The 2009 influenza pandemic was due to a swine-origin influenza strain that was a reassortant of human, avian, and swine genes. The viral ribonucleoprotein is composed of a North American avian-derived PA and PB2, a human H3N2-derived PB1, and a classical swine-derived NP (3
). In general, the activity of avian polymerase complexes in mammalian cells is very poor, but some mutations are known to enhance activity, including PB2 158G, 627K, and 701N (5
). The 2009 pH1N1 viruses replicate and transmit efficiently in humans but have the avian-type low-pathogenicity residues 158E, 627E, and 701D in PB2, suggesting the involvement of other residues for efficient replication of pH1N1 in the mammalian host. Two residues in PB2 have been identified which contribute to pH1N1 polymerase activity: residue 591R, which locates close to residue 627 in the three-dimensional structure (19
), and residue 271A, which is highly conserved among human viruses and enhances avian polymerase activity in mammalian cells (2
). However, in this study using a reporter gene assay, we found that the pH1N1 PA has a stronger impact on polymerase activity than the pH1N1 PB2 in mammalian cells. Although both the PA and PB2 of the pH1N1 virus are of avian virus origin, the avian Nan virus polymerase containing Cal PA was over 20 times more active than the Nan complex containing Cal PB2 at higher temperatures (A, lanes 7 and 9). Interestingly, the human Aichi and the highly pathogenic VN1203 and 1918 virus polymerase components function as efficiently with Cal PA as with their homologous complexes, unlike with Cal PB2, which resulted in significantly reduced activity (). Replacement of Cal PA with that of the Nan, Aichi, VN1203, or 1918 virus resulted in reduced activity of the Cal polymerase complex, indicating that Cal PA contains residues that, together with Cal PB2, complement polymerase activity in the mammalian host.
Enhanced activity of the Nan polymerase complex provided by Cal PA was observed at 37°C and 39°C but not at 34°C (A, lane 9). However, the additional inclusion of Cal PB2 enhanced activity at 34°C (lane 12), suggesting that Cal PB2 contains residues required for the complex to be active at lower temperatures. Because the Aichi, VN1203, and 1918 virus complexes containing Cal PA were active at lower temperatures, it is likely that one or more PB2 residues present in the 2009 pH1N1, seasonal human virus, and highly pathogenic avian viruses is necessary for activity at lower temperatures. In fact, we previously showed that the PB2 E627K mutation strongly activates the Nan polymerase complex even at 34°C (2
). Cal PB2 591R, which is considered to compensate for the lack of 627K, may allow for low-temperature activity of the pH1N1. Replacement of Cal PB2 with that of Nan reduced activity slightly at 37 or 39°C (about 2- to 2.5-fold) but strongly at 34°C (25-fold; A, lanes 1 and 2), which clearly indicates that PB2 from a conventional avian virus does not contain the residue(s) required for low-temperature activity. The underlying mechanism for the temperature dependence of the influenza polymerase is unknown, although interactions with host inhibitory or enhancing factors could be an interesting subject for future research (20
The role of PA in host adaptation is not as well characterized as the role of PB2. However, a recent study indicates the PA mutation T97I increases polymerase activity of an avirulent avian virus in a reporter gene assay, as well as virulence in a mouse model (28
). In our study, we have shown that multiple residues, especially 85I, 186S, and 336M in Cal PA, contribute to the enhancement of avian polymerase activity in mammalian cells, which is essential for mammalian host adaptation. The addition of each mutation to Nan PA significantly increased Nan polymerase activity (). However, individual mutations in Cal PA at positions 85, 186, or 336 to Nan residues only slightly decreased Cal polymerase activity, supporting the idea that multiple residues in Cal PA contribute to enhanced Cal polymerase activity in mammalian cells. PA possesses the endonuclease activity required for the “cap-snatching” mechanism (4
), and the crystal structures of two domains of PA have been determined (4
). The amino acids that increased polymerase activity, 85I, 186S, and 336M, are all surface exposed on their domains. 85I and 186S are located in the domain containing the endonuclease active site. While 186S is located on the opposite side of the structure from the endonuclease active site, it is part of a proposed bipartite nuclear localization signal, residues 124 to 139 and 186 to 247 (23
). The residue 85I is located on the surface adjacent to the endonuclease site. The branched, hydrophobic isoleucine at this position in the pH1N1 PA is very different from the polar neutral threonine in most other strains and could potentially affect the endonuclease activity of PA. The residue 336M is located on the opposite end of PA from the PB1-binding site, and no clear function is linked to this residue. However, because of its location at the surface of the molecule, it could be involved in interactions with cellular proteins that inhibit or enhance polymerase activity. It is likely that multiple mutations contribute to the highly active Cal polymerase through multiple complementary unknown mechanisms.
