Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.
- MeSH
- elektronová kryomikroskopie MeSH
- genové produkty gag chemie genetika ultrastruktura MeSH
- HEK293 buňky MeSH
- HIV-1 chemie genetika ultrastruktura MeSH
- kapsida chemie ultrastruktura MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- myši MeSH
- proteinové domény MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- tomografie elektronová MeSH
- virion chemie genetika ultrastruktura MeSH
- virové plášťové proteiny chemie genetika ultrastruktura MeSH
- virus myší leukemie chemie genetika ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- srovnávací studie MeSH
The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.
- MeSH
- mutageneze MeSH
- myši MeSH
- proteinové domény MeSH
- sekundární struktura proteinů MeSH
- virové plášťové proteiny chemie genetika MeSH
- virus myší leukemie chemie genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies.
- MeSH
- chromatografie afinitní MeSH
- klonování DNA MeSH
- kyselina myristová chemie metabolismus MeSH
- myši MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- Retroviridae - proteiny chemie genetika izolace a purifikace metabolismus MeSH
- retrovirové infekce virologie MeSH
- virus myší leukemie chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
