Abstract
BackgroundGenetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood.Methodology/Principal FindingsHere, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated.Conclusions/SignificanceOverall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes.
Highlights
Influenza A virus is a member of the family Orthomyxoviridae, and classified into subtypes by antigenic differences of the two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA) [1]
Our results revealed that the PB2 subunit of H5N1 has a strong inhibitory effect on the RNP activity when introduced into the polymerase of other influenza strains
VN PB2 alone or VN PB2 - VN PB1 significantly increased the activity of SW polymerase (Figure 1, lanes, 27 and 29). These results indicate that the PB2 subunits of these two H5N1 strains differ in their properties when reconstituted into hybrid RNP with other influenza strains, and HK PB2 has a strong inhibitory effect on RNP activity
Summary
Influenza A virus is a member of the family Orthomyxoviridae, and classified into subtypes by antigenic differences of the two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA) [1]. These results are consistent with recent RNP reconstitution studies suggesting that PA plays a major role in increasing activities of hybrid polymerase [14,47,49] Such a tendency could not be observed in hybrid WSN polymerase, suggesting that the PA subunit cannot always overcome the restriction of genetic reassortment. Four residues at positions 108, 508, 524 and 627 of the PB2 subunit appear to be important determinants that are involved in the accumulation of functional RNP and in modulating the polymerase activity These results may suggest a possible mechanism by which the generation of replicative reassortant virus of influenza is highly restricted. The role of the RNA polymerase in the genetic reassortment is not well understood
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