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. 1998 Jan;72(1):497-503.
doi: 10.1128/JVI.72.1.497-503.1998.

The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells

J W Rossen  1 , R de Beer, G J Godeke, M J Raamsman, M C Horzinek, H Vennema, P J Rottier
Affiliations

The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells

J W Rossen et al. J Virol. 1998 Jan.

Abstract

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK(MHVR) cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK(MHVR) cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.

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Figures

FIG. 1
FIG. 1
Release of TGEV and MHV-A59 from TM-treated LMR cells. Parallel cultures of LMR cells grown on filters were infected with TGEV or MHV-A59 from the apical side at 16 h p.s. To some cultures, 2 μg of TM per ml was added at 1 h p.i., and these drug concentrations were maintained throughout the experiment. Cells were labeled with 35S labeling mix from 4.5 to 7.5 h p.i., and each culture medium was harvested and analyzed. Viral proteins were immunoprecipitated from the apical (lanes A) and basolateral (lanes B) medium with anti-MHV or anti-TGEV serum. Indicated on the left are the positions of TGEV nucleocapsid (N) and S proteins and of glycosylated and unglycosylated forms of the membrane protein (M and M′, respectively). Indicated on the right are the positions of the cleaved form of the spike protein (S1/S2) and the N and M proteins of MHV-A59. Note that the high-molecular-mass protein (∼250 kDa) detected in the basolateral medium is an unidentified cellular protein nonspecifically coimmunoprecipitated only from the basolateral medium of LMR cells (36).
FIG. 2
FIG. 2
Immunoisolation of TGEV particles from the medium of TM-treated LMR cells. LMR cells grown on filters were infected with TGEV from the apical side at 16 h p.s. In some cultures, 2 μg of TM per ml (B and D) was present from 1 h p.i. onward. Cells were labeled with 35S labeling mix from 4.5 to 7.5 h p.i. and lysed, and viral proteins were immunoprecipitated from lysates (A and B). (C and D) Viral particles were immunoisolated from the apical (lanes A) or basolateral (lanes B) medium with a MAb to the TGEV spike protein (αS) or anti-TGEV serum (αT); a control sample was processed without antibodies (−). The high-molecular-mass protein (∼250 kDa) found in the basolateral medium in panel D (and in longer exposures of panel C; not shown) is an unidentified cellular protein nonspecifically coimmunoprecipitated only from the basolateral medium of LMR cells (36). The exposure times of the gels in panels A through D were 7, 84, 3, and 84 h, respectively. Indicated on the left are the positions of the glycosylated and unglycosylated forms of the spike (S and S′, respectively) and membrane (M and M′, respectively) proteins and the nucleocapsid (N) protein. Molecular mass markers (in kilodaltons) are indicated on the right.
FIG. 3
FIG. 3
Release of MHV-A59 from TM-treated MDCKMHVR cells. Filter-grown MDCKMHVR cells were infected with MHV-A59 from the apical side at 16 h p.s. In some cultures, 0.5 μg of TM per ml was present from 1 h p.i. onward. Cells were labeled with 35S labeling mix from 6 to 9 h p.i., and viral proteins were immunoprecipitated from apical (lanes A) and basolateral (lanes B) media with anti-MHV serum. Indicated on the left are the positions of the uncleaved (S) and cleaved (S1/S2) forms of the spike protein and the membrane (M) and nucleocapsid (N) proteins. Molecular mass markers (in kilodaltons) are indicated on the right.
FIG. 4
FIG. 4
