Extent of terminal complementarity modulates the balance between transcription and replication of vesicular stomatitis virus RNA.

Abstract

We compared the templae properties of a subgenomic RNA that onaed the authentic 5' and 3' ends of the vesicular stomat virus genome with those of RNAs in which the wild-tpe termini were engineered to extend their complementarity from 8 to 51 nucleotldes as seen in defective interfering RNAs. The RNA with authentic 5' and 3' ends dirted abundant t rpton but low replication. In contrast, RNAs with complement termini derived from either end of the genome replicated wellibut tra ibed poorly or not at all.These results have for undtandig the mechanisms of RNA replication and tra iption; they explain the replicative dominance of defective interfering RNAs and demonstrate that the extent ofterminal complementarity rather than its exact sequence is a major determinant of whether the template predominantiy directs nscription or replication.During infection the negative-stranded RNA genome of vesicular stomatitis virus (VSV) directs two distinct RNA synthetic processes: transcription offive discrete capped and polyadenylylated mRNAs and replication of a perfect fulllength copy of the genome. The same core RNA-dependent RNA polymerase is thought to execute both processes using as template a ribonucleoprotein complex ofthe viral genomic RNA. Transcription is the initial and predominant RNA synthetic event; synthesis of nucleocapsid (N) protein to encapsidate nascent genomic RNA is required for replication and is one trans-acting factor that modulates the balance between transcription and replication (1).To examine the role oftemplate sequences in the regulation of these two RNA synthetic processes, we established a system in which the entire replication cycle of a VSV defective interfering (DI) particle was reconstructed from cDNA clones (2). The DI RNA chosen for this work (DI-T) is a member ofthe copy-back family ofDI RNAs in which the 5' 2163 nt are derived from the wild-type (wt) VSV 5' terminus, but the 3' terminus is a 45-nt complement of the 5' terminus (3-6). DI genomes of this type have a replicative advantage over wt VSV and interfere with its replication (4). Deletion of internal regions of the DI-T genomic RNA and replacement with heterologous sequences have shown that the inverted repeats of the 5' end were sufficient to signal RNA replication (A. K. Pattnaik and G.W.W., unpublished data). Since the natural role of the 5' terminal sequence is to act exclusively as an origin of replication during synthesis of full-length'VSV negative strands, its presence at each end of the DI RNA has been proposed to account at least in part for the replicative advantage ofthis class ofDI particle and hence for its ability to interfere with the replication of wt VSV (4).In the work reported here, we took advantage of the ability to recover replicable RNAs from cDNA clones to compare the template properties ofRNA analogues that contained the authentic VSV 5' and 3' termini with those of RNAs engineered to have increasing extents of terminal complementarity. The results showed that RNAs that had the 3' end of the VSV geno...

Sequence analysis of a functional polymerase (L) gene of bovine respiratory syncytial virus: determination of minimal trans-acting requirements for RNA replication.

“…3 B, UC) especially in lanes 4-7, which probably represents a fraction of T7 primary transcript derived from vTF7-3-driven transcription of BLT δ7 that remained uncleaved at its HDV ribozyme site. Similar observations were also made by Wertz et al (1994) who worked with a vesicular stomatitis virus genome analogue and reported suboptimal cleavage at the HDV ribozyme sequence. Since the TRIzol protocol separates RNA from DNA, our total RNAs were not treated with DNase prior to Northern blot hybridization.…”

Functional Characterization of the Genomic Promoter of Borna Disease Virus (BDV): Implications of 3′-Terminal Sequence Heterogeneity for BDV Persistence

“…Terminal complementarity has also been shown to affect transcription and replication by vesicular stomatitis virus (49,50). In contrast to these examples, formation of a panhandle does not appear to play any role in the replication of respiratory syncytial virus (13) or Sendai virus (44).…”

“…In the case of other prototypic mononegaviruses such as Rous sarcoma virus, vesicular stomatitis virus, and rabies virus, this terminal complementarity includes the very first nucleotides at the 5Ј and 3Ј ends of their genomes and antigenomes (25,46,49). However, for BDV the best-fit alignment of the published sequences showed that the first three or four nucleotides remained unpaired (32).…”

Genome trimming: A unique strategy for replication control employed by Borna disease virus

“…For members of Orthomyxoviridae (14,15) and Bunyaviridae (16,17), the formation of panhandle structures is required for efficient initiation of viral genome replication. In the case of the Mononegavirales, it is unclear whether the panhandle structure is indeed formed during viral replication (18) or whether the terminal complementarity of the genome simply reflects conserved structures of the genomic and antigenomic promoters (19).…”