Non coding extremities of the seven influenza virus type C vRNA segments: effect on transcription and replication by the type C and type A polymerase complexes

Bernadette Crescenzo-Chaigne,Cyril Barbezange,Sylvie Van Der Werf

doi:10.1186/1743-422x-5-132

Bernadette Crescenzo-Chaigne, Cyril Barbezange + Show 1 more

Open Access

https://doi.org/10.1186/1743-422x-5-132

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Abstract

BackgroundThe transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. The plasmid-based system for the in vivo reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases.ResultsThe NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex.ConclusionIn the context of a PB2-like reporter vRNA template, the sequence upstream the polyU stretch plays a role in the transcription/replication process by the type C polymerase complex.

Highlights

The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions
To further analyze the type specificity of the interactions between the polymerase complex and the 3' and 5' ends of the vRNA and its consequences on transcription and replication, we extended our study to reporter vRNA templates harboring the complete 3' and 5' NC regions of the seven genomic segments of type C influenza virus
(page number not for citation purposes) http://www.virologyj.com/content/5/1/132 virus polymerase complexes using a transient transcription/replication assay [23]. Because this approach based on the CAT reporter gene activity showed major differences for the PB2-like vRNA template, we investigated the sequence requirements for optimal transcription versus replication of the PB2-like template by measuring the levels of messenger RNAs (mRNAs) and vRNA by real-time RT-PCR

Summary

Introduction

The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. For each genomic viral RNA, the coding region is flanked by non-coding (NC) sequences at both ends of the segment These terminal nucleotide sequences are involved in the transcription and replication of viral RNA [1,2] which further require the P and NP proteins. The messenger RNAs (mRNAs) are products of the transcription process They are capped at the 5' end with a 10 to 13 nucleotides (nt) sequence of nonviral origin derived from newly synthesized host nuclear RNAs through a so-called capsnatching mechanism. At their 3' end they possess a poly(A) sequence that results from termination of RNA synthesis at a polyU sequence localized 17 to 22 nt upstream of the 5' end of the genomic vRNA template. Initiation of the synthesis of cRNAs and vRNAs is primer-independent and anti-termination occurs at the polyU sequence during cRNA synthesis (for review [3])

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