Amino acid substitutions contributing to α2,6-sialic acid linkage binding specificity of human parainfluenza virus type 3 hemagglutinin-neuraminidase.

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.

Plaque formation assay for human parainfluenza virus type 1.

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.

A single amino acid mutation at position 170 of human parainfluenza virus type 1 fusion glycoprotein induces obvious syncytium formation and caspase-3-dependent cell death.

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.

Synthesis and evaluation of 4-O-alkylated 2-deoxy-2,3-didehydro-N-acetylneuraminic acid derivatives as inhibitors of human parainfluenza virus type-3 sialidase activity.

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.