Adaptive strategies of the influenza virus polymerase for replication in humans

Abstract

Transmission of influenza viruses into the human population requires surmounting barriers to cross-species infection. Changes in the influenza polymerase overcome one such barrier. Viruses isolated from birds generally contain polymerases with the avian-signature glutamic acid at amino acid 627 in the PB2 subunit. These polymerases display restricted activity in human cells. An adaptive change in this residue from glutamic acid to the human-signature lysine confers high levels of polymerase activity in human cells. This mutation permits escape from a species-specific restriction factor that targets polymerases from avian viruses. A 2009 swine-origin H1N1 influenza A virus recently established a pandemic infection in humans, even though the virus encodes a PB2 with the restrictive glutamic acid at amino acid 627. We show here that the 2009 H1N1 virus has acquired second-site suppressor mutations in its PB2 polymerase subunit that convey enhanced polymerase activity in human cells. Introduction of this polymorphism into the PB2 subunit of a primary avian isolate also increased polymerase activity and viral replication in human and porcine cells. An alternate adaptive strategy has also been identified, whereby introduction of a human PA subunit into an avian polymerase overcomes restriction in human cells. These data reveal a strategy used by the 2009 H1N1 influenza A virus and identify other pathways by which avian and swine-origin viruses may evolve to enhance replication, and potentially pathogenesis, in humans.

Fig. 1.

Identification of an adaptive strategy utilized by the polymerase of 2009 A(H1N1) in human cells. (A) Cell-based activity assays for influenza polymerases from human, avian, and 2009 A(H1N1) primary isolates containing WT or mutant PB2. Activity assays were performed in 293T cells and normalized to PB2 K627 for comparison. The identity of PB2 amino acid 627 for the naturally occurring (WT) and mutant variants for each isolate are indicated. Western blot analyses were performed on lysates used in the polymerase activity assays with the indicated antibodies. (B) Structural model of the SR polymorphism in the PB2 627 domain. Structures of the human and avian PB2 627 domains (27) are shown along with a model of the 2009 A(H1N1) domain. (C) The SR polymorphism contributes to polymerase activity for 2009 A(H1N1) isolates in human cells. Assays were performed as in A using WT PB2 or the GQ mutant (S590G/R591Q). Polymerase proteins were detected by Western blot analysis. For all activity assays, n = 3 ± standard deviation.

Fig. 2.

The SR polymorphism enhances activity of an avian influenza polymerase. (A) Polymerase activity assays were performed with the avian S009 polymerase containing WT or mutant PB2. The SR polymorphism was introduced into PB2 SR (G590S/Q591R). The human-signature lysine was introduced into PB2 K (E627K). Activity was measured for polymerases expressed in human (293T) and avian (DF1) cells and normalized to WT PB2. n = 3 ± standard deviation. PB1, PB2, and PA were detected by Western blot. (B) Primer extension analysis of viral RNAs produced by WT or mutant polymerase in 293T cells. Migration of molecular weight standards is indicated in base pairs. (C) Electrostatic surface potentials were mapped onto models of S009 PB2 627 domains and colored from −4 kT/e (red) to 4 kT/e (blue).

Fig. 3.

The SR polymorphism enhances replication in restrictive cells. Multicycle replication kinetics were determined for WT and mutant viruses in human, porcine and avian cells. Recombinant virus encoding WT or mutant forms of the S009 polymerase was used to infect A549 cells (MOI = 0.01) or PK (15) and DF1 cells (MOI = 0.001). Virus yield at the indicated time points was determined by plaque assay. Data represent the mean of three independent infections ± standard deviation.

Fig. 4.

Acquisition of a human PA rescues activity of reassortant avian polymerases in human cells. Activity assays of reassortant polymerases were performed in 293T cells. Subunits of the S009 polymerase were replaced with those from human virus as indicated. Activity was normalized to WT S009 polymerase. n = 3 ± standard deviation.