RESUMEN
Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.
Asunto(s)
Cápside/metabolismo , Virus de la Leucemia Murina de Moloney/metabolismo , Nucleocápside/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Animales , Cápside/química , Membrana Celular/metabolismo , Cristalografía , Histidina/metabolismo , Procesamiento de Imagen Asistido por Computador , Membrana Dobles de Lípidos/metabolismo , Ratones , Microscopía Electrónica , Datos de Secuencia Molecular , Nucleocápside/química , ARN/metabolismoRESUMEN
To analyze contacts made by Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins in immature and mature virus particles, we have employed a cysteine-specific crosslinking approach that permits the identification of retroviral Gag protein interactions at particular residues. For analysis, single cysteine creation mutations were made in the context of protease-deficient or protease-competent parental constructs. Cysteine creation mutations were chosen near the N- and C-termini of CA and at a site adjacent to the M-MuLV Glu-Ala Fv1 N/B host range determination sequence. Analysis of immature virions showed that PrGag proteins were crosslinked at C-terminal CA residues to form dimers while crosslinking of particle-associated N-terminal and N/B region mutant proteins did not yield dimers, but showed evidence of linking to an unknown 140- to 160-kDa partner. Analysis of mature virions demonstrated that both N- and C-terminal CA residues participated in dimer formation, suggesting that processed CA N- and C-termini are free to establish interprotein associations. Interestingly, N/B region mutant residues in mature virus particles did not crosslink to form dimers, but showed a novel crosslinked band, consistent with an interaction between the N/B tropism determining region and a cellular protein of 45-55 kDa.
Asunto(s)
Cápside/química , Cápside/metabolismo , Virus de la Leucemia Murina de Moloney/crecimiento & desarrollo , Virus de la Leucemia Murina de Moloney/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Células COS , Cápside/genética , Reactivos de Enlaces Cruzados/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dimerización , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Peso Molecular , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , Mutación/genética , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteínas/metabolismo , Transfección , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Ensamble de VirusRESUMEN
We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.