Polymerase activity of hybrid ribonucleoprotein complexes generated from reassortment between 2009 pandemic H1N1 and seasonal H3N2 influenza A viruses

Abstract

Background: A novel influenza virus (2009 pdmH1N1) was identified in early 2009 and progressed to a pandemic in mid-2009. This study compared the polymerase activity of recombinant viral ribonucleoprotein (vRNP) complexes derived from 2009 pdmH1N1 and the co-circulating seasonal H3N2, and their possible reassortants.

Results: The 2009 pdmH1N1 vRNP showed a lower level of polymerase activity at 33°C compared to 37°C, a property remenisence of avian viruses. The 2009 pdmH1N1 vRNP was found to be more cold-sensitive than the WSN or H3N2 vRNP. Substituion of 2009 pdmH1N1 vRNP with H3N2-derived-subunits, and vice versa, still retained a substantial level of polymerase activity, which is probably compartable with survival. When the 2009 pdmH1N1 vRNP was substituted with H3N2 PA, a significant increase in activity was observed; whereas when H3N2 vRNP was substituted with 2009 pdmH1N1 PA, a significant decrease in activity occurred. Although, the polymerase basic protein 2 (PB2) of 2009 pdmH1N1 was originated from an avian virus, substitution of this subunit with H3N2 PB2 did not change its polymerase activity in human cells.

Conclusions: In conclusion, our data suggest that hybrid vRNPs resulted from reassortment between 2009 pdmH1N1 and H3N2 viruses could still retain a substantial level of polymerase activity. Substituion of the subunit PA confers the most prominent effect on polymerase activity. Further studies to explore the determinants for polymerase activity of influenza viruses in associate with other factors that limit host specificity are warrant.

Figure 1

Polyermase activity of vRNP complexes of 2009 pandemic H1N1 at 33°C and 37°C. (a) 293T cells, 37°C and 33°C. (B) A549 cells, 37°C and 37°C. Polymerase activity as reflected by the normalized relative light units ratio (mean ± standard deviation, n = 3) at 37°C compared to 33°C, * represents statistical significance at p < 0.03, and # represents statistical significance at p < 0.01.

Figure 2

Polyermase activity of vRNP complexes of 2009 pandemic H1N1, seasonal H3N2 and WSN H1N1. (a) 293T cells, 37°C. (b) 293T cells, 33°C. (c) A549 cells, 37°C. (d) A549 cells, 33°C. Polymerase activity as reflected by the normalized relative light units was expressed as relative activity (mean ± standard deviation, n = 3) compared to the reference strain WSN H1N1, * represents statistical significance at p < 0.01 compared to WSN H1N1, and # represents statistical significance at p < 0.05 compared to H3N2.

Figure 3

Polymerase activity of 2009 pdmH1N1 vRNP substituted with H3N2 PB1, PB2, PA and NP. Recombinant vRNPs were transfected into (a) 293T and (b) A549 cells at an incubation temperature of 37°C. Polymerase activity as reflected by the normalized relative light units was expressed as relative activity (mean ± standard deviation, n = 3) compared to the parent pdmH1N1 vRNP, * represents statistical significance at p < 0.05.

Figure 4

Polymerase activity of H3N2 vRNPs substituted with 2009 pdmH1N1 PB1, PB2, PA and NP. Recombinant vRNPs were transfected into (a) 293T and (b) A549 cells at an incubation temperature of 37°C. Polymerase activity as reflected by the normalized relative light units was expressed as relative activity (mean ± standard deviation, n = 3) compared to the parent H3N2 vRNP, * represents statistical significance at p < 0.05.