Regulation of RNA Synthesis by the Genomic Termini of Vesicular Stomatitis Virus: Identification of Distinct Sequences Essential for Transcription but Not Replication

The 11,161-nucleotide (nt) nonsegmented negative-sense RNA genome of vesicular stomatitis virus (VSV), tightly encapsidated by its nucleocapsid protein (N), forms the active template for transcription and replication by the viral polymerase (18). The same core polymerase, a complex of the phosphoprotein (P) and large subunit (L), is required for both RNA synthetic processes, but other than the requirement for a constant supply of N protein during replication (2, 34), the mechanism by which these two discrete activities are regulated is unknown. The viral genome comprises a 47-nt leader region (Le), five transcriptional units for the N, P, M (matrix), G (glycoprotein), and L mRNAs, and a 59-nucleotide trailer region (Tr), arranged in the order 3′ Le-N-P-M-G-L-Tr 5′. The 3′ Le is thought to act as a promoter for transcription of the viral mRNAs and also as a promoter for replication to yield the positive-sense antigenome. The 3′ end of the positive-strand antigenome, which is the complement of the 59-nt genomic 5′ Tr (TrC), is thought to act as the promoter for replication of progeny negative-sense genomes. Thus, Le is used as a promoter for two discrete RNA synthetic processes, transcription and replication, whereas TrC acts exclusively as a promoter of replication. Consistent with the use of Le and TrC as promoters of replication, they share common sequence elements and are identical at 25 of 46 positions for VSV (Indiana). The areas of identity are localized at nt 1 to 8, 13 to 18, 30 to 33, whereas significant sequence divergence occurs at nt 9 to 12, 19 to 29, and 34 to 46 (Fig. ​(Fig.1A).1A). While it is generally accepted that the short terminal regions of all the Mononegavirales contain cis-acting signals essential for encapsidation, replication, transcription, and assembly (7, 9, 12, 14, 16, 31–33, 53), the requirements at the termini for transcription and replication are poorly defined; this in part is due to the multifunctional nature of the termini, making dissection of their roles complex. FIG. 1 Examination of the roles of the VSV genomic termini in transcription and in replication. (A) Structures of VSV genomic analog and mutant VSV replicons showing the four possible permutations of the genomic termini. Negative-sense genomic analogs of the ... Viral transcription, which is the predominant RNA synthetic activity in infected cells, occurs first. Available evidence suggests that the polymerase uses a single entry site at the 3′ end of the genome (17) and transcribes a 47-nt leader RNA which is neither capped or polyadenylated (10, 11, 26) and five capped and polyadenylated mRNAs whose relative abundance decreases with distance from the 3′end: N > P > M > G > L (1, 3, 47). At each of these gene junctions, a conserved sequence signals termination and polyadenylation of the upstream mRNA and transcription of the downstream mRNA (4, 5, 39, 43). During replication, in a reaction that requires continuous N protein synthesis (34), the polymerase initiates at the exact 3′ end of the negative-sense genome and generates a complete genome-length positive strand, ignoring the transcriptive stop and start signals. In turn, the 3′ end of the positive strand (TrC) promotes synthesis of the genome-length negative strand. Replication is asymmetric, producing an approximately 5 to 10-fold excess of negative-sense genomes over positive-sense antigenomes in infected cells (24, 38, 40, 42, 48). This led to the suggestion that TrC is a more effective promoter of replication than Le. Support for this notion came from studies of defective interfering (DI) particle RNAs, which outcompete wild-type virus at the level of replication. The most efficiently replicating class of DI RNAs are generated by a copyback mechanism so that the 3′ terminus is complementary to the 5′ terminus, and thus the promoters for both positive- and negative-strand synthesis are TrC. However, such a copyback arrangement of the termini increases the extent of complementarity between the termini, which enhances levels of replication and could account for the replicative advantage of these DI RNAs (49). To examine the cis-acting requirements at the termini of the VSV genome for transcription and replication, we made a series of alterations to the Le and Tr regions of subgenomic replicons and of infectious virus and determined the effects on RNA synthesis. The alterations included compensatory changes at both termini to distinguish the effects on RNA synthesis of altering either the sequence or the extent of complementarity between the termini. The results reported here define the 15 most terminal nucleotides at each end of the genome as essential for replication and show that both the primary sequence and the extent of complementarity between the natural termini regulate RNA synthesis. These studies identify distinct sequences at the termini essential for transcription but not replication, and they show that Le functions as a less efficient promoter of replication than TrC, at least in part because of its essential role in transcription.