Computational analysis and determination of a highly conserved surface exposed segment in H5N1 avian flu and H1N1 swine flu neuraminidase

Abstract

Background: Catalytic activity of influenza neuraminidase (NA) facilitates elution of progeny virions from infected cells and prevents their self-aggregation mediated by the catalytic site located in the body region. Research on the active site of the molecule has led to development of effective inhibitors like oseltamivir, zanamivir etc, but the high rate of mutation and interspecies reassortment in viral sequences and the recent reports of oseltamivir resistant strains underlines the importance of determining additional target sites for developing future antiviral compounds. In a recent computational study of 173 H5N1 NA gene sequences we had identified a 50-base highly conserved region in 3'-terminal end of the NA gene.

Results: We extend the graphical and numerical analyses to a larger number of H5N1 NA sequences (514) and H1N1 swine flu sequences (425) accessed from GenBank. We use a 2D graphical representation model for the gene sequences and a Graphical Sliding Window Method (GSWM) for protein sequences scanning the sequences as a block of 16 amino acids at a time. Using a protein sequence descriptor defined in our model, the protein sliding scan method allowed us to compare the different strains for block level variability, which showed significant statistical correlation to average solvent accessibility of the residue blocks; single amino acid position variability results in no correlation, indicating the impact of stretch variability in chemical environment. Close to the C-terminal end the GSWM showed less descriptor-variability with increased average solvent accessibility (ASA) that is also supported by conserved predicted secondary structure of 3' terminal RNA and visual evidence from 3D crystallographic structure.

Conclusion: The identified terminal segment, strongly conserved in both RNA and protein sequences, is especially significant as it is surface exposed and structural chemistry reveals the probable role of this stretch in tetrameric stabilization. It could also participate in other biological processes associated with conserved surface residues. A RNA double hairpin secondary structure found in this segment in a majority of the H5N1 strains also supports this observation. In this paper we propose this conserved region as a probable site for designing inhibitors for broad-spectrum pandemic control of flu viruses with similar NA structure.

Figure 1

Double hairpin forming conserved RNA structure from 3' terminal. Double hairpin structure from RNA secondary structure prediction server of 3'-terminal 50 base region. The strain used for here is A/duck/Laos/NCVD-35/2007(H5N1) and the g_R value of this sequence is most widely distributed among the whole database.

Figure 2

Comparative graphical representation of aa segment variability and ASA. In this graph Average Solvent Accessibility (ASA) (in brown) is compared with GSWM generated amino acid segment variability (in blue). The y-axis represents both the variability and solvent accessibility. The x-axis represents the sliding window middle position number.

Figure 3

Distribution of conserved sequence stretches on neuraminidase surface. Surface penetration of portions of the highly conserved sequences determined from Figure 2. Last 16 aa region corresponding to point F of Figure 2 is coloured blue. 16-aa segment corresponding to point A in Figure 2 is shown here in cherry red colour, that of point B in pink, point C in dark chocolate, point D in deep salmon and point E in orange. These are large stretches of which only parts are visible on the surface, but much lesser in extent than the last 16 aa stretch corresponding to point F of Figure 2.