Thermal aggregation of SARS-CoV membrane protein

Abstract

SARS-CoV membrane protein could be detected easily using Western blotting in non-denaturing condition but not regular denaturing treatment. Boiling treatment, causing the aggregation of SARS-CoV membrane protein in the stacking gels, results in the failure to detect the membrane protein in the separating gels. Aggregated membrane proteins could not be dissociated by 1% Triton-X 100, 6M urea, or 2% SDS. The region with amino acid residues from 51 to 170 is responsible for thermal aggregation of SARS-CoV membrane protein. Hydrophobic regions with amino acid residues from 61 to 90, from 91 to 100, from 136 to 170, are essential for this protein aggregation. Thermal aggregation of SARS-CoV membrane protein is not unique among structural proteins of coronaviruses. However, SARS-CoV membrane protein seems to be more sensitive to heat treatment, since the membrane protein of MHV-JHM, another member of the Coronaviridae, would not aggregate after the same treatment. Therefore, if SARS-CoV membrane protein needs to be analyzed using SDS-PAGE, boiling should be avoided. Thermal aggregation of SARS-CoV membrane protein may be one of the reasons for the inactivation of this virus by heat. The unusual property of SARS-CoV membrane protein aggregation induced by heat also provides a model for the study of protein aggregation.

Fig. 1

SARS-CoV membrane protein could not be easily detected using Western blotting with boiling treatment. (A) Membrane proteins in either wild type or mutation in individual Cys residue could easily be detected in non-denaturing (nd) but not denaturing (d) treatment. Protein transiently expressed in Vero E6 cells were equally aliquoted and treated in different conditions. Serum of one SARS patient was used as first antibody in WB analysis. Proteins marked by the thick arrow are the glycosylated SARS-CoV membrane protein, the same size as the membrane protein from purified SARS-CoV particles (virions). (B) Boiling but not 2-ME treatment is responsible for the inefficient detection of membrane protein in WB analysis. Sample preparation and detection are the same as that of (A). (C) Thermal effect on the SARS-CoV membrane protein. Sample preparation and detection are the same as that of (A).

Fig. 2

Thermal aggregation of SARS-CoV membrane protein. (A) Effect of boiling treatment on SARS-CoV membrane protein in the presence of 6M urea or 2% SDS. Sample preparation and detection are the same as those of Fig. 1 except samples were treated with either 6 M urea or 2% SDS before boiling treatment. There are many distinct protein bands other than unglycosylated and glycosylated M proteins. These protein bands may represent the cellular proteins interacting with M protein during non-denaturing condition. The interaction between cellular proteins and M protein is disrupted in the presence of denaturants (6 M urea or 2% SDS). (B) Recombinant membrane protein (M^*) was detected in stacking gel after boiling treatment. Sample preparation and detection are the same as those of Fig. 1 except whole SDS-PAGE including stacking gel was transferred to perform the WB assay. The empty arrow indicates the membrane protein aggregates.

Fig. 3

Domains important for the thermal aggregation of SARS-CoV membrane protein. (A) Various recombinant fusion proteins (M^*, M51-221^*, M1-170^*, M51-170^*, and MΔ51-170^*) were transiently expressed in the cells. Proteins were collected and heated at different temperatures (20, 60, 80, and 100 °C) for 10 min. After that, the samples were analyzed by WB using anti-V5 monoclonal antibody. The proteins marked by the thin arrow are unglycosylated recombinant membrane proteins while the other ones marked by the thick arrow are glycosylated recombinant proteins. The protein smaller than unglycosylated membrane protein (marked by dotted line) containing the C-terminus of recombinant membrane protein may be derived by cleavage from precursor recombinant membrane proteins or translation by internal initiation. In any case, it was not produced by heat treatment since its concentration did not increase after heat treatment. (B) Same as (A), different recombinant fusion proteins (MΔ47-60^*, MΔ61-90^*, MΔ91-100^*, MΔ101-135^*, and MΔ136-170^*) were used.

Fig. 4

Characteristics of thermal aggregation of SARS-CoV membrane protein. (A) Two proteins (M51-170^* and MΔ51-170^* in the left panel while M^* and MΔ136-170^* in the right panel) were transiently expressed in the cells and protein samples heated at 100 °C for 0, 10, or 30 min were detected by anti-V5 monoclonal antibody. MΔ51-170^* (left panel) and MΔ136-170^* (right panel) were marked by the arrows. (B) Aggregation of M^* protein was induced by treatment at 100 °C for 10 min. After cooling to the room temperature, different denaturants (6 M urea, 1% TX-100, or 2% SDS in final concentration) were used to dissociate the aggregated membrane proteins into monomers.