Stachyflin is a novel compound having H1 and H2 subtype-specific anti-influenza A virus activity. Stachyflin has no inhibition on H3 subtype influenza A or influenza B viruses. The susceptibility of the reassortant viruses between H1 and H3 subtype influenza A viruses to Stachyflin indicated that its target was virus-encoded hemagglutinin (HA). The results of the timing of Stachyflin addition against in vitro virus infection and virus-mediated hemolysis assay suggested that the drug inhibited the HA-mediated virus-cell fusion process. More directly, Stachyflin interfered with HA conformational change induced by low pH or heat treatment. The effect of Stachyflin could not be eliminated by washing of the Stachyflin-treated virus, which caused very specific virucidal effect. This is a remarkable property among small molecules which inhibit low-pH induced HA conformational change. From these findings, we concluded that the mechanism of Stachyflin action is to inhibit HA conformational change which is necessary for virus-cell membrane fusion. Stachyflin may be used as a tool for a study of molecular mechanism of low-pH induced HA conformational change, and offers potential as a pharmaceutical agent.

No abstract

A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and six amplified genomic DNA clones of bovine PrP were characterized. These clones possessed specific characteristics observed in other animal PrP genes. However, the bovine PrP was divided into two types by the number of repeats. One possessed four octapeptide repetitive sequences, like other animal PrP genes, and consisted of 256 amino acids; the other had five such repetitive sequences and 264 amino acids. The amino acid sequence of the former bovine PrP agreed with that of sheep PrP up to the 165th amino acid from the N-terminus. Bovine PrP cDNA introduced into mouse L-929 cells were stably expressed. The expression level of recombinant bovine PrP in the cells judged by immunofluorescence was higher than that of authentic mouse PrP. The recombinant PrP comigrated with authentic bovine PrP in SDS-polyacrylamide gel electrophoresis, suggesting that the recombinant product was fully glycosylated in L-929 cells. Distinct bundles of the intermediate filaments were frequently seen at the perinuclear region of the cells.

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1-14, 1999). Stachyflin-resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA-expressing cells and octadecylrhodamine (R18)-labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin-resistant phenotype of HA protein. The molecular mechanism of anti-HA conformational change activity of Stachyffin is discussed.