Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

] Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i IOCB and Gilead Research Center Flemingovo nám 2 166 10 Prague Czech Republic [2] Department of Biotechnology Institute of Chemical Technology Prague Technická 5 166 28 Prague Czech Republic

] Molecular Medicine Partnership Unit European Molecular Biology Laboratory Universitätsklinikum Heidelberg Heidelberg Germany [2] Department of Infectious Diseases Virology Universitätsklinikum Heidelberg Im Neuenheimer Feld 324 69120 Heidelberg Germany

] Structural and Computational Biology Unit European Molecular Biology Laboratory Meyerhofstrasse 1 69117 Heidelberg Germany [2] Molecular Medicine Partnership Unit European Molecular Biology Laboratory Universitätsklinikum Heidelberg Heidelberg Germany

Department of Biochemistry and Microbiology Institute of Chemical Technology Prague Technická 5 166 28 Prague Czech Republic

Structural and Computational Biology Unit European Molecular Biology Laboratory Meyerhofstrasse 1 69117 Heidelberg Germany

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