The PA 85I, 186S, and 336M mutations identified by reporter gene assay actually enhanced viral polymerase activity when introduced into viruses, confirming that these mutations in an avian PA can enhance avian virus polymerase activity in mammalian host cells. The 85I and 336M mutations strongly enhanced viral protein synthesis at an early stage of infection (). The virus containing the 85I mutation grew faster and to a higher titer than WT virus at 37 and 39°C, which is consistent with the reporter gene assay results. However, viruses containing the 186S or 336M mutations did not show enhanced multistep growth in A549 cells. While the 336M mutation enhanced polymerase activity more than the 85I mutation as determined by the reporter gene assay or viral protein synthesis, the enhanced polymerase activity by the 336M mutation did not directly reflect the multistep growth of the mutant virus. This result may suggest that the mutation at residue 336 caused a deficiency in a process other than transcription, possibly through unbalanced genome replication, impaired transport of nucleocapsids, or defective virus assembly.
When we examined pathogenicity in vivo
, we found that none of the mutations resulted in lethal infection, in sharp contrast to the PB2 627K mutation (2
), which indicates that these PA residues are host adaptation but not pathogenicity factors in the mammalian host, as expected from the fact that wild-type pH1N1 viruses are not pathogenic to mice. However, the 336M mutation increased morbidity, as evidenced by weight loss and lung pathology, as well as the lung viral titer at late times after infection. Unexpectedly, the 85I mutation, which increased multistep growth in cultured cells, did not enhance in vivo
virus growth or morbidity in mice. These data may support the idea that pathogenicity is not simply reflected by better virus growth as determined in cultured cells but also by the host response, including elevated cytokine release and inflammation, which can then affect lung pathology and virus growth.
Sequence data analysis indicates that few influenza strains contain one or some of the PA residues 85I, 186S, and 336M (). Although almost all of the pH1N1 viruses contain these three key residues, very few other influenza isolates have these PA residues, regardless of the host. Not one non-pH1N1 strain has all three residues in PA. The majority of the strains containing PA 85I isolated prior to emergence of the 2009 pandemic are avian viruses (13 out of 15 non-pH1N1 viruses). PA 336M can be found in both avian and human isolates. Seventeen human isolates and 12 avian isolates contained PA 336M. Interestingly, multiple highly pathogenic H5N1 human isolates contain PA 336M (A/Hong Kong/483/97, A/Hong Kong/485/97, A/Hong Kong/532/97, and A/Hong Kong/542/97). Although A/Hong Kong/483/97 and A/Hong Kong/485/97 contain PB2 627K, two strains, A/Hong Kong/532/97 and A/Hong Kong/542/97, were pathogenic to mice (15
), lack the previously identified virulence markers in polymerase PB2 158G, 271A, 627K, and 701N and PA 97I genes, and have the PA 336M mutation revealed in this study. Some H5N1 viruses isolated from Hong Kong chickens around the same time also contain PA 336M. Further study is needed to determine the contribution of PA mutations to the highly pathogenic phenotype of H5N1 viruses containing PB2 627E. It is possible that the enhancement of polymerase activity by PA 336M contributes to host adaptation and pathogenicity of these H5N1 viruses, although it is unlikely that this single residue explains the highly pathogenic phenotype of the viruses.
It is not clear when and how the PA 85I, 186S, and 336M mutations were introduced into the swine virus PA gene, as surveillance of swine influenza virus prior to the 2009 pandemic was extremely limited. Also, despite efficient viral replication in miniature pigs, infection with the pH1N1 was asymptomatic, which may explain the lack of detection of the virus in pigs before emergence of the pandemic (13
). This pandemic highlights the need for continued and expanded influenza virus surveillance, especially in swine, which continue to play a major role in the emergence of pandemic influenza A viruses. It should be noted that identification of the polymerase mutations that affect host adaptation provides critical information for identifying potential future pandemic viruses.