The direct transmission of avian H5N1 influenza A viruses to humans suggests a new mechanism for influenza outbreaks. However, potential mutations in avian viruses that confer increased pathogenicity or transmissibility in humans and other mammalian hosts are not fully examined. In this study, we identified PB2 residue 271 as a key amino acid for polymerase function and virus growth in mammalian cells. Our data show that a single mutation at 271 from the avian virus-conserved T to the human virus-conserved A enhances polymerase activity in a reporter gene assay, as well as the growth of a recombinant virus containing an otherwise-avian polymerase both in vitro and in vivo. Our results indicate a role for PB2 residue 271 in the mammalian adaptation of avian influenza A viruses.
PB2 residues 627 and 701 have been shown previously to enhance viral pathogenicity and transmission in mammalian hosts. The role of PB2 627 is the most extensively studied. A single mutation from E to K dramatically improves the function of an avian polymerase complex and enhances viral growth in mammalian cells, especially at lower temperatures (28
). Some H5N1 viruses with E at this position show low pathogenicity in mice, while those with K are highly pathogenic (15
). In vivo experiments have further revealed that 627K allows for the growth of H5N1 viruses in the upper respiratory tracts of mice (16
), and viruses with PB2 627E show reduced transmission in a guinea pig model compared to that of viruses containing 627K (42
). These studies suggest that enhanced replication at lower temperatures contributes to enhanced growth in the upper airway of infected animals or humans and thus improves transmission. Almost all human viruses have 627K, whereas avian viruses have 627E in PB2 (3
). The analysis of the polymerase activity of the avian Nan polymerase complex using a reporter gene assay indicates that the introduction of the E627K mutation enhanced activity at both low and high temperatures in 293T cells, while the T271A mutation enhanced activity only at higher temperatures (37 and 39°C) (Fig. ). This result suggests that the mechanism of the enhancement of polymerase activity differs between the mutations at 271 and 627. It is highly likely that enhanced polymerase activity by the 271A mutation contributes to increased virus growth in the mouse lung (Fig. ). Interestingly, the T271A mutation did not increase polymerase activity at low temperature as determined in vitro, although the growth of the T271A mutant virus in cultured cells was more efficient than that of WT virus at 34°C (Fig. ). It is not clear how the T271A mutation enhances virus growth in cultured cells at 34°C. It is possible that the slight increase in polymerase activity detected in the reporter gene assay (Fig. ) is sufficient for enhanced virus growth in tissue culture (Fig. ). Another possibility is that, in addition to its role in polymerase activity, the highly conserved host-specific amino acid 271 also plays an important role in virus assembly and spread in mammalian cells.
The PB2 mutation D701N also has been implicated in the adaptation of H5N1 viruses to mammalian hosts (6
). Studies using an avian-adapted H7N7 strain and its mouse-adapted variant showed that the single mutation D701N in PB2 improved polymerase activity 3-fold in a reporter gene assay in 293T cells (10
). Other studies using recombinant A/Panama/2007/99 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses showed that the D701N mutation enhances transmission between guinea pigs (42
). A recent study also has shown that the D701N mutation enhances the binding of PB2 to importin α1 and correspondingly increases the level of PB2 in the nucleus in mammalian cells, suggesting that the adaptation of the viral polymerase to the nuclear import machinery plays an important role in the interspecies transmission of influenza virus (12
Of the 10 highly conserved PB2 residues we examined in this study, only two, E627K and T271A, enhanced the polymerase activity of an otherwise-avian complex in mammalian cells. None of the other mutations significantly enhanced polymerase activity in mammalian 293T cells, except the A588I mutation, which increased activity about 3-fold (Fig. ). It is not clear if other conserved residues play roles in adaptation to mammalian hosts. Although these residues did not enhance polymerase activity in vitro, these residues could be involved in interactions with mammalian host proteins during the viral life cycle and thus are required for the efficient replication and assembly of the virus in mammalian host cells. They also may affect the interaction of PB2 with other influenza virus proteins in mammalian cells. Since other structural components also contain host-specific residues, it is of interest to further determine the role of these conserved residues in virus growth.
