Expression and characterization of soluble human parainfluenza virus type 1 hemagglutinin–neuraminidase glycoprotein

• Published in: Journal of virological methods Vol. 98; no. 1; pp. 53 - 61
• Main Authors: Wang, Z.Michael, Tong, L.Leah, Grant, Deborah, Cihlar, Tomas
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01-10-2001; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Animal viral diseases; Animals; Baculoviridae - genetics; Baculovirus expression system; Biological and medical sciences; Cell Line; Fundamental and applied biological sciences. Psychology; Genetic Vectors; HN glycoprotein; HN protein; HN Protein - biosynthesis; HN Protein - chemistry; HN Protein - genetics; Human parainfluenza virus 1; Human parainfluenza viruses; Humans; Hydrogen-Ion Concentration; Infectious diseases; Insecta; Medical sciences; Microbiology; Molecular Sequence Data; Neuraminidase - metabolism; Nickel; Parainfluenza Virus 1, Human - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Sodium Chloride; Viral diseases; Virology; Human parainfluenza viruses; Baculovirus expression system; HN glycoprotein; Hemagglutinin; Insecta; Hydrophobicity; Baculovirus; Paramyxoviridae; Characterization; Baculoviridae; Human parainfluenza virus 1; Pathogenic; Antiviral; Cell; Clone; Availability; Human; Enzyme; Glycoprotein; Virus; Paramyxovirinae; Mononegavirales; Treatment; Arthropoda; Glycosidases; Exo-α-sialidase; Hydrolases; Invertebrata; O-Glycosidases; Paramyxovirus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(01)00355-X

Human parainfluenza virus types 1 (hPIV-1), 2, and 3 represent significant respiratory pathogens for which no antiviral treatment is currently available. To characterize the biochemical functions of the hPIV-1 hemagglutinin–neuraminidase (HN) glycoprotein, a potential target for antiviral therapy, we cloned and expressed a soluble portion of hPIV-1 HN (amino acid residues 137–575), lacking the
N-terminal hydrophobic membrane anchorage region, in insect cells using the baculovirus secretion expression system. The expressed HN protein was purified through cation-exchange chromatography followed by metal affinity chromatography, using the 6×His epitope introduced at the carboxyl terminus of the recombinant protein.
N-terminal amino acid sequence analysis of purified HN indicated that the honeybee melittin secretion signal peptide was correctly removed during post-translational processing. Further characterization revealed that the purified HN protein was
N-glycosylated and exhibited neuraminidase activity whose characteristics resembled those of the native HN protein of hPIV-1 virions. The establishment of this expression and purification system has allowed us to further explore the biochemical characteristics of paramyxovirus HN and to obtain material that could be suitable for X-ray crystallography studies.