Release of MHV-A59 ts mutant Albany 18 from LMR cells and MDCKMHVR cells. Filter-grown LMR (A and B) and MDCKMHVR (C) cells were infected with MHV-A59 ts mutant Albany 18 from the apical side at 16 h p.s. After the 1-h inoculation period, cells were further incubated at 33°C (permissive temperature) or 39°C (nonpermissive temperature), as indicated. Cells were labeled with 35S labeling mix from 8.5 to 11.5 h p.i., and viral proteins were immunoprecipitated from cell lysates (A) or immunoisolated in the absence of any detergent from the apical (lanes A) or basolateral (lanes B) medium (B and C) with MAb against S (αS) and M (αM) proteins and polyclonal antisera against MHV and vesicular stomatitis virus (vsv). Indicated on the left are the positions of the 150-kDa form of the spike protein (S/gp150) and the membrane (M) and nucleocapsid (N) proteins. Molecular mass markers (in kilodaltons) are indicated on the right. Note that the exposure times for the gels of experiments performed at 33°C were about three times as long as those for the gels of experiments done at 39°C, except for panel A, where only half the amount of sample was loaded for the experiment performed at 39°C compared to that for the experiment performed at 33°C.
FIG. 4
FIG. 4
Release of MHV-A59 ts mutant Albany 18 from LMR cells and MDCKMHVR cells. Filter-grown LMR (A and B) and MDCKMHVR (C) cells were infected with MHV-A59 ts mutant Albany 18 from the apical side at 16 h p.s. After the 1-h inoculation period, cells were further incubated at 33°C (permissive temperature) or 39°C (nonpermissive temperature), as indicated. Cells were labeled with 35S labeling mix from 8.5 to 11.5 h p.i., and viral proteins were immunoprecipitated from cell lysates (A) or immunoisolated in the absence of any detergent from the apical (lanes A) or basolateral (lanes B) medium (B and C) with MAb against S (αS) and M (αM) proteins and polyclonal antisera against MHV and vesicular stomatitis virus (vsv). Indicated on the left are the positions of the 150-kDa form of the spike protein (S/gp150) and the membrane (M) and nucleocapsid (N) proteins. Molecular mass markers (in kilodaltons) are indicated on the right. Note that the exposure times for the gels of experiments performed at 33°C were about three times as long as those for the gels of experiments done at 39°C, except for panel A, where only half the amount of sample was loaded for the experiment performed at 39°C compared to that for the experiment performed at 33°C.
FIG. 4
FIG. 4
Release of MHV-A59 ts mutant Albany 18 from LMR cells and MDCKMHVR cells. Filter-grown LMR (A and B) and MDCKMHVR (C) cells were infected with MHV-A59 ts mutant Albany 18 from the apical side at 16 h p.s. After the 1-h inoculation period, cells were further incubated at 33°C (permissive temperature) or 39°C (nonpermissive temperature), as indicated. Cells were labeled with 35S labeling mix from 8.5 to 11.5 h p.i., and viral proteins were immunoprecipitated from cell lysates (A) or immunoisolated in the absence of any detergent from the apical (lanes A) or basolateral (lanes B) medium (B and C) with MAb against S (αS) and M (αM) proteins and polyclonal antisera against MHV and vesicular stomatitis virus (vsv). Indicated on the left are the positions of the 150-kDa form of the spike protein (S/gp150) and the membrane (M) and nucleocapsid (N) proteins. Molecular mass markers (in kilodaltons) are indicated on the right. Note that the exposure times for the gels of experiments performed at 33°C were about three times as long as those for the gels of experiments done at 39°C, except for panel A, where only half the amount of sample was loaded for the experiment performed at 39°C compared to that for the experiment performed at 33°C.
FIG. 5
FIG. 5
Release of MHV-A59-like particles from mTAL cells. To express the M and E genes of MHV-A59 in mTAL cells, filter-grown cells were infected at 16 h p.i. with recombinant SFVs, vS1mM and vS1mM5, expressing the MHV-A59 M gene (M) and the MHV-A59 M and E genes (M + E), respectively. Cells were labeled with 35S labeling mix from 6 to 9 h p.i., and viral proteins were immunoprecipitated from the apical (lanes A) and basolateral (lanes B) media with anti-MHV serum. The positions of M proteins are bracketed on the left. Molecular mass markers (in kilodaltons) are indicated on the right.
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