Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system.
An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.
Mena
I
I
Centro Nacional de Microbiología Virología e Inmunología Sanitarias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
de la Luna
S
S
Albo
C
C
Martín
J
J
Nieto
A
A
Ortín
J
J
Portela
A
A
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014764:Viral Proteins
D012367:RNA, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases