Computational studies on the substrate interactions of influenza A virus PB2 subunit

Abstract

Influenza virus, which spreads around the world in seasonal epidemics and leads to large numbers of deaths every year, has several ribonucleoproteins in the central core of the viral particle. These viral ribonucleoproteins can specifically bind the conserved 3' and 5' caps of the viral RNAs with responsibility for replication and transcription of the viral RNA in the nucleus of infected cells. A fundamental question of most importance is that how the cap-binding proteins in the influenza virus discriminates between capped RNAs and non-capped ones. To get an answer, we performed molecular dynamics simulations and free energy calculations on the influenza A virus PB2 subunit, an important component of the RNP complexes, with a cap analog m7GTP. Our calculations showed that some key residues in the active site, such as Arg355, His357, Glu361 as well as Gln406, could offer significant hydrogen bonding and hydrophobic interactions with the guanine ring of the cap analog m7GTP to form an aromatic sandwich mechanism for the cap recognition and positioning in the active site. Subsequently, we applied this idea to a virtual screening procedure and identified 5 potential candidates that might be inhibitors against the PB2 subunit. Interestingly, 2 candidates Cpd1 and Cpd2 have been already reported to have inhibitory activities to the influenza virus cap-binding proteins. Further calculation also showed that they had comparatively higher binding affinities to the PB2 subunit than that of m7GTP. We believed that our findings could give an atomic insight into the deeper understanding of the cap recognition and binding mechanism, providing useful information for searching or designing novel drugs against influenza viruses.

Figure 1. Illustratively showing the binding mode of the PB2 cap binding domain in the presence of the cap analog m7GTP.

The protein structure was shown in a ribbon diagram, while m7GTP is in ball-and-stick representations. The secondary structure elements of the PB2 cap binding domain are labeled with α-helices in red and β-strands in yellow. The cap analog m7GTP is colored according to its atomic types.

Figure 2. Energy contributions (kcal/mol) for each residue to the binding free energies of the PB2-m7GTP complex.

Only the residues that have positive contributions to the binding free energies are listed in this figure. Among these residues, His357 has the most contributions owe to having both hydrogen bonding and π-π stacking interactions with the cap analog m7GTP.

Figure 3. A 2D snapshot from MD simulations to show the binding interactions of the cap analog m7GTP with the key residues in the cap binding domain.

The hydrogen bond is shown in green dashed line with the arrow pointed to the hydrogen acceptor. The π-π stacking and π-cation interactions are presented in green benzene ring symbol. The residues without any dashed line or benzene ring symbol have only van der Waals interactions with m7GTP.

Figure 4. The cap-binding pockets for the influenza A virus PB2 subunit.

The residues around the cap-binding pocket are colored so that those aromatic amino acids forming the cap sandwich around the cap analogue m7GTP are in blue, those binding the functional groups of the guanine residue are in orange, those stabilizing the 7-methyl group are in yellow and those binding the triphosphate moiety are green.