Alkaline phosphatase fusions to the respiratory syncytial virus F protein as an approach to analyze its membrane topology.
Manoil and Beckwith (1985) have constructed a transposon, TnphoA, that permits the generation of hybrid proteins composed of alkaline phosphatase (AP) lacking its signal peptide fused to amino-terminal sequences of other proteins. This transposon has been used to localize export signals and analyze membrane topology of bacterial proteins. We have applied this approach to the membrane fusion protein (F) of respiratory syncytial virus (RSV). The transposon TnphoA and a plasmid directing bacterial expression of the F gene were used to construct F-AP hybrids. These hybrids yielded AP activity, indicating the presence of viral sequences that promoted protein transport through the cytoplasmic membrane. Sequence analysis showed that TnphoA was inserted at four different positions within the F1 subunit. Deletion of the hydrophobic F1 amino-terminus (fusion-related domain) resulted in AP transport to the periplasm, suggesting that the hydrophobic amino-terminus of the F2 subunit is sufficient to promote protein export. Some hybrids were apparently cleaved at or near the F2/F1 junction. The periplasmic localization of an uncleaved hybrid strongly suggested that the fusion-related domain of the F protein, when in the uncleaved F0 precursor, can be moved across the bacterial cytoplasmic membrane. Although these results apply to the recombinant F protein, they agree with the presumed signal sequence and membrane topology of the native F glycoprotein. Thus, this method may be useful in determining membrane topology and in localizing important domains of viral proteins.
Martin-Gallardo
A
A
Praxis Biologics Inc., Rochester, NY 14623.
Deich
R A
RA
Fien
K A
KA
Metcalf
B J
BJ
Anilionis
A
A
Paradiso
P R
PR
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D004251:DNA Transposable Elements; D015707:HN Protein; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins / Q000235:genetics*; D014764:Viral Proteins
D000956:Antigens, Viral; D004251:DNA Transposable Elements; D015707:HN Protein; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; C116204:attachment protein G; D000469:Alkaline Phosphatase