The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model

Abstract

Adaptation of influenza A viruses to a new host species usually involves the mutation of one or more of the eight viral gene segments, and the molecular basis for host range restriction is still poorly understood. To investigate the molecular changes that occur during adaptation of a low-pathogenic avian influenza virus subtype commonly isolated from migratory birds to a mammalian host, we serially passaged the avirulent wild-bird H5N2 strain A/Aquatic bird/Korea/W81/05 (W81) in the lungs of mice. The resulting mouse-adapted strain (ma81) was highly virulent (50% mouse lethal dose ‫؍‬ 2.6 log 10 50% tissue culture infective dose) and highly lethal. Nonconserved mutations were observed in six viral genes (those for PB2, PB1, PA, HA, NA, and M). Reverse genetic experiments substituting viral genes and mutations demonstrated that the PA gene was a determinant of the enhanced virulence in mice and that a Thr-to-Iso substitution at position 97 of PA played a key role. In growth kinetics studies, ma81 showed enhanced replication in mammalian but not avian cell lines; the PA 97I mutation in strain W81 increased its replicative fitness in mice but not in chickens. The high virulence associated with the PA 97I mutation in mice corresponded to considerably enhanced polymerase activity in mammalian cells. Furthermore, this characteristic mutation is not conserved among avian influenza viruses but is prevalent among mouse-adapted strains, indicating a host-dependent mutation. To our knowledge, this is the first study that the isoleucine residue at position 97 in PA plays a key role in enhanced virulence in mice and is implicated in the adaptation of avian influenza viruses to mammalian hosts.

Reassortment and Mutation of the Avian Influenza Virus Polymerase PA Subunit Overcome Species Barriers

“…The PA I97 mutation arose following the serial passage of a low-pathogenicity avian H5N2 virus in mice and contributed to the high pathogenicity of the newly derived virus (42). The PA I97 mutation increased polymerase activity and replication in mice but not chickens, suggesting that it might influence pathogenicity in a species-specific fashion.…”

“…2B). S009 lacks the enhancing I85, I97, and S186 mutations (3,42), raising the possibility that other novel polymorphisms in PA might also contribute to the enhanced polymerase activity. Together, these data suggest that there are multiple strategies by which mutations in PA can contribute to enhanced polymerase activity, virus replication, and, possibly, viral host tropism.…”

Virulence of pandemic (H1N1) 2009 influenza A polymerase reassortant viruses

“…17 Therefore to address this, we generated various polymerase point mutant pandemic viruses (PB2, 627K/701N; PB1, expression of PB1-F2 protein; PA, 97I) by an established reverse genetics system. 16 We found in our work that expression of the 627K or 701N mutation in PB2 or driving the PB1-F2 protein expression enhanced reporter gene activity in vitro compared with that of the wild-type (WT) CA04 polymerase complex. However, the individual or even the simultaneous incorporation of these virulence markers did not essentially increase acid (aa) substitutions could be observed aside from the 97I.…”

“…10 In addition to the specific contributions of the surface glycoprotein hemagglutinin (HA) and the non-structural 1 (NS1) protein, each of the polymerase genes have been continuously implicated to harbor molecular determinants for host range and virulence such as the presence of lysine (K) and/or asparagine (N) residue at positions 627 and 701 in PB2, [11][12][13] expression of the alternatively-spliced PB1-F2 protein, 14,15 and the isoleucine (I) substitution in PA at position 97. 16 The absence of these characteristic molecular factors in the genetic make-up of the pandemic (H1N1) 2009 virus may partially contribute to its low virulence among humans.…”

“…However, the individual or even the simultaneous incorporation of these virulence markers did not essentially increase acid (aa) substitutions could be observed aside from the 97I. 16 Therefore to verify whether the 97I mutation alone was responsible for increased virulence, we generated two reverse-mutants reflecting modifications in PA at this specific codon (Av/W81 PA 97I and Av/ma81 PA 97T ) in the CA04 background and tested their pathogenicity in groups of mice intranasally inoculated with 10 4.5 TCID 50 of respective viruses. Survival rates were monitored for 13 d. Consequently, virulence phenotype of the revertant virus Av/ma81 PA 97T was attenuated allowing 100% survival of infected mice (17.6% mean body weight loss) whereas Av/W81 (H5N2) was enhanced with the introduction of the PA 97I inducing 60% mortality rates (Fig.…”