A statistical strategy to identify recombinant viral ribonucleoprotein of avian, human, and swine influenza A viruses with elevated polymerase activity

Abstract

Objectives: Reassortment of influenza A viruses can give rise to viral ribonucleoproteins (vRNPs) with elevated polymerase activity and the previous three pandemic influenza viruses contained reassorted vRNPs of different origins. These suggest that reassorted vRNP may be one of the factors leading to a pandemic virus. In this study, we reconstituted chimeric vRNPs with three different viral strains isolated from avian, human and swine hosts. We applied a statistical strategy to identify the effect that the origin of a single vRNP protein subunit or the interactions between these subunits on polymerase activity.

Design: Eighty one chimeric vRNPs were reconstituted in 293T cells at different temperatures. Polymerase activity was determined by luciferase reporter assay and the results were analysed by multiway anova and other statistical methods.

Results: It was found that PB2, PB1, NP, PB2-PB1 interaction, PB2-PA interaction and PB1-NP interaction had significant effect on polymerase activity at 37°C and several single subunits and interactions were identified to lead to elevation of polymerase activity. Furthermore, we studied 27 out of these 81 different chimieric vRNPs in different combinations via fractional factorial design approach. Our results suggested that the approach can identify the major single subunit or interaction factors that affect the polymerase activity without the need to experimentally reproduce all possible vRNP combinations.

Conclusions: Statistical approach and fractional factorial design are useful to identify the major single subunit or interaction factors that can modulate viral polymerase activity.

Keywords: Fractional factorial design; influenza virus; polymerase; reassortment.

Figure 1

Relative polymerase activity of 81 recombinant viral ribonucleoprotein (vRNP) at 33°C. Data are expressed as mean relative polymerase activity in relative to the polymerase activity of wild‐type human origin vRNP complex (HHHH). Error bars represent one standard deviation (n = 3).

Figure 2

Relative polymerase activity of 81 recombinant viral ribonucleoproteins (vRNPs) at 37°C. Data are expressed as mean relative polymerase activity in relative to the polymerase activity of wild‐type human origin vRNP complex (HHHH). Error bars represent one standard deviation (n = 3).

Figure 3

Relative polymerase activity of 81 recombinant viral ribonucleoprotein (vRNPs) at 40°C. Data are expressed as mean relative polymerase activity in relative to the polymerase activity of wild‐type human origin vRNP complex (HHHH). Error bars represent one standard deviation (n = 3).

Figure 4

Mean relative polymerase activity of wild‐type vRNP complexes of three different origins at different temperatures. Polymerase activity of vRNPs of wild‐type human origin (black), avian origin (dark gray), swine origin (light gray), or no vRNP (mock, unfilled) reconstituted in human 293T cells at 33, 37, or 40°C was compared. The normalized data are expressed as mean relative polymerase activity in relative to wild‐type human origin polymerase activity at the respective temperatures. Error bars represent one standard deviation (n = 3; *P < 0·05; **P < 0·001, by t‐test).