Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

Brazilian Journal of Microbiology (2011) 42: 340-345 ISSN 1517-8382 STRUCTURAL ANALYSIS OF HUMAN RESPIRATORY SYNCYTIAL VIRUS P PROTEIN: IDENTIFICATION OF INTRINSICALLY DISORDERED DOMAINS Fernando M. Simabuco1, John M. Asara2, Manuel C. Guerrero2, Towia A. Libermann2, Luiz F. Zerbini2, Armando M. Ventura1* 1 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil.; 2Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA. Submitted: February 23, 2010; Returned to authors for corrections: May 12, 2010; Approved: June 21, 2010. ABSTRACT Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex. Key words: Human respiratory syncytial virus; P protein; intrinsically disordered domains; oligomerization INTRODUCTION The P protein interacts with two other viral proteins: the N protein, which interacts with the viral RNA (vRNA) and forms The Human Respiratory Syncytial Virus (HRSV) is an the nucleocapsid; and the L protein, which is the major subunit enveloped non-segmented negative sense RNA virus that of the vRNA polymerase (3). One of P protein functions is to belongs to the Paramyxoviridae family, Pneumovirinae allow specificity of the N protein to the vRNA encapsidation, subfamily (3). HRSV is considered the most important and another is to confer stability of the L protein in the pathogen leading to respiratory disease in infants and neonates ribonucleo-complex (3). It is also known that P protein is able worldwide, which might present serious symptoms, like to oligomerize in tetramers and the oligomerization domain is pneumonia and bronchiolitis (6). HRSV genome has about located in the central region (9). 15,000 nucleotides and contains 10 genes encoding 11 proteins. Intrinsically disordered proteins lack stable secondary and The P protein or Phosphoprotein has 241 amino acids and is tertiary structure under physiological conditions and in the phosphorilated in serines located at positions 116, 117, 119, absence of a biding ligand (5). Previous studies by Karlin et al. 232 and 237 (12,15). (7) demonstrated that N and P proteins of Paramyxovirinae *Corresponding Author. Mailing address: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Professor Lineu Prestes, 1374, São Paulo-SP, Brasil 05508-900.; Tel.: 55 11 3091-7276 Fax: 55 11 3091-7354.; E-mail: 340 Simabuco, F.M. et al. Human Respiratory Syncytial Virus P protein subfamily members have intrinsically disordered domains that (Amersham) to separate MBP from P protein fragments. might be related with protein functions. A model was proposed Digested MBP-P protein was applied onto a HiTrap DEAE for the Measles virus replication process based on the Fast Flow column (Amersham). The elution was performed flexibility of disordered domains (1), and similar studies have using a NaCl gradient combining Buffer A (100 mM TrisHCl, not yet been made for the Pneumovirinae subfamily. pH 8.2, 25 mM NaCl and 1mM EDTA) and Buffer B (Buffer A We describe here in silico predictions of intrinsically disordered regions for the HRSV P protein. The protein was plus 1 M NaCl). Fractions were collected and analyzed in SDSPAGE. expressed in bacteria and purified as previously described (13) Western Blot analysis under non-denaturating conditions and a degradation pattern of the recombinant protein was was performed after PAGE without SDS. The proteins were observed during the purification steps, however, the protein transferred fragments were still able to form oligomers. Furthermore, we (Amersham), which was blocked and incubated with anti-P show by mass spectrometry analysis that the degraded protein monoclonal antibody C771 (11). The detection of the primary regions are part of the predicted intrinsically disordered regions antibody was performed using an anti-mouse peroxidase- and the degradation is not occurring at the oligomerization conjugated antibody (KPL). Native P protein was extracted, domain, previously characterized by Llorente et al. (9,10). under non-denaturating conditions, from HEp-2 cells infected to Hybond-ECL Nitrocellulose membrane with HRSV at a multiplicity of infection (MOI) of 10. MATERIALS AND METHODS Mass spectrometry analysis Prediction of disordered domains For the mass spectrometry analysis of the degraded P The approaches used here for predicting intrinsically protein fragments, the following protocol was performed. After disordered regions are similar to the ones described by Karlin SDS-PAGE, the Coomassie blue stained gel bands were et al. (7). First, the HRSV (strain A2) P protein sequence excised, washed with acetonitrile 50% and cut. Cysteine (AAC14897) was submitted to Predictor Of Naturally residues were reduced with 10 mM dithiothreitol and alkylated Disordered Regions (PONDR) server (http://www.pondr.com/) with 10 mM iodoacetamide. Gel slices were then washed and using VL3 method. In a second approach, P protein sequence digested with 250 ng of modified trypsin (Promega) in 50 mM was submitted to Hydrophobic Cluster Analysis (HCA) using ammonium bicarbonate (pH 8.3) overnight at 37°C. Peptides the DRAWHCA software (http://www.lmcp.jussieu.fr/~soyer/ were extracted from the gel pieces with 20 mM ammonium www-hca/hca-form.html). The secondary bicarbonate, followed by 40% acetonitrile and 2% formic acid. structure (http://bioinf.cs.ucl.ac.uk/psipred/ An aliquot of peptide mixture was applied onto a nanoflow by PSIPRED prediction of psiform.html) was also used to show the presence or lack of reversed-phase secondary structure. spectrometry system at a flow rate of about 300 nL/min. Data P protein was expressed in bacteria fused with Maltose Binding Protein (MBP) and purified using amylose resin column. After digestion with enterokinase to cleave the fused proteins, we observed a degradation pattern for P protein as previously reported (13). Despite the degradation, anion exchange chromatography (AEC) was performed using an Fast Protein Liquid Chromatography chromatography tandem mass dependent (...truncated)