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. 2000 Sep;74(17):8127-34.
doi: 10.1128/jvi.74.17.8127-8134.2000.

Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells

K Narayanan  1 , A Maeda, J Maeda, S Makino
Affiliations

Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells

K Narayanan et al. J Virol. 2000 Sep.

Abstract

Coronavirus contains three envelope proteins, M, E and S, and a nucleocapsid, which consists of genomic RNA and N protein, within the viral envelope. We studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (MHV). Coimmunoprecipitation analyses demonstrated an interaction between N protein and M protein in infected cells. Pulse-labeling experiments showed that newly synthesized, unglycosylated M protein interacted with N protein in a pre-Golgi compartment, which is part of the MHV budding site. Coimmunoprecipitation analyses further revealed that M protein interacted with only genomic-length MHV mRNA, mRNA 1, while N protein interacted with all MHV mRNAs. These data indicated that M protein interacted with the nucleocapsid, consisting of N protein and mRNA 1, in infected cells. The M protein-nucleocapsid interaction occurred in the absence of S and E proteins. Intracellular M protein-N protein interaction was maintained after removal of viral RNAs by RNase treatment. However, the M protein-N protein interaction did not occur in cells coexpressing M protein and N protein alone. These data indicated that while the M protein-N protein interaction, which is independent of viral RNA, occurred in the M protein-nucleocapsid complex, some MHV function(s) was necessary for the initiation of M protein-nucleocapsid interaction. The M protein-nucleocapsid interaction, which occurred near or at the MHV budding site, most probably represented the process of specific packaging of the MHV genome into MHV particles.

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Figures

FIG. 1
FIG. 1
Interaction between N protein and M protein in MHV-infected cells. (A) DBT cells were infected with MHV-JHM, and intracellular proteins were labeled with Tran[35S] label from 8 to 8.5 h p.i. (lanes 2 to 4) or with [3H]glucosamine from 6.5 to 8.5 h p.i. (lane 5). The intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lane 2), an anti-M protein monoclonal antibody (lanes 3, 5) or an anti-H2K monoclonal antibody (lane 4), and viral proteins were analyzed by SDS–15% PAGE. Lane 1, 14C-labeled protein size marker. (B) MHV-JHM-infected DBT cells were pulse-labeled with Tran[35S] label for 5 min at 8 h p.i., and intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lane 2). Lane 1, 14C-labeled protein size marker. Ab, antibody.
FIG. 2
FIG. 2
Specific M protein-mRNA 1 interaction in MHV-infected cells. MHV-JHM-infected DBT cells were labeled with 32Pi from 6 to 8 h p.i. in the presence of actinomycin D, and cytoplasmic protein lysates were prepared. The intracellular (i.c.) proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lane 2), an anti-M protein monoclonal antibody (lane 3), or an anti-H2K monoclonal antibody (lane 4). MHV-specific RNAs were extracted from the immunoprecipitated samples and analyzed by agarose-formaldehyde gel electrophoresis. Lane 1, virus-specific RNAs extracted from 32P-labeled MHV-infected cells at 8 h p.i.
FIG. 3
FIG. 3
Interaction between N protein and M protein after RNase A treatment. MHV-JHM-infected DBT cells were labeled with Tran[35S] label from 8 to 8.5 h p.i., and cytoplasmic lysates were prepared. Equal volumes of the lysates were either treated with RNase A (lane 2) or mock treated (lane 1) for 15 min at room temperature. The intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody and analyzed by SDS–15% PAGE.
FIG. 4
FIG. 4
Analysis of interaction between expressed N protein and M protein. (A) DBT cells that were infected with SinN pseudovirions (lanes 1 and 2) or SinM pseudovirions (lanes 3 and 4) were labeled with Tran[35S] label from 5 to 5.5 h p.i. The intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lanes 1 and 4) or an anti-M protein monoclonal antibody (lanes 2 and 3), and viral proteins were analyzed by SDS–15% PAGE. (B) Equal volumes of 35S-labeled intracellular protein lysates from DBT cells, infected with SinM pseudovirions alone and SinN pseudovirions alone, were mixed, and intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lane 1), an anti-M protein monoclonal antibody (lane 2), or an anti-H2K monoclonal antibody (lane 3). The viral proteins were analyzed by SDS–12% PAGE. (C) DBT cells were coinfected with SinM pseudovirions and SinN pseudovirions, and intracellular proteins were labeled with Tran[35S] label from 5 to 5.5 h p.i. The intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lane 1), an anti-M protein monoclonal antibody (lane 2), or an anti-H2K monoclonal antibody (lane 3). Viral proteins were analyzed by SDS–15% PAGE. The marked protein bands (indicated by arrows and an asterisk) are non-MHV proteins.
FIG. 5
FIG. 5
Interaction between the nucleocapsid and M protein in the absence of S and E proteins. DBT cells were infected with DIssA/LA16 at 39.5°C. At 3.5 h post-DIssA/LA16 infection, cells were superinfected with either SinM pseudovirions (lanes 1 to 3) or SinLacZ pseudovirions (lanes 4 to 6) and incubated at 39.5°C. The intracellular proteins were labeled with Tran[35S] label from 8.5 to 9 h post-DIssA/LA16 infection, and cytoplasmic lysates were prepared. The intracellular proteins were immunoprecipitated with an anti-N protein monoclonal antibody (lanes 1 and 4), an anti-M protein monoclonal antibody (lanes 2 and 5), or an anti-H2K monoclonal antibody (lanes 3 and 6). The viral proteins were analyzed by SDS–12% PAGE. The 14C-labeled protein size marker is shown on the left of the gel. The marked protein bands (indicated by arrows and an asterisk) are non-MHV proteins.
FIG. 6
FIG. 6
Specific interaction of M protein with DIssA RNA in the absence of S and E proteins. DBT cells were infected with DIssA/LA16 at 39.5°C. At 3.5 h post-DIssA/LA16 infection, cells were superinfected with either SinM pseudovirions (lanes 1 and 2) or SinLacZ pseudovirions (lanes 3 and 4) and incubated at 39.5°C. At 9 h post-DIssA/LA16 infection, cytoplasmic lysates were prepared and separated into two groups. Intracellular RNAs (lanes 1 and 3) were extracted from one group of lysates. An anti-M protein monoclonal antibody was added to another group, and immunoprecipitation was performed. RNAs were extracted from the immunoprecipitated samples (lanes 2 and 4). Extracted RNAs were separated by 1% agarose gel electrophoresis, and DIssA RNA was detected by Northern blot analysis. The part of the autoradiogram that contains DIssA RNA is indicated.
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