Structure of the gene N:gene NS intercistronic junction in the genome of vesicular stomatitis virus

Abstract

It is known that in the vesicular stomatitis virus genome, the N gene and the NS gene are adjacent and found in the orientation 3′-N-NS-5′ on the single-stranded negative-sense genome RNA. The junction between the two genes is the site of several processes during synthesis of mRNAs from the genome RNA template—specifically, polyadenylation and termination of N mRNA synthesis and the beginning of NS mRNA synthesis, with capping and methylation of the NS mRNA. This paper presents the primary structure of the intercistronic region together with the flanking sequences in the two genes. This nucleotide sequence was determined by copying N mRNA into a double-stranded DNA, digesting the DNA with restriction nucleases and isolating a DNA fragment corresponding to a sequence close to but distinct from the 3′ terminus of N mRNA. This fragment was then annealed to genome RNA and used as a primer for reverse transcription through the end of the N gene and into the start of the NS gene. The sequence of this complementary DNA, and thus of the genome RNA across the junction, was obtained by the dideoxynucleoside triphosphate chain termination method. The structure of the junction (as the genome RNA strand), proceeding 3′ to 5′, consists of N gene heteropolymeric mRNA coding sequences, followed by 3′-UUUUUUUGA-5′, followed by NS gene heteropolymeric mRNA coding sequences. The oligo(U) tract is thought to be involved in initiating synthesis of the 3′ terminal poly(A) tract of N mRNA. Several other nearby sequences may be involved as control or signal entities in processing events. The only part of the junction not representedin the mature mRNAs is the sequence of 5′-CU-3′, which is complementary to the sequence 3′-GA-5′ shown above.