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Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an -helical domain involved in oligomerization

¹ Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain
² Fundación CNIC Carlos III, Instituto de Salud Carlos III, Melchor Fernández Almagro 3, 28029 Madrid, Spain
³ Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS and Universités Aix-Marseilles I and II, ESIL, 163 Avenue de Luminy, Case 925, 13288 Marseille Cedex 9, France
⁴ Laboratoire de Virologie Moléculaire et Structurale, UMR 2472/1157 CNRS-INRA, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France

Correspondence
José A. Melero
jmelero{at}isciii.es

Human respiratory syncytial virus (HRSV) phosphoprotein (P), an essential cofactor of the viral polymerase, is much shorter (241 aa) than and has no sequence similarity to P of other paramyxoviruses. Nevertheless, bioinformatic analysis of HRSV P sequence revealed a modular organization, reminiscent of other paramyxovirus Ps, with a central structured domain (aa 100–200), flanked by two intrinsically disordered regions (1–99 and 201–241). To test the predicted structure experimentally, HRSV P was purified from cell extracts infected with recombinant vaccinia virus or HRSV. The estimated molecular mass of P by gel filtration (500 kDa) greatly exceeded the theoretical mass of a homotetramer, proposed as the oligomeric form of native P. Nevertheless, the profile of cross-linked products obtained with purified P resembled that reported by others with P purified from bacteria or mammalian cells. Thus, the shape of HRSV P probably influences its elution from the gel filtration column, as reported for other paramyxovirus Ps. Digestion of purified HRSV P with different proteases identified a trypsin-resistant fragment (X) that reacted with a previously characterized monoclonal antibody (021/2P). N-terminal sequencing and mass spectrometry analysis placed the X fragment boundaries (Glu-104 and Arg-163) within the predicted structured domain of P. Cross-linking and circular dichroism analyses indicated that fragment X was oligomeric, with a high -helical content, properties resembling those of the multimerization domain of Sendai and rinderpest virus P. These results denote structural features shared by HRSV and other paramyxovirus Ps and should assist in elucidation of the HRSV P structure.

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