Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

Jardetzky TS (2012) Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence. PLoS Pathog 8(8): e1002855. doi:10.1371/journal.ppat.1002855 Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence Ping Yuan 0 Reay G. Paterson 0 George P. Leser 0 Robert A. Lamb 0 Theodore S. Jardetzky 0 Felix A. Rey, Institut Pasteur, France 0 1 Department of Structural Biology, Stanford University School of Medicine, Stanford, California, United States of America, 2 Department of Molecular Biosciences, Northwestern University , Evanston , Illinois, United States of America, 3 Howard Hughes Medical Institute, Northwestern University , Evanston, Illinois , United States of America Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase b-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an autoinhibited state of HN, the requirement for proteolytic activation of HN0 and associated reduced virulence. - Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This research was supported in part by NIH research grants to T.S.J. (GM-61050) and R.A.L. (AI-23173). G.P.L. is a Specialist and R.A.L. is an Investigator of the Howard Hughes Medical Institute. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Newcastle disease virus (NDV) belongs to the large and diverse family of paramyxoviruses, which is responsible for many human and animal diseases [1]. The paramyxoviruses include other viruses such as mumps virus, measles virus, Sendai virus, respiratory syncytial virus (RSV), metapneumovirus (MPV), parainfluenza viruses (PIV) 15, Nipah virus and Hendra virus. NDV infects birds and many different strains have been isolated worldwide that vary in pathogenicity and virulence. Highly virulent strains can cause a contagious disease with respiratory, neurological and digestive tract pathology, with the most severe infections leading to substantial economic losses in the poultry industry, despite aggressive vaccination programs. Highly virulent velogenic viscerotropic NDV strains, known as exotic NDV (END) strains, not endemic in the US, have caused severe disease outbreaks, such as the 1971 outbreak that required the killing of over 12 million chickens at a cost of $56 million with additional costs over a 4 year cleanup. A more recent END outbreak in 20022003 required the culling of over 3 million chickens in California at a cost of over $161 million. Continuing concerns about the severe economic impact of NDV outbreaks has led to the classification of NDV strains with an intracerebral pathogenicity index of .0.7, or containing a fusion protein with a multibasic cleavage site, as a U.S. Department of Agriculture Select Agent [2,3]. Recently it has been shown that NDV is able to selectively kill tumor cells, suggesting it could be useful as an oncolytic agent, and NDV is also being investigated as a potential vaccine vector (reviewed in [4]). Paramyxoviruses are enveloped, negative-sense, single-stranded RNA viruses. Virions consist of a nucleocapsid, a matrix protein, and an envelope formed by a lipid membrane, typically with two glycoproteins displayed on the surface [1]. For virus penetration into target cells, the lipid envelope must fuse with a cell membrane. Membrane fusion, for nearly all paramyxoviruses, is triggered at the cell surface in a receptor-dependent, pHindependent manner, unlike the pH-dependent influenza virus hemagglutinin mechanism [1,5,6]. For most members of the virus family, two viral glycoproteins are required to mediate this entry process the fusion (F) glycoprotein and an attachment protein referred to as hemagglutinin-neuraminidase (HN), hemagglutinin (H), or glycoprotein (G), depending on the virus [1,5,6]. Activation of the F protein requires virus-specific (homotypic) interactions with the attachment glycoprotein for viral entry, except for RSV and MPV [7,8]. Newcastle disease virus (NDV) can cause severe disease in birds, with the most virulent strains causing sudden death, even in vaccinated populations in the poultry industry. Highly virulent exotic NDV (END) strains have caused largescale outbreaks in the US in 1971 and 2003, requiring the culling of 12 million and 3 million chickens, respectively. Additional economic costs were associated with containment and cleanup. NDV strains vary greatly in their virulence and ability to cause such outbreaks. Two proteins at the surface of the virus, the hemagglutinin-neuraminidase (HN) and the fusion (F) protein, activate NDV entry into cells and variations in both proteins are linked to differences in strain-specific virulence. Certain avirulent strains of NDV, such as NDV Ulster, express a longer HN protein with a C-terminal segment that must be proteolytically cleaved to fully activate the protein. Here we demonstrate that the extra C-terminal 45 amino acids of NDV Ulster HN adopt a well-defined structure, not present in the shorter HN proteins from virulent strains, that blocks two key receptor binding regions necessary for attachment to cells and virus entry. The results clarify how this unique evolutionary adaptation suppresses HN functions in avirulent NDV strains, consistent with an important role for this region in modulating NDV pathogenicity. The HN attachment proteins are found in a subset of the paramyxoviruses, including NDV, mumps virus, parainfluenza virus 5 (PIV5), Sendai virus, and human parainfluenza viruses 14 (hPIV14) [1]. HN protein binds to the rec (...truncated)