Identification of a New Membrane-associated Polypeptide Specified by the Coronavirus Infectious Bronchitis Virus

Smith, A.C.; Boursnell, M. E. G.; Binns, M. M.; Brown, T. D. K.; Inglis, Stephen

doi:10.1099/0022-1317-71-1-3

Cited by 39 publications

(34 citation statements)

References 30 publications

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“…With antibodies prepared against the whole E protein, positive but weak staining of unfixed cells infected with infectious bronchitis virus was observed (40). Surface staining was also reported for MHV-(49) and TGEV-infected cells, as well as for insect cells expressing the TGEV E protein by a baculovirus vector (12).…”

Section: Discussionmentioning

confidence: 79%

“…Consistent with their membrane association (12,40,48,49), the proteins are generally quite hydrophobic in nature, particularly in their N-terminal half (see Fig. 1).…”

mentioning

confidence: 68%

“…The MHV-A59 E protein was reported to be acylated on the basis of a biochemical assay (49), but attempts to directly label the TGEV protein with palmitic acid failed (12). By immunofluorescence, the E protein was observed in infected cells in a granular (IBV) (40) or punctate (BCV and MHV-A59) (1, 49) pattern, as well as at the plasma membrane (12,40,49). The cell surface staining with a C-terminus-specific antibody led Godet et al (12) to suggest a C exo N endo membrane topology for the TGEV E protein, i.e., with its C and N termini exposed luminally and cytoplasmically, respectively.…”

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confidence: 99%

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Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

Raamsman

Locker

Hooge

et al. 2000

J Virol

174

204

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The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.Coronaviruses, a family of viruses belonging to the newly established order of the Nidovirales (for reviews, see references 8 and 37) have enveloped virions containing a nonsegmented, plus-stranded RNA genome. The RNA is packaged by the nucleocapsid (N) protein into a helical nucleocapsid. The surrounding envelope contains three, and sometimes four, membrane proteins. The spike (S) protein, a type I glycoprotein, occurs as trimers that constitute the characteristic surface projections. These function primarily in virus entry, being responsible for binding to the receptor on the target cell and for mediating fusion of viral and cellular membranes. The membrane (M) protein is a triple-spanning glycoprotein. It is the most abundant envelope protein component having essential functions in virus assembly. The hemagglutinin-esterase protein is present in only a subset of coronaviruses. The type I glycoprotein occurs in virions in disulfide-linked homodimeric form. Its biological role in the vi...

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Section: Discussionmentioning

confidence: 79%

“…Consistent with their membrane association (12,40,48,49), the proteins are generally quite hydrophobic in nature, particularly in their N-terminal half (see Fig. 1).…”

mentioning

confidence: 68%

mentioning

confidence: 99%

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Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

Raamsman

Locker

Hooge

et al. 2000

J Virol

174

204

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“…The envelope (E) protein, due to its small size and low level in virions, has not been well characterized. However, it is associated with the virion envelope (20,30).Evidence from studies of other coronaviruses suggests the coronavirus E protein is likely to play an important role in virus assembly. When the E and M proteins from either mouse hepatitis virus (MHV) or transmissible gastroenteritis virus (TGEV) are expressed together in cells from cDNA, virus-like particles (VLPs), roughly the same size and shape as virions, are released from the cells (3, 25, 37).…”

mentioning

confidence: 99%

Infectious Bronchitis Virus E Protein Is Targeted to the Golgi Complex and Directs Release of Virus-Like Particles

Corse

Machamer

2000

J Virol

221

336

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The coronavirus E protein is a poorly characterized small envelope protein present in low levels in virions. We are interested in the role of E in the intracellular targeting of infectious bronchitis virus (IBV) membrane proteins. We generated a cDNA clone of IBV E and antibodies to the E protein to study its cell biological properties in the absence of virus infection. We show that IBV E is an integral membrane protein when expressed in cells from cDNA. Epitope-specific antibodies revealed that the C terminus of IBV E is cytoplasmic and the N terminus is translocated. The short luminal N terminus of IBV E contains a consensus site for N-linked glycosylation, but the site is not used. When expressed using recombinant vaccinia virus, the IBV E protein is released from cells at low levels in sedimentable particles that have a density similar to that of coronavirus virions. The IBV M protein is incorporated into these particles when present. Indirect immunofluorescence microscopy showed that E is localized to the Golgi complex in cells transiently expressing IBV E. When coexpressed with IBV M, both from cDNA and in IBV infection, the two proteins are colocalized in Golgi membranes, near the coronavirus budding site. Thus, even though IBV E is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly.Coronaviruses are enveloped positive-strand RNA viruses. In contrast to many of the well-studied enveloped viruses that bud from the plasma membrane of cells, coronaviruses acquire their membrane envelope by budding into the lumen of Golgi and pre-Golgi compartments. After budding, virions are thought to move in vesicles through the secretory pathway and to exit the cell when these vesicles fuse with the plasma membrane (11, 34). The specific compartment into which coronaviruses bud is the cis-Golgi network (CGN), also known as the endoplasmic reticulum-Golgi intermediate compartment (15). The mechanism of budding-site selection is unclear. Just as enveloped viruses that bud from the plasma membrane must direct the accumulation of their envelope proteins at the cell surface, coronaviruses must localize their envelope proteins to the membranes of the cis-Golgi network. The possible role of the targeting of the individual membrane proteins in coronavirus budding-site selection has been studied by expressing them from cDNA in cultured cells.The coronavirus avian infectious bronchitis virus (IBV) has three known membrane proteins. The spike (S) protein is a large glycoprotein involved in target cell recognition and fusion (6). When IBV S is expressed alone, it is transported to the plasma membrane (36), and so it is unlikely that S alone is responsible for determining the site of virus budding. The matrix (M) protein is a glycoprotein with three transmembrane domains; its large C terminus is thought to bind to the nucleocapsid during budding (17, 32). IBV M is found in the cis-Golgi network and cis-Golgi complex when expressed alone (23), and thus it reache...

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“…Overnight cultures of E. coil strain DH5 carrying pUEXbased recombinants were diluted 100-fold in 2YT broth, grown at 30 °C to an optical density at 550 nm (OD550) of about 0.2 and then incubated for 2 h and 20 min at 42 °C. Fusion proteins were recovered from 100 ml cultures of bacterial cells as described by Smith et at_ (1990), and analysed on 10~ SDS-polyacrylamide gels. For preparative purposes gels were stained for 10 min with 0-05% Coomassie blue in water, followed by destaining in water for 10 to 20 min.…”

Section: Preparation Of Bacterial Fusion Proteinsmentioning

confidence: 99%

Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

Jin

Elliott

1992

Journal of General Virology

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Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits. Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation. Two of these antisera were specific for either the amino-or carboxy-terminal regions of the L protein. The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein. Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase. Antigenically authentic L protein was expressed. Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity. Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein. The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay. These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability. Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.

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Identification of a New Membrane-associated Polypeptide Specified by the Coronavirus Infectious Bronchitis Virus

Cited by 39 publications

References 30 publications

Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

Infectious Bronchitis Virus E Protein Is Targeted to the Golgi Complex and Directs Release of Virus-Like Particles

Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

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