RNA Polymerase of Influenza Virus: Role of NP in RNA Chain Elongation1

Abstract

Ribonucleoprotein (RNP) cores of influenza virus A/PR/8/34 were dissociated into RNA polymerase (PB1-PB2-PA complex)-associated genome RNA and nuclear protein (NP) fractions by CsCl centrifugation. The RNA polymerase-RNA complexes were capable of catalyzing the endonucleolytic cleavage of capped RNA, the initiation of primer-dependent RNA synthesis, and the synthesis of small-sized RNA, but were unable to synthesize template-sized RNA. By adding the NP protein to the RNA polymerase-RNA complexes, RNP (RNA polymerase-RNA-NP) complexes were reconstituted; they synthesized template-sized transcripts as did native RNP cores. These observations are consistent with the model where viral RNA polymerase is composed of the three P proteins while NP is essential for the elongation of RNA chains. RNP was completely dissociated into RNA-free proteins (PB1, PB2, PA, and NP) and a protein-free genome RNA fraction by centrifugation in cesium trifluoroacetate (CsTFA) and glycerol. By mixing the protein and RNA fractions, primer-dependent RNA-synthesizing activity was regained. These complexes, however, produced only small-sized RNA, presumably due to incorrect assembly of NP on viral RNA.

The influenza virus RNA synthesis machine

“…The large differences in cRNA and 115,116 and in vivo, 88,102 although replication of short RNA templates can proceed in the absence of NP. 61,117 The incorporation of NP into new vRNPs is facilitated by the activity of cellular factors UAP56 and Tat-SF1, that may act as chaperones. 118,119 Viral polymerase should also be required stoichiometrically, as the end product is not vRNA but a vRNP.…”

The M1 and NP proteins of influenza A virus form homo- but not heterooligomeric complexes when coexpressed in BHK-21 cells.

“…In the infected cell newly synthesized NPs are imported into the nucleus where they take part in a RNA elongation\RNP assembly process (Davey et al, 1985 ;Honda et al, 1988 ;Krug et al, 1989). According to a favoured assembly model one or a few NPs bind to newly synthesized viral RNA, which will then stimulate further NP associations by cooperative NP-NP and NP-RNA interactions (Yamanaka et al, 1990).…”

Mutational analysis of influenza virus promoter elements in vivo

“…These conserved segments of 13 and 12 nucleotides in length have been observed to form dsRNA panhandle structures (Hsu et al, 1987;Seong & Brownlee, 1992;Fodor et al, 1993) which have recently been analysed in vitro in more detail using internally deleted model RNAs (Baudin et al, 1994;Tiley et al, 1994). In the virion, the panhandle formed at the ends of all RNA segments binds specifically to the viral RNA polymerase complexes, while the interiors of the segments associated with viral nucleoprotein (NP) stay singlestranded (Compans et al, 1972;Honda et al, 1988;Martin et al, 1992). Upon infection these viral RNPs initially serve as templates for the synthesis of viral mRNAs by a specific cap-snatching mechanism (Plotch et al, 1979;Braam et al, 1983) and later direct synthesis of full-length complementary RNAs (cRNAs), probably dependent on the absence or presence of newly synthesized NP protein, respectively (Shapiro & Krug, 1988).…”