RESUMO
The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.
Assuntos
Pareamento de Bases , Vírus da Influenza A/genética , Regiões Promotoras Genéticas , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/genética , Animais , Western Blotting , Bovinos , Linhagem Celular , Vírus da Influenza A/crescimento & desenvolvimento , Mutagênese Sítio-Dirigida , Hibridização de Ácido Nucleico , Plasmídeos , RNA Complementar/análise , RNA Mensageiro/análise , RNA Viral/análise , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells.
Assuntos
Vírus da Influenza A/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Genes Virais , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/patogenicidade , Camundongos , RNA Polimerase Dependente de RNA , Análise de Sequência de Proteína , Especificidade da Espécie , TransfecçãoRESUMO
The poly(A) tail of influenza virus mRNAs is synthesized by the viral RNA polymerase by reiterative copying of a U5-7 sequence near the 5' end of the viral RNA (vRNA) template. We have engineered a vRNA molecule by replacing its viral U6 poly(A) site with a negative-sense eukaryotic polyadenylation signal. The vRNA was transcribed by the viral RNA polymerase and the transcription product was processed by the cellular 3' end processing machinery in vivo. According to the current model, 3' end processing of eukaryotic pre-mRNAs is coupled to cellular RNA polymerase II (pol II) transcription; thus only RNAs synthesized by pol III are believed to be polyadenylated efficiently. Our results show that the cellular polyadenylation machinery is nevertheless able to recognize and process RNA transcripts that are not synthesized by pol II, indicating that synthesis by pol II is not an absolute requirement for 3' end processing in vivo.
