Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-03-06
2000-11-21
Stucker, Jeffrey
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 71, 435 772, C12P 2106, G01N 3353
Patent
active
06150131&
DESCRIPTION:
BRIEF SUMMARY
1. INTRODUCTION
The present invention relates to assays for the identification of compounds that block palmitylation of influenza virus hemagglutinin (HA), and inhibit infectious virus formation. The invention also relates to the use of such compounds as antiviral agents for the treatment of flu.
2. BACKGROUND OF THE INVENTION
The hemagglutinin (HA) of influenza virus is the major surface antigen and is one of the best characterized membrane glycoproteins. It has receptor-binding and fusion activity, which are both necessary for the initiation of viral infection. The protein contains a large ectodomain that carries receptor and fusion activities, a stretch of hydrophobic amino acids which constitutes the transmembrane domain, and a short cytoplasmic tail. This cytoplasmic tail contains 10-11 amino acids, depending on the subtype of the HA (Ward, 1981, Curr. Top. Microbiol. Immunol. 94/95:1-74; FIG. 1). Comparison of the HA sequences in this region reveals that 5 amino acids are highly conserved and that they are identical in 11 of the 14 known subtypes (Doyle et al., 1985, J. Cell Biol. 100:704-714; Kawaoka et al., 1990, Virology 179:759-767; Nobusawa et al., 1991, Virology 182:475-485; Simpson & Lamb, 1992, J. Virol. 66:790-803). Two of these conserved residues are cysteines (positions 560 and 563) and a third highly conserved cysteine is located in the membrane-anchoring domain (position 553) (FIG. 1). It as previously been shown that the conserved cysteines of subtype H2, H3 and H7 HAs are post-translationally modified by covalent addition of palmitic acids (Naeve & Williams, 1990, EMBO J. 9:3857-3866; Naim et al., 1992, J. Virol. 66:7585-7588; Schmidt & Lambrecht, 1985, J. Gen. Virol. 66:2635-2647; Steinhauer et al., 1991, Virology 184:445-448). The levels of palmitylation have been quantified for the A/Japan/305/57 HA (H2), whose 17 carboxy-terminal amino acids are identical to those of the A/WSN/33 HA (H1). The cysteines at positions 560 and 563 (FIG. 1) appear to be highly modified, with position 563 incorporating at least half of the fatty acid label, whereas position 553 incorporates only about 10% of the total palmitate (Naim et al., 1992, J. Virol. 66:7585-7588).
The extent and significance of palmitylation of viral proteins is not yet fully understood (reviewed in McIlhinney, 1990, TIBS 15:387-391). The fact that HeLa cells are refractory to influenza virus growth has been ascribed to a defect in palmitylation of this cell line, but other mechanisms responsible for the abortive infection in HeLa cells cannot be ruled out (Portincasa et al., 1992, Res. Virol. 143:401-406). Experiments using hydroxylamine to remove lipid from viral proteins have suggested that the fatty acid moiety is important for membrane fusion (Schmidt & Lambrecht, 1985, J. Gen. Virol. 66:2635-2647). Another report used the antibiotic cerulenin to inhibit acylation and has implicated the lipid in viral release (Schlesinger & Malfer, 1982, J. Biol. Chem. 257:9887-9890). However, these interpretations are not definitive since hydroxylamine might affect the protein structure and cerulenin is known to exert a general toxic effect. For vesicular stomatitis virus (VSV) it has been reported that the elimination of the palmitylation site in the G protein has no effect on membrane fusion or glycoprotein incorporation into virions (Whitt et al., 1989, J. Virol. 63:3569-3578). Studies with alphaviruses have shown that the elimination of either one of the two palmitylation sites in the carboxy terminus of the glycoprotein E2 decreases the efficiency of virus budding (Ivanova & Schlesinger, 1993, J. Virol. 67:2546-2551), and that a mutant with changes in both palmitate addition sites was not viable (Gaedigk-Nitschko & Schlesinger, 1991, Virology 183:206-214).
There is one report which suggests that the A/Japan/305/57 HA (H2) requires palmitate for membrane fusion (Naeve & Williams, 1990, EMBO J. 9:3857-3866). However, this finding has not been supported by other workers using either H2, H3 or H7 subtype HAs (Naim et al., 1992, J. Viro
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Naeve and Williams, 1990, "Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion", EMBO J 9(12):3857-3866.
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Nelson Brett
Stucker Jeffrey
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