Functional Characterisation of the Genomic and Antigenomic Promoters of Sendai Virus

Abstract

A natural Sendai virus internal deletion defective interfering (DI) RNA, previously shown to encode a truncated NP protein and previously cloned under the control of the T7 RNA polymerase promoter, was expressed from plasmid and shown to replicate in cell tissue culture when the viral proteins NP, P, and L were coexpressed from cloned genes. The efficient replication was dependent on the total length of the RNA to be a multiple of 6 nucleotides, showing that the "rule of six" applied for a DI RNA that has conserved the end sequences of the nondefective viral RNA. Compared to the copy-back H4 DI RNA, the replication efficiency of the internal deletion DI RNA was reproducibly 20-fold lower. Reciprocal exchanges between the minus-strand 3'-end primary sequences of the two DI RNAs showed that the replication efficiency of the derivatives obtained directly correlated with the origin and the extent of the primary sequence. Moreover, some of the derivatives exhibited a replication efficiency comparable to that of the copy-back DI RNA with, however, the ability to transcribe a functional mRNA similar to the internal deletion DI RNA. This indicated that the transcription ability of a viral RNA was not sufficient to explain a low replication efficiency.

A Novel Role for Viral-Defective Interfering Particles in Enhancing Dendritic Cell Maturation

“…Copy-back DI particles lack the genomic promoter that is replaced by the antigenomic promoter preventing transcription but allowing replication. Copy-back DI particles replicate up to 20 times faster than species having both genomic and antigenomic promoters, due to specific sequences within their promoters that confer higher replication abilities (41,42,44). Our studies show that viruses with enhanced DC activation abilities have increased levels of antigenomic promoter-flanked copy-back DI particles (Fig.…”

“…DI particles are enriched in coinfected cells as they replicate more efficiently than standard virus genomes due to their smaller length and differential promoter sensitivities (41,42). They, thereby, interfere with standard virus protein production by monopolizing the viral replication machinery.…”

Suppression of the Sendai Virus M Protein through a Novel Short Interfering RNA Approach Inhibits Viral Particle Production but Does Not Affect Viral RNA Synthesis

“…Nucleocapsid RNAs from infected cells were prepared as described before (8). Infected cells were disrupted in lysis buffer 1 (0.6% NP-40, 50 mM Tris-HCl [pH 8.0], 10 mM NaCl) (37).…”

“…Northern blot assays were performed as described before (8). For nucleocapsid RNA analysis, 32 P-labeled 5Ј ex-riboprobes of the plus and minus polarity were used (37).…”

Genetic manipulation of non-segmented negative-strand RNA viruses

“…This was aimed at the elucidation of cis-and trans-acting transcription factors and involved promoter mutagenesis (Engelhorn et al, 1993;Kfilin et al, 1994;Calain & Roux, 1995;Pattnaik et al, 1995;Wertz et al, 1995;Yu et al, 1995) as well as the analysis of particular properties of the virus polymerases (Jacques et al, 1994;Schnell & Conzelmann, 1996) and the virus assembly process (Kaptur et al, 1995;Mebatsion et al, 1995;Stillman et al, 1995). The approach appears to be applicable to most of the negative-strand RNA viruses, including the segmented bunyaviruses (Dunn et al, 1995;Lopez et al, 1995).…”