Bharata, Tanmay A ; Menendez, Luis R ; Hagena, Wim J ; Luxd, Vanda ; Igonete, Sebastien ; Schorba, Martin ; Schur, FlorianIST Austria ; Kraüsslich, Hans Georg ; Briggsa, John A
The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
The authors thank Leonardo Trabuco for help with running MDFF, Maria Anders for preparing amprenavir-inhibited virus, Marie-Christine Vaney for help with X-ray data processing and structure refinement, Ahmed Haouz and Patrick Weber (robotized crystallization facility Proteopole, Institut Pasteur) for help in crystal screening, and the European Molecular Biology Laboratory (EMBL) Information Technology Services Unit and Frank Thommen for technical support. This study was supported by Deutsche Forschungsgemeinschaft Grants BR 3635/2-1 (to J.A.G.B.) and KR 906/7-1 (to H.-G.K.) and a Federation of European Biochemical Societies long-term fellowship (to T.A.M.B.). The laboratory of J.A.G.B. acknowledges financial support from EMBL and the Chica und Heinz Schaller Stiftung.
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Bharata T, Menendez L, Hagena W, et al. Cryo electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly. PNAS. 2014;111(22):8233-8238. doi:10.1073/pnas.1401455111
Bharata, T., Menendez, L., Hagena, W., Luxd, V., Igonete, S., Schorba, M., … Briggsa, J. (2014). Cryo electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly. PNAS, 111(22), 8233–8238. https://doi.org/10.1073/pnas.1401455111
Bharata, Tanmay, Luis Menendez, Wim Hagena, Vanda Luxd, Sebastien Igonete, Martin Schorba, Florian Schur, Hans Kraüsslich, and John Briggsa. “Cryo Electron Microscopy of Tubular Arrays of HIV-1 Gag Resolves Structures Essential for Immature Virus Assembly.” PNAS 111, no. 22 (2014): 8233–38. https://doi.org/10.1073/pnas.1401455111.
T. Bharata et al., “Cryo electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly,” PNAS, vol. 111, no. 22, pp. 8233–8238, 2014.
Bharata T, Menendez L, Hagena W, Luxd V, Igonete S, Schorba M, Schur F, Kraüsslich H, Briggsa J. 2014. Cryo electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly. PNAS. 111(22), 8233–8238.
Bharata, Tanmay, et al. “Cryo Electron Microscopy of Tubular Arrays of HIV-1 Gag Resolves Structures Essential for Immature Virus Assembly.” PNAS, vol. 111, no. 22, National Academy of Sciences, 2014, pp. 8233–38, doi:10.1073/pnas.1401455111.