Host Determinant Residue Lysine 627 Lies on the Surface of a Discrete, Folded Domain of Influenza Virus Polymerase PB2 Subunit

Abstract

Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain) exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design experiments to elucidate the effects of mutations on polymerase–host factor interactions.

Author Summary

There is worldwide concern that currently circulating avian influenza viruses will cross the species barrier and become highly pathogenic, human transmissible strains with pandemic potential. This could result from residue changes in several influenza proteins, either by point mutations, or through shuffling of the segmented avian and mammalian viral genomes. Numerous studies have highlighted potentially important residues for inter-species transmission, and several are found in the influenza polymerase that replicates and transcribes the viral genome. The polymerase PB2 subunit contains a number of such positions, notably residue 627, which is glutamic acid in avian viruses but lysine in human-adapted strains. Experiments have shown that the polymerase mutations affect the efficiency of viral replication in different host species, but the molecular mechanisms are unknown. As a first step towards resolving this enigma, we have identified a novel domain of PB2, containing many host determinant sites, and determined its atomic structure by X-ray crystallography. The species-specific residues are all located on the domain surface, suggesting they could be involved in interactions with viral proteins or host factors. The 627 position is solvent-exposed in both the lysine and glutamic acid variants, respectively either reinforcing or disrupting a striking positively charged surface patch. The identification and structural characterisation of biochemically well-behaved domains of PB2 provides new tools for understanding the phenomenon of inter-species transmission that is of global health importance.