Biological and Structural Characterization of a Host-Adapting Amino Acid in Influenza Virus

Abstract

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.

The influenza virus RNA synthesis machine

“…2). In addition to the subunit interfaces mentioned above, the atomic structures of domains of the PA and PB2 subunits have been determined 47,55,56,[63][64][65][66][67] but no information on the structure of the polymerase active site in the PB1 subunit is yet available. The data reported allowed the identification of the polymerase cap-binding site in PB2 and established that the endonuclease responsible for cap-snatching resides at the N-terminal region of PA, in agreement with previous genetic contacts detected in the cryo-EM structure of the mini-RNP.…”

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

“…Intriguingly, fatal viral infections of humans with avian viruses of the H5N1 subtype that have retained the avian like PB2-E627 have been reported 9 . Moreover, the 2009 H1N1 pandemic virus also harbours PB2-E627, but additionally possesses a lysine at position 591 (PB2-E591K), which is able to partially, but not completely, compensate for the lack of the PB2-E627K mutation 10,11 , suggesting that other adaptive factors may account for the efficient replication of these viruses in humans. Other mutations in PB2, known to have a role in host adaptation, are the exchange of aspartic acid with asparagine at position 701 (PB2-D701N), which can confer high pathogenicity to avian H5N1 and H7N7 viruses in mice 4,12 and the substitution of threonine with alanine at position 271 (PB2-T271A), which has been suggested to enhance polymerase activity in mammalian cells 13 .…”

Gene Composerin a structural genomics environment

“…For the example given here of the H1N1 RNA-dependent RNA polymerase subunit PB2, specific DNAs were difficult if not impossible to obtain for this work and therefore gene synthesis was the only option. Multiple versions of the protein were created and expression and purification were accomplished (Raymond et al, 2011;Yamada et al, 2010). Purified protein entered into crystallization trials and a structure model was determined (Fig.…”

“…7). The structure and its impact on our understanding of cross-species transmission are discussed elsewhere (Yamada et al, 2010).…”

“…Structure model of influenza A RNA-dependent RNA polymerase subunit PB2 (PDB entry 3kc6; Yamada et al, 2010). The expressed construct was designed using Gene Composer as described in this paper.…”