title: Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Kuo
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA.
authors.forename: Kelley R
authors.initials: KR
authors.lastname: Hurst-Hess
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA.
authors.forename: Cheri A
authors.initials: CA
authors.lastname: Koetzner
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA Paul.Masters@health.ny.gov.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Masters
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017934:Coronavirus / Q000502:physiology*; D018352:Coronavirus Infections / Q000821:virology*; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000502:physiology; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D016415:Sequence Alignment; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D014763:Viral Matrix Proteins