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Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras - PubMed

  • ️Fri Jan 01 2016

Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

Lili Kuo et al. J Virol. 2016.

Abstract

The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought.

Importance: The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Figures

FIG 1
FIG 1

Coronavirus M- and N-protein domain structure. (A) Schematics of the M and N proteins with a summary of currently assigned interactions. Tm, transmembrane domain; NTD (N1b), amino-terminal RNA-binding domain; SR, serine- and arginine-rich region; CTD (N2b), carboxy-terminal RNA-binding domain; B, spacer region; N3, carboxy-terminal domain. (B) Alignment of the MHV and SARS-CoV (SCoV) M proteins. Tm domains are as modeled in the work of Rottier et al. (29). Vertical bars between the ectodomain (ecto), Tm domains, and endodomain (endo), functional crossover boundaries in the constructed chimeras; filled circles, nonfunctional crossover boundaries within the M endodomain. (C) Alignment of the carboxy termini of the MHV and SARS-CoV N proteins. Vertical bar, the functional crossover boundary in chimeras; broken line, boundary between spacer B and domain N3. The GenBank accession numbers for the sequences shown are AY700211 for MHV A59 and AY278741 for SARS-CoV strain Urbani.

FIG 2
FIG 2

Construction of an MHV chimera containing the entire M protein of SARS-CoV. (A) Schematics of wild-type (wt) virus and chimeras MN1, MN2, and MN3 containing mutant M and N proteins. Shading represents the sequence of SARS-CoV substituted for that of MHV. (B) Western blots of lysates from mouse 17Cl1 cells infected with wild-type MHV or MN3 virus; mock, uninfected 17Cl1 cells. Additional controls, designated mock (V) and SCoV (V), were protein fractions from TRIzol extracts of mock-infected and SARS-CoV-infected Vero cells, respectively. Blots were probed with polyclonal anti-MHV N antibody (left), monoclonal anti-SARS-CoV M antibody (middle), or monoclonal anti-MHV M antibody (right). (C) Plaques of the MN3 mutant (passage 2 stock) at 33, 37, or 39°C compared with those of the isogenic wild-type virus (passage 3 stock). Plaque titrations were carried out on L2 cells; monolayers were stained with neutral red at 72 h postinfection and were photographed 18 h later. Infectious titers measured at all three temperatures were 8.6 × 107 PFU/ml for the wild type and 2.3 × 106 PFU/ml for MN3.

FIG 3
FIG 3

Effect of E-protein mutations on SARS-CoV M chimeras. (A) Alignment of MHV and SARS-CoV E proteins showing independent reverting mutations that enhance the growth of the MN3 chimera at 39°C. Solid bar, the Tm domain; the circled mutation of MN3rev3 (F20S), the mutation chosen for incorporation into subsequent constructs. The GenBank accession numbers for the sequences shown are AY700211 for MHV A59 and AY278741 for SARS-CoV strain Urbani. (B) Schematics of wild-type virus and chimeras containing mutant E, M, and N proteins. Shading represents the sequence of SARS-CoV substituted for that of MHV. (C) Plaques of wild-type, MN3, MN3rev3, MN6, and MN7 viruses at 37 and 39°C (passage 4 stock for the wild type, passage 2 stocks for the mutants). Plaque titrations were carried out on L2 cells; monolayers were stained with neutral red at 72 h postinfection and were photographed 18 h later. The infectious titers (numbers of PFU per milliliter) measured at each temperature are indicated.

FIG 4
FIG 4

Intramolecular M-protein chimeric substitutions. (A) Schematics of wild-type virus and chimeras MN8, MN9, and MN10 containing mutant E, M, and N proteins. Shading represents the sequence of SARS-CoV substituted for that of MHV. (B) Plaques of wild-type, MN8, MN9, and MN10 viruses at 37°C (passage 4 stock for the wild type, passage 3 stocks for the mutants). Plaque titrations were carried out on L2 cells; monolayers were stained with neutral red at 72 h postinfection and were photographed 18 h later. The infectious titers (numbers of PFU per milliliter) measured are indicated. (C) Schematics of lethal substitutions in chimeras MN4A to MN4C, MN5A, and MN5B, made in attempts to define a carboxy-terminal subregion of the M endodomain sufficient for interaction with domain N3. MN5A and MN5B also contained the MHV E-gene mutation F20S.