The results of our in vitro reporter gene assay (Fig. ) and sequence analysis (Tables and ) support the idea that PB2 residue 271 contributes to the 2009 novel swine-origin influenza A (H1N1) virus human pandemic. The S-OIV possesses PB2 and PA genes of North American avian virus origin; a PB1 gene of human H3N2 virus origin; HA, NP, and NS genes of classical swine virus origin; and NA and M genes of Eurasian avian-like swine virus origin (5
). Previous studies as well as our data in Fig. clearly indicate that polymerase complexes containing avian virus PB2 show poor activity in mammalian host cells. However, the polymerase activity of the S-OIV is much higher than that of the Nan complex (Fig. ). The avian-origin S-OIV PB2 contains both 627E and 701D, the same residues found in Nan and most other avian viruses. However, the S-OIV contains PB2 271A, which we showed enhances polymerase activity and virus growth in mammalian hosts (Fig. , , and ). In fact, the activity of the Cal polymerase complex at 34°C was reduced 6-fold by mutation at 271 from A to the avian virus-conserved residue T (Fig. ). In addition, the 271A mutation significantly enhanced virus spread in cultured mammalian cells, especially at lower temperature (Fig. ). Therefore, 271A in S-OIV is likely to contribute to its efficient transmission through both enhanced polymerase activity and virus growth in mammalian hosts.
PB2 271A has been maintained in the majority of swine isolates since the introduction of triple-reassortant viruses around 1998 (Table ), and all pandemic S-OIV isolates have 271A/627E/701D. However, the polymerase activity of the Cal complex with 271T still is higher than that of the Nan polymerase complex. In addition, the A271T mutation in the Cal polymerase complex did not significantly affect polymerase activity at higher temperatures, suggesting that additional unidentified residues in PB2 or other polymerase components also contribute to the high polymerase activity of the Cal complex in mammalian cells.
Unlike the E627K mutation, the T271A mutation by itself was not sufficient to cause lethal infection by WSN-based viruses containing Nan NP and polymerase proteins. However, the virus titer in lungs of mice infected with the 271A mutant was much higher than that of WT virus-infected mice (Fig. ). Additionally, the double mutant 271A/627K showed a higher lung virus titer than the E627K mutant, especially at 6 days postinfection, when the virus titer in lungs infected with the 271A/627K mutant was almost 10-fold higher than that of 627K mutant-infected mouse lungs. Pathological analysis of lungs showed the most severe lesions in 271A/627K mutant-infected mice with massive inflammatory cell infiltration, severe hemorrhage, and alveolar destruction (Fig. ). These results suggest the contribution of the T271A mutation to the pathogenicity of influenza virus in mice. However, the fact that the 271A mutation by itself cannot cause lethal infection, despite the high viral titer in lungs, suggests that virus growth in the mouse lung does not necessarily correlate with virus pathogenicity. Pathological analysis of infected lungs indicates that both the T271A and E627K mutants induce massive cell infiltration. However, unlike 271A, infection with the 627K mutant was associated with severe hemorrhage, suggesting that the 627K virus causes more severe damage in lung tissue (Fig. ). We also noticed an earlier and more severe cytopathic effect in 627K virus-infected A549 and MDCK cells compared to that of cells infected with the T271A mutant (data not shown). These observations suggest that, in addition to enhanced virus growth, lung tissue damage strongly influences the pathogenicity of influenza A viruses.
While our studies have shown that residue 271 is important for the mammalian adaptation of influenza virus, the mechanism of action of 271 still needs to be determined. Studies of 627 mutants suggest that residue 627 affects the interaction of PB2 with NP (36
). Another study suggests that the E-to-K mutation at 627 facilitates escape from an inhibitory factor that restricts the function of avian-derived polymerase in human cells (31
). Additional studies by Kuzuhara et al. (21
) have found that a charged “basic groove” in 627K-containing PB2 binds RNA, especially the 5′ vRNA promoter, with high affinity compared to that for 627E, suggesting that the RNA-binding activity of PB2 contributes to enhanced polymerase activity (21
). PB2 627E and PB2 627K possess distinct electrostatic properties (45
) and may interact differentially with host inhibitory factors (31
) and/or viral RNA (21
) as a result. It is not known whether PB2 residue 271 also is involved in the interactions of PB2 with NP or RNA. Early cross-linking studies suggested that two separate sequences of PB2 at residues 242 to 282 and residues 538 to 577 are involved in cap binding (17
). However, the crystal structure of a PB2 fragment containing residues 318 to 483 includes a PB2 cap-binding domain within the solved structure (14
). It is possible that residue 271 locates close to the cap binding domain in the three-dimensional structure and thus affects the interaction of PB2 with the cap structure. Future structural studies of the PB2 domain containing residue 271 will help to elucidate the function and mechanism of the action of residue 271 of PB2 and its effect on host adaptation.