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Point mutations affecting the directed transport of the influenza hemagglutinin
23
OF THE
POINT MUTATIONS AFFECTING INFLUENZA HEMAGGLUTININ
THE DIRECTED
TRANSPORT
W. GARIEM, K. KURODA, W. SCHUY, Ch. WILL, H. NARUSE, Ch. SCHOLTISSER, and H.-D. KLENK. Institut fiirVirolcgie, Justus-
Liebig-Universit& Giessen D-6300 Giessen Mutants of fowl plague virus have been analyzed which show temperature sensitive (ts) defects in the intracellular transport of HA. The transport is blocked either between rough E.R. and Golgi apparatus or between Golgi apparatus and plasma membrane as indicated by cell fractionation and immune fluoresanalysis of the hemagglutinin sequence cence studies. Comparative and wild type revealed that the defect with mutants, revertants, in transport is due to point mutations which are located in the stem region of the spike.
24
MATURATION OF THE LARGE GLYCOPROTEIN (VP84) OF RESPIRATORY SYNCYTIAL VIRUS BRUCE F. FERNIE, GAIL DAPOLITO, PAUL COTE, JR. AND JOHN L. GERIN, Division of Molecular Virology & Immunology, Georgetown University, 5640 Fishers Lane, Rockville, MD 20852-1770, U.S.A. contains Respiratory syncytial virus, like other paramyxoviruses, two glycoproteins; the fusion (F) protein, MW 68K and the large glycoprotein (G), Mw 84K. A comparison of the synthesis of these two proteins revealed striking differences. G protein appears to be extensively glycosylated with N- and 0- linked carbohydrate. It appears on the cell surface and is released much faster than F protein (TL of maximum release 50 and 80 min. Although the MW of mat&e G is 80-90K, the protein backbone respectively). is much smaller. Collins -et al (J. Virol. (1984) 49:578) reported that the putative mRNA for G protein encodes a 36K protein. We have used kinetic studies to show that G is synthesized through a series of precursors having a 'ladder-like' appearance on SDS/PAGE and terminating with a major, stable Endo H treatment 1) has no effect on mature G.2) precursor, MW 45K. eliminates the precursor proteins, and 3) produces a glycosylated 26K protein that is presumably the G protein backbone with attached proximal Analysis of G protein synthesis in BSC-1 monkey glucosamine residues. cells obtained from C. Pringle indicate that intracellular G has a MW of only 47K while released G found in the culture fluid has a MW of 84K. This explains the results of Wunner and Pringle (Virology (1976) 73:228) who found two glycoproteins of MW 40-50K but little 84K G in partially purified virus or infected cells. These two proteins probably represent the large (Fl) component of the fusion protein and the form of G we have just The mechanism of conversion from 47K to 84K G will be examined identified. and the possibility that G matures via a dimerization process discussed. 12
OF THE
POINT MUTATIONS AFFECTING INFLUENZA HEMAGGLUTININ
THE DIRECTED
TRANSPORT
W. GARIEM, K. KURODA, W. SCHUY, Ch. WILL, H. NARUSE, Ch. SCHOLTISSER, and H.-D. KLENK. Institut fiirVirolcgie, Justus-
Liebig-Universit& Giessen D-6300 Giessen Mutants of fowl plague virus have been analyzed which show temperature sensitive (ts) defects in the intracellular transport of HA. The transport is blocked either between rough E.R. and Golgi apparatus or between Golgi apparatus and plasma membrane as indicated by cell fractionation and immune fluoresanalysis of the hemagglutinin sequence cence studies. Comparative and wild type revealed that the defect with mutants, revertants, in transport is due to point mutations which are located in the stem region of the spike.
24
MATURATION OF THE LARGE GLYCOPROTEIN (VP84) OF RESPIRATORY SYNCYTIAL VIRUS BRUCE F. FERNIE, GAIL DAPOLITO, PAUL COTE, JR. AND JOHN L. GERIN, Division of Molecular Virology & Immunology, Georgetown University, 5640 Fishers Lane, Rockville, MD 20852-1770, U.S.A. contains Respiratory syncytial virus, like other paramyxoviruses, two glycoproteins; the fusion (F) protein, MW 68K and the large glycoprotein (G), Mw 84K. A comparison of the synthesis of these two proteins revealed striking differences. G protein appears to be extensively glycosylated with N- and 0- linked carbohydrate. It appears on the cell surface and is released much faster than F protein (TL of maximum release 50 and 80 min. Although the MW of mat&e G is 80-90K, the protein backbone respectively). is much smaller. Collins -et al (J. Virol. (1984) 49:578) reported that the putative mRNA for G protein encodes a 36K protein. We have used kinetic studies to show that G is synthesized through a series of precursors having a 'ladder-like' appearance on SDS/PAGE and terminating with a major, stable Endo H treatment 1) has no effect on mature G.2) precursor, MW 45K. eliminates the precursor proteins, and 3) produces a glycosylated 26K protein that is presumably the G protein backbone with attached proximal Analysis of G protein synthesis in BSC-1 monkey glucosamine residues. cells obtained from C. Pringle indicate that intracellular G has a MW of only 47K while released G found in the culture fluid has a MW of 84K. This explains the results of Wunner and Pringle (Virology (1976) 73:228) who found two glycoproteins of MW 40-50K but little 84K G in partially purified virus or infected cells. These two proteins probably represent the large (Fl) component of the fusion protein and the form of G we have just The mechanism of conversion from 47K to 84K G will be examined identified. and the possibility that G matures via a dimerization process discussed. 12