RESUMO
Human parainfluenza virus type 3 (hPIV3) recognizes both α2,3- and α2,6-linked sialic acids, whereas human parainfluenza virus type 1 (hPIV1) recognizes only α2,3-linked sialic acids. To identify amino acid residues that confer α2,6-linked sialic acid recognition of hPIV3, amino acid residues in or neighboring the sialic acid binding pocket of the hPIV3 hemagglutinin-neuraminidase (HN) glycoprotein were substituted for the corresponding residues of hPIV1 HN. Hemadsorption assay with sialyl linkage-modified red blood cells indicated that amino acid residues at positions 275, 277, 372, and 426 contribute to α2,6-linked sialic acid recognition of the HN3 glycoprotein.
Assuntos
Proteína HN/química , Mutação de Sentido Incorreto , Vírus da Parainfluenza 3 Humana/enzimologia , Ácidos Siálicos/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Eritrócitos/química , Eritrócitos/metabolismo , Proteína HN/genética , Proteína HN/metabolismo , Haplorrinos , Humanos , Vírus da Parainfluenza 3 Humana/genética , Ácidos Siálicos/genética , Ácidos Siálicos/metabolismo , Especificidade por Substrato/genéticaRESUMO
Human parainfluenza virus type 1 (hPIV1) generally does not show visible plaques in common cell lines, including Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells, by plaque formation assays for human parainfluenza virus type 3 (hPIV3) and Sendai virus. In several conditions of the plaque formation assay, complete elimination of serum proteins in the overlay medium was necessary for visualization of hPIV1-induced plaque formation in LLC-MK(2) cells. We developed a plaque formation assay for hPIV1 isolation and titration in LLC-MK(2) cells using an initial overlay medium of bovine serum albumin-free Eagle's minimum essential medium containing agarose and acetylated trypsin for 4-6 d followed by a second overlay staining medium containing agarose and neutral red. The assay allowed both laboratory and clinical hPIV1 strains to form large plaques. The plaque reduction assay was also performed with rabbit anti-hPIV1 antibody as a general evaluation model of viral inhibitors to decrease both the plaque number and size. The results indicate that the plaque formation assay is useful for hPIV1 isolation, titration, evaluation of antiviral reagents and epidemiologic research.
Assuntos
Vírus da Parainfluenza 1 Humana/crescimento & desenvolvimento , Ensaio de Placa Viral , Animais , Linhagem Celular , Meios de CulturaRESUMO
An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.
Assuntos
Caspase 3/metabolismo , Proteína HN/genética , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 1 Humana/metabolismo , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Animais , Caspase 3/genética , Morte Celular , Linhagem Celular , Transformação Celular Viral , Células Gigantes/fisiologia , Proteína HN/metabolismo , Humanos , Cinética , Macaca mulatta , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Neuraminidase/antagonistas & inibidores , Ligação Proteica/fisiologia , Proteínas Virais de Fusão/metabolismo , Replicação Viral/fisiologiaRESUMO
The X-ray crystal structure of the paramyxoviral surface glycoprotein haemagglutinin-neuraminidase (HN) from Newcastle Disease virus was used as a template to design inhibitors of the HN from human parainfluenza virus type-3 (hPIV-3). 4-O-Alkylated derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), accessed from 8,9-O-isopropylidenated-Neu5Ac2en1Me, were found to inhibit the sialidase (neuraminidase) activity of hPIV-3 (strain C243) in the range of 3-30muM. This is comparable or improved activity compared to the parent 4-hydroxy compound.