In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA.

Abstract

A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection ofHeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.

Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes

“…Recently, this has been confirmed by successful RNA polymerase purification (Honda et al, 1990) and reconstitution of the infectious nucleocapsid structure (Luytjes et al, 1989;Yamanaka et al, 1991). The establishment of a transfection system enabled genetic manipulation to be used to gain a detailed understanding of the structure and function of individual virus proteins in transcription and replication.…”

Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system

“…The encapsidated RNAs can be transcribed in vitro by the virus-specific replicase and expressed in vivo when transfected into influenza virus-infected cells. In vitro studies on reconstituted RNPs have demonstrated that the sequences conserved at the 3' ends of cRNA and vRNA molecules are the minimum promoters for virusspecific RNA synthesis (Parvin et al, 1989;Luo et at., 1991;Yamanaka et al, 1991;Li & Palese, 1992;Seong & Brownlee, 1992a, b;Piccone et al, 1993). It is, however, not clear whether the viral promoter has to be located at the extreme 3' end of the RNA template.…”

“…In the case of influenza virus, several procedures that allow in vitro reconstitution of influenza virus-like ribonucleoprotein (RNP) complexes using synthetic RNAs and purified viral proteins have been described (Parvin et al, 1989;Luytjes et al, 1989;Yamanaka et al, 1991 ;Seong & Brownlee, 1992a;Martin et al, 1992). The encapsidated RNAs can be transcribed in vitro by the virus-specific replicase and expressed in vivo when transfected into influenza virus-infected cells.…”

The position 4 nucleotide at the 3' end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens.

“…It is also possible that the non-coding region of vRNA segments may also play a role in down-regulation, although it is difficult to find consensus elements in these heterogeneous sequences. In fact, a number of studies have investigated the potential role of non-coding nucleotides using an in vivo reporter gene system (Kim et al, 1997 ;Li & Palese, 1992 ;Neumann & Hobom, 1993 ;Piccone et al, 1993 ;Yamanaka et al, 1991), an in vitro transcription system (Fodor et al, 1993(Fodor et al, , 1994, or using transfectant viruses which carry mutations in the endogenous RNA genome (Bergmann & Muster, 1996 ;Luo et al, 1992 ;Zheng et al, 1996). It has been shown that genetic determinants of replication control also involve non-conserved nucleotides of the terminal sequences (Bergmann & Muster, 1996 ;Luo et al, 1992 ;Zheng et al, 1996).…”

“…Sequence analysis of genomic RNAs of influenza A viruses revealed highly conserved sequences of 12 nt at the 3h terminus and 13 nt at the 5h terminus, which are partially complementary to each other and form a panhandle structure (Desselberger et al, 1980 ;Hsu et al, 1987 ;Robertson, 1979 ;Skehel & Hay, 1978 ;Winter & Fields, 1980). The importance of these conserved sequences in transcription and replication of influenza virus RNAs has been investigated in vivo (Li & Palese, 1992 ;Neumann & Hobom, 1993 ;Piccone et al, 1993 ;Yamanaka et al, 1991 ;Zheng et al, 1996) and in vitro (Fodor et al, 1994. Within this conserved sequence motif, a unique natural variation, U or C, is observed at position 4 of the 3h end of the vRNA.…”