for NS activity.

Characterization of the mutations responsible for the electrophoretic mobility differences in the NS proteins of vesicular stomatitis virus New Jersey complementation group E mutants.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus, New Jersey serotype, classified in complementation group E contain lesions in the NS gene, which manifest as marked electrophoretic mobility differences of the mutant NS proteins in SDS-polyacrylamide gels. We have cloned full-length cDNA copies of the mutant NS mRNAs, and have determined their nucleotide sequences. tsE1 and tsE3 had single nucleotide changes, and tsE2 had two nucleotide changes, compared to the wild-type NS gene. Three of the mutations were clustered in a region of 18 nucleotides. All the nucleotide differences resulted in amino acid substitutions, which in each case changed the charge of the amino acid concerned. Analysis of the wild-type and mutant NS protein sequences by the method of Chou & Fasman indicated that single amino acid substitutions can radically alter the predicted secondary structure, and these data are discussed in relation to the observed electrophoretic mobility differences.

Characterization of double-stranded RNA genetic elements associated with biological control of chestnut blight: organization of terminal domains and identification of gene products.

We have determined the organization within the terminal domains of the major large double-stranded RNA genetic elements associated with the hypovirulent strain EP713 of the chestnut blight pathogen Cryphonectria (Endothia) parasitica. Only the polyadenylated strand contained long open reading frames. Furthermore, only RNA of the same polarity as the polyadenylated strand was detectable in a single-stranded form, indicating that the polyadenylated strand is the coding or plus strand. The organization of the 5'-proximal portion of the plus strand consisted of a 495 nucleotide non-coding leader sequence followed by two overlapping open reading frames. The first, ORF1, extended 957 nucleotides while the second, ORF2, began 68 nucleotides upstream of the ORF1 termination codon and extended at least 1412 nucleotides. No open reading frames of significant size were detected within 0.8 kb of the poly(A) tail. In vitro translation of synthetic transcripts containing ORF1 yielded a polypeptide of Mr 29 kd. The ORF1 product was also detected in lysates of the hypovirulent strain but was absent in lysates of the isogenic virulent strain. It represents the first protein to be identified as a gene product encoded by a hypovirulence-associated double-stranded RNA genetic element.