FIG 5
FIG 5

Analysis of MN8 mutant virions. MN8 and wild-type virions were purified by equilibrium centrifugation on continuous gradients of 20 to 30% iodixanol that were collected in 15 fractions, as detailed in Materials and Methods. (A) Infectious titers determined for viral peak fractions 4 through 11 for MN8 (shaded bars) and the wild type (open bars). The densities of all fractions were measured by refractometry (triangles, MN8; circles, wild type). (B, C) Western blots of virion proteins in each fraction probed with polyclonal anti-N (MHV) and monoclonal anti-M ectodomain (MHV) antibodies. (D, E) Northern dot blots of serial 2-fold dilutions of purified virion RNA detected with a probe specific for genomic RNA. (F) Amounts of total protein and genomic RNA (gRNA) and infectivity for wild-type and MN8 virions. Chemiluminescence was quantitated for N protein, M protein, and gRNA and summed over fractions 3 through 12; values are expressed relative to those for the wild type. Total infectivity (numbers of PFU) was summed over fractions 4 through 11.

FIG 6
FIG 6

Deficiency of S protein in MN8 virions. (A) Viral peak fractions from iodixanol gradients (fractions 7 to 10 for the wild type and 4 to 7 for MN8) were separated in a 12% SDS-polyacrylamide gel and analyzed by Western blotting by probing with a polyclonal antibody specific for the carboxy terminus of S protein, as well as with polyclonal anti-N and monoclonal anti-M antibodies. At the right is a longer exposure of MN8 fraction 5 to allow visualization of S. (B) Western blot of lysates from mouse 17Cl1 cells infected with wild-type or MN8 virus, separated in an 8% SDS-polyacrylamide gel, and probed with polyclonal anti-S antibody; mock, uninfected 17Cl1 cells. (C) Western blot of wild-type and MN8 virions separated in a 15% SDS-polyacrylamide gel and probed with polyclonal anti-S antibody. (D) Western blot of purified wild-type, MN8, MN3, and MN3rev3 virions separated in an 8% SDS-polyacrylamide gel and probed with polyclonal anti-S antibody. S0, 180-kDa uncleaved S protein; S2, 90-kDa carboxy-terminal cleavage product of S; SC, carboxy-terminal fragment of S. (E) Schematic of the lethal substitution in chimera MN11, which did not rescue the S-protein deficiency in MN8.

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References

    1. Masters PS, Perlman S. 2013. Coronaviridae, p 825–858. In Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Racaniello VR, Roizman B (ed), Fields virology, 6th ed, vol 1 Lippincott Williams & Wilkins, Philadelphia, PA.
    1. Perlman S, Netland J. 2009. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7:439–450. doi:10.1038/nrmicro2147. - DOI - PMC - PubMed
    1. Vennema H, Godeke GJ, Rossen JW, Voorhout WF, Horzinek MC, Opstelten DJ, Rottier PJ. 1996. Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. EMBO J 15:2020–2028. - PMC - PubMed
    1. Bos EC, Luytjes W, van der Meulen HV, Koerten HK, Spaan WJ. 1996. The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology 218:52–60. doi:10.1006/viro.1996.0165. - DOI - PMC - PubMed
    1. Corse E, Machamer CE. 2000. Infectious bronchitis virus E protein is targeted to the Golgi complex and directs release of virus-like particles. J Virol 74:4319–4326. doi:10.1128/JVI.74.9.4319-4326.2000. - DOI - PMC - PubMed

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