Mutations in influenza A virus PB2 residues 701 and 702 alter viral growth and importin-α subtype dependence

Abstract

 Influenza A viruses are known to spread across different species. Recent human cases of avian influenza infection in China are of serious public health concerns. Polymerase basic protein 2 (PB2) is the viral polymerase subunit that have a critical role in cross-species transmission. Many comprehensive studies about adaptive mutations in PB2 (such as E627K, D701N and K702R) have been taken to investigate the fitness of avian influenza viruses in mammalian systems. However, the limited polymorphisms of PB2 residues 701 and 702 in natural isolates restricted the understandings of the roles played by these residues. In order to study PB2-701/702 mutations other than D701N and K702R, a site-directed random mutagenesis approach targeting PB2 residues 701 and 702 was used to establish a mutant plasmid library. Recombinant mutant viruses were rescued in human embryonic kidney 293T cells and passaged in either Madin-Darby canine kidney (MDCK) cells or embryonated chicken eggs, so as to study the viability of recombinant viruses in different host systems. A total of 31 different polymorphisms (including wild-type A/Puerto Rico/8/1934) were isolated by plaque purifications in MDCK cells or chicken fibroblast DF-1 cells. The polymerase activity assay in human (293T cells) or avian (DF-1 cells) system demonstrated distinct phenotypes at 33oC (nasal temperature), 37oC (human core temperature) and 39oC (avian core temperature). Furthermore, difference in virus replication was observed at early time-point in MDCK cells and DF-1 cells, implicating that PB2-701/702 mutations determined the fitness at early stage of infection. Higher temperature reduced the virus replication of PB2-701/702 mutants but not for wild-type in mammalian and avian systems. Structural predictions of 701/702-loop suggested a positive correlation between the surface charge distribution and polymerase activity in 293T cells (Pearson r, p<0.0001). The polymerase activity of PB2-701/702 mutants with additional PB2 K627E mutation was reduced in 293T cells, especially in the vRNPs with PB2-701N/702G and PB2-701S/702R. The silencing of importin-α isoforms modulated the polymerase activity of PB2-701/702 mutants. The effect of temperature change was shown in the polymerase activity upon importin-α silencing, where the polymerase activity of PB2-701/702 mutants was reduced in importin-α1 or –α7-silenced cells at 37oC and 33oC but not at 39oC. The silencing of importin-α1 and –α7 impaired the growth rate of PB2-701/702 mutants, yet the effect of importin-α1 silencing was less prominent in the infection of PB2-701S/702R. The virus replication of PB2-701A/702E, PB2-701H/702G and PB2-701S/702F has enhanced in importin-α4-silenced A549 cells at 72 h.p.i.. The intracellular localization of PB2, PB1 and NP had no difference between PB2-701/702 mutants and wild-type in the transfected or infected cells. The precipitation of importin-α5 and –α7 was increased with PB2-701A/702K in importin-α4-silenced cells, whereas PB2-701S/702R showed the enhanced binding of importin-α7. In this study, a panel of PB2-701/702 mutants was used to determine the plasticity of PB2. Differential phenotypes of PB2-701/702 mutants were observed in viral replication in mammalian and avian systems. Distinct dependence of human importin-α isoforms in viral transcription and replication of PB2-701/702 mutants has no relationship with the intracellular localization of PB2, PB1 and NP.