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-l ike chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expr ession of the four influenza virus core proteins (nucleoprotein, PA, P B1 and PB2) was performed by transfection of four PGEM recombinant pla smids, 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, demons trating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at th e 3' end were also expressed, resulting in CAT activity. This showed t hat the influenza virus polymerase can recognize its promoter when loc ated 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 as sembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.