Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier

Abstract

Avian influenza A viruses, such as the highly pathogenic avian H5N1 viruses, sporadically enter the human population but often do not transmit between individuals. In rare cases, however, they establish a new lineage in humans. In addition to well-characterized barriers to cell entry, one major hurdle which avian viruses must overcome is their poor polymerase activity in human cells. There is compelling evidence that these viruses overcome this obstacle by acquiring adaptive mutations in the polymerase subunits PB1, PB2, and PA and the nucleoprotein (NP) as well as in the novel polymerase cofactor nuclear export protein (NEP). Recent findings suggest that synthesis of the viral genome may represent the major defect of avian polymerases in human cells. While the precise mechanisms remain to be unveiled, it appears that a broad spectrum of polymerase adaptive mutations can act collectively to overcome this defect. Thus, identification and monitoring of emerging adaptive mutations that further increase polymerase activity in human cells are critical to estimate the pandemic potential of avian viruses.

Fig 1

Host range of influenza A viruses. Wild water birds represent the natural reservoir of influenza A viruses, from which they can be transmitted to a wide variety of other hosts, including horses, cats, dogs, whales, seals, wild flying birds, chicken, pigs, and humans. Only recently, influenza A virus has also been detected in bats, although the origin is unclear.

Fig 2

Illustration of the influenza A virus replication cycle. Influenza A virus particle binds to the cellular receptor and enters the cell by endocytosis. The viral ribonucleoproteins (vRNPs) are released into the cytoplasm upon acidification of the endosome. vRNPs are transported into the nucleus, where transcription and replication occur. Replication is supported by the viral nuclear export protein NEP. For genome replication, negative-sense viral RNA is transcribed into plus-sense cRNA that is complexed by the viral polymerase and NP to form the cRNP and serves as a template for vRNA synthesis. After export from the nucleus, vRNPs are assembled into new viral particles at the plasma membrane and released from the cell.

Fig 3

Experimental systems for identification of adaptive mutations of influenza A viruses. Several experimental systems are used to identify adaptive mutations that facilitate host adaptation. These include the following. (A) Bioinformatics analysis of bird- and human-derived influenza virus genome sequences. Such analyses are frequently used to identify unique nucleotide changes that occur only in humans but not in avian species that are selected in the specific host. (B) The polymerase reconstitution assay allows functional analysis of adaptive mutations in the polymerase subunits or NP by reverse genetics. Expression plasmids for the polymerase subunits PB2, PB1, and PA as well as NP and a viral genome analog, harboring a reporter gene instead of the viral protein, are transfected into cells, and reporter gene activity can be measured 24 h later. With this approach, the activities of polymerases from different influenza virus strains can be compared and analyzed in human and avian cells. (C) Different animal models, including mice and ferrets, are used to determine pathogenicity and airborne transmissibility of bird- and human-derived influenza A virus strains. (D) Biochemical analysis can be performed with virion-derived RNPs or recombinant proteins from different expression systems to study polymerase activity in vitro.

Fig 4

Described mutations increasing polymerase activity in mammalian cells. Published data were analyzed to screen for predicted host-adaptive amino acids (Bioinformatics) and mutations experimentally shown to increase activity of an avian influenza virus polymerase in the context of mammalian cells or to increase pathogenicity in a viral infection (Experimental) (115, 116). Functional domains are indicated in green (interaction with viral proteins), red (involved in nuclear localization), purple (involved in nuclear export), orange (interaction with cellular proteins), yellow (MxA resistance), and blue (RNA binding).