Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport.
Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.
Garten
W
W
Institut für Virologie, Philipps-Universität, Marburg, Germany.
Will
C
C
Buckard
K
K
Kuroda
K
K
Ortmann
D
D
Munk
K
K
Scholtissek
C
C
Schnittler
H
H
Drenckhahn
D
D
Klenk
H D
HD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000235:genetics
D006389:Hemagglutinins, Viral; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D012367:RNA, Viral