Expression of the S1 and S2 subunits of murine coronavirus JHMV spike protein by a vaccinia virus transient expression system.
The spike (S) protein of murine coronavirus JHMV, variant cl-2, comprises two polypeptides, N-terminal S1 (with an N-terminal signal peptide) and C-terminal S2 (with a C-terminal transmembrane domain). In order to express these subunits, we constructed three different vaccinia virus transfer vectors (VV-TVs) containing cDNAs encoding the S1 protein without a transmembrane domain (pSFS1utt), the S1 protein with a C-terminal transmembrane domain derived from S2 (pSFS1tmd) or the S2 protein with an N-terminal signal peptide derived from S1 (pSFssS2). The S1 and S2 proteins were expressed in DBT cells by infection with vaccinia virus and transfection of these VV-TVs. In cells transfected with the pSFS1utt and pSFS1tmd, 96K and 106K proteins, respectively, were detected by Western blotting. The ssS2 protein expressed by pSFssS2 was 96K, which was slightly larger than the authentic S2 protein. The S1utt and S1tmd proteins were shown by binding studies using a panel of monoclonal antibodies to be antigenically indistinguishable from the authentic S1 protein. The S1tmd and ssS2 proteins were detected on the cell surface by immunofluorescence, whereas the S1utt protein was not. However, when the S1utt protein was expressed together with the ssS2 protein, the S1utt was detected on the cell membrane. This suggested that the S1utt was associated with ssS2 on the cell membrane. These observations indicate that the expressed S1 and S2 proteins associated in a similar manner to the authentic S1 and S2 proteins produced in DBT cells infected with cl-2. However, cell fusion was not observed in cells expressing either S1 or S2 nor in cells coexpressing both S1 and S2, although the whole S protein expressed by VV-TV did induce fusion.
Kubo
H
H
National Institute of Neuroscience, NCNP, Tokyo, Japan.
Taguchi
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F
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D005822:Genetic Vectors; D008562:Membrane Glycoproteins; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis; D064370:Spike Glycoprotein, Coronavirus; D014616:Vaccinia virus; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D000956:Antigens, Viral; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus