Summary auto-generated
This study uses an in vivo mutagenesis system to analyze influenza virus RNA promoter elements. Researchers inserted influenza viral sequences flanking a chloramphenicol acetyltransferase (CAT) reporter gene into plasmids under control of RNA polymerase I, then transfected these into cells and superinfected with influenza virus. The viral polymerase recognized and transcribed the synthetic vRNA-like transcripts, converting them to mRNA and enabling CAT expression. By systematically introducing mutations into the conserved terminal sequences of the viral RNA (which form double-stranded panhandle structures), the researchers identified critical features required for promoter recognition. They found that a guanine residue at position 5 in the 5' non-template strand is essential for RNA polymerase binding, while mismatches at other positions have minor importance. The bulged nucleotide at position 10 appears to function as a flexible joint between rigid structural elements rather than being directly recognized by polymerase. Several mutations actually enhanced expression levels above wild-type, and these improvements were maintained during serial virus passages, indicating selection for the improved promoter sequences.
Key findings
- A guanine residue at position 5 of the vRNA 5' non-template strand is critical for viral RNA polymerase binding and promoter function
- The double-stranded panhandle structure formed by complementary vRNA terminal sequences serves as a recognition substrate for viral polymerase, with specific base pairs and mismatches playing different roles
- Mutations introducing three substitutions (G3A, U5C, C8U) dramatically increased CAT expression levels 20-100 fold over wild-type, and these enhanced promoters were selected for during serial virus passages
- The bulged nucleotide at position 10 acts as a flexible joint in the RNA structure rather than a direct polymerase recognition element
- Viral RNA replication and mRNA synthesis showed similar improvements with the enhanced promoter mutants, indicating the mutations improve both RNA synthesis activities
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Abstract
RNA polymerase I transcription in vivo in transiently DNA-transfected cells has been used to express influenza virus vRNA molecules coding for chloramphenicol acetyltransferase (CAT) in an antisense orientation. Influenza virus superinfection provided viral RNA polymerase and other proteins required for transcriptional conversion of minus-strand vRNA into plus-strand viral mRNA molecules expressing CAT activity. This system has been used for analysis of the vRNA sequences which cooperatively constitute the vRNA promoter structure via nucleotide exchanges as well as deletions and insertions of both terminal segments. Several mutants caused greatly enhanced expression over wild-type levels, which was transmitted during serial passage of progeny virus. The data obtained for the mutations in various promoter elements support a model implicating double-stranded vRNA promoter structures in binding of viral polymerase, and in consecutive steps during initiation of RNA synthesis.