Sendai virus intra-host population dynamics and host immunocompetence influence viral virulence during in vivo passage

Peña, José; Chen-Harris, Haiyin; Allen, Jonathan E.; Hwang, Mona; Elsheikh, Maher; Mabery, Shalini; Bielefeldt-Ohmann, Helle; Zemla, Adam T.; Bowen, Richard A.; Borucki, Monica K.

To help characterize an effect of identified mutations on virus phenotypic changes a homology-based structural model of HN protein from SeV strain Z (GI:546225829) was constructed using the AS2TS system (Zemla et al. 2005) . The HN protein of human parainfluenza 3 virus (hPIV3) was identified as the closest Protein Data Bank (PDB) structural template for the modeling. It was experimentally solved at the resolution of 1.65 angstroms (Ã… ) (PDB entry: 4MZA) (Xu et al. 2013) , and the level of sequence identity between HN from Sendai and hPIV3 is 55%. Examples of other identified structural homologs that were leveraged during the modeling process include: HN from Newcastle Disease virus (NDV) (PDB entry: 1E8U; resolution: 2.00 Ã… ; sequence identity: 27%) (Connaris et al. 2002) , HN from Simian virus 5 (Sv5) (4JF7; 2.50 Ã… ; 24%) (Welch et al. 2013) , Nipah virus (NiV) (3D11; 2.31; 22%), Hendra virus (HeV) (2VSK; 2.00; 20%), Measles virus (MeV) (2ZB6; 2.60; 16%). Using available templates a set of initial structural models was created. To assess regions of sequence-structure conservation or variability in the modeled Sendai HN protein an extensive search for similar fragments in other proteins from PDB was performed using the StralSV algorithm (Zemla et al. 2011) , which identifies protein fragments that exhibit structural similarities despite low primary amino acid sequence similarity. Results from these searches were used for modeling of missing loop or termini regions in the constructed preliminary models. For the construction of the final structural model the coordinates from the PDB chain 4mza_B were used as a primary template. The conformation of side-chain atoms was predicted using SCWRL (Krivov, Shapovalov, and Dunbrack 2009 ) when residue-residue correspondences did not match. Residues that were identical in the template and HN protein were copied from the template onto the model. The structural and stereochemical quality of the model was checked using a contact-dot algorithm in the MolProbity software package (Chen et al. 2010) , and the final constructed model was finished with relaxation using UCSF Chimera (Pettersen et al. 2004) .