Avian influenza H5-containing virus-like particles (VLPs): host-cell receptor specificity by STD NMR spectroscopy.

H5N1 influenza virus is a highly contagious pathogen infecting poultry and other birds. The emergence of a human pandemic influenza virus from an avian progenitor appears to involve a switch in preferential binding of the influenza virus hemagglutinin (HA) from a(2,3)-linked N-acetylneuraminic acid containing glycans (the major form in the avian enteric tract) to a(2,6)-linked N-acetylneuraminic acid containing glycans (the major form in the human upper respiratory tract). Avian influenza viruses such as the H3 virus HA receptor typically contain the amino acids Gln226 and Gly228, which create a narrow binding pocket favoring a(2,3)-linked N-acetylneuraminic acid containing glycans. A number of other studies have also investigated HA–glycan recognition. Interestingly, the X-ray crystal structure of the reassembled HA of the 1918 Spanish influenza virus shows that its avianlike narrow binding pocket still allows highaffinity binding of a(2,6)-linked N-acetylneuraminic acid containing glycans. The mutation of only one amino acid (Asp190) within the HA binding site appears to determine the preference of the avianlike 1918 virus for human a(2,6)linked N-acetylneuraminic acid containing glycans. A mutation of Asp190 to Glu190 in the HA of the H5N1 strain could potentially switch its binding preference to a(2,6)linked glycans and consequently result in the emergence of a human pandemic virus. This fact emphasizes an urgent need to investigate the Nacetylneuraminic acid containing glycan recognition requirements by influenza virus HA that may lead to the development of novel antiinfluenza drugs that bind to the viral HA protein and consequently prevent the entry of the virus into human host cells. Rapid access to structural information would provide a detailed understanding of how virus particles interact with host cells on a molecular level and the determinants that prevent interspecies transmission of influenza viruses. Herein we present the first saturation transfer difference (STD) NMR spectroscopic study analyzing H5containing virus-like particles (VLPs) derived from the highly pathogenic avian H5N1 influenza strain in a complex with a(2,6)and a(2,3)-linked N-acetylneuraminides to mimic an in vitro or in vivo virus–host-cell interaction. We recently reported the production of H5 pseudotyped virus particles. To provide larger quantities of suitable VLPs for NMR studies, we have now successfully engineered heterologous H5 influenza VLPs by coexpression of pCDNA–synH5 coding for the hemagglutinin (H5) of H5N1 influenza virus and pCMV-dR8.91-expressing HIV-Gag-pol protein. Coexpression leads to spontaneous assembly of chimeric H5-VLPs, which contain the HA protein. The hemagglutinin, detected as uncleaved HA-0 precursor and HA-1/HA-2 cleaved mature forms, was incorporated on the surface of the viral particles at high levels. Expression of the viral HA protein was characterized by Western blot using specific C-terminal flag-tag M2 antibodies (Figure 1) and detection of VLPs by electron microscopy (Figure 2). In this study we investigated the capacity of H5-VLPs for competitive selection from a mixture of a(2,6)and a(2,3)sialyllactose (6’-SL and 3’-SL, respectively) of a preferred ligand (and therefore linkage) by means of STD NMR spectroscopy. It has been previously demonstrated that STD NMR spectroscopy can be utilized to investigate ligand interactions with whole virus particles, platelets, and intact cells. The large size of viruses and cells makes them attractive for studies with STD NMR spectroscopy because the inherently large line width enables saturation of the particle without affecting ligand signals. Additionally, the larger correlation time of bulky virus particles results in efficient spin diffusion and consequently stronger saturation transfer. To ensure the stability of the influenza H5-VLPs, NMR experiments were performed without prior purification, and 10% D2O was added for locking purposes. The H NMR spectrum of the influenza H5-VLPs (Figure 3a) shows the signals of the 20% sucrose cushion [*] Dr. T. Haselhorst, Dr. T. Islam, F. J. Rose, Prof. M. von Itzstein Institute for Glycomics, Griffith University, Gold Coast Campus Queensland, 4222 (Australia) Fax: (+61)7-555-29040 E-mail: m.vonitzstein@griffith.edu.au Homepage: http://www.griffith.edu.au/glycomics Dr. J.-M. Garcia, J. C. C. Lai, Prof. J. S. M. Peiris Hong Kong University Pasteur Research Centre Ltd., Dexter H.C. Man Building 8 Sassoon Road, Pokfulam, Hong Kong (China)