RESUMO
The HIV-1 capsid is an ordered protein shell that houses the viral genome during early infection. Its expansive surface consists of an ordered and interfacing array of capsid protein hexamers and pentamers that are recognized by numerous cellular proteins. Many of these proteins recognize specific, assembled capsid interfaces not present in unassembled capsid subunits. We used protein-engineering tools to capture diverse capsid assembly intermediates. We built a repertoire of capsid assemblies (ranging from two to 42 capsid protein molecules) that recreate the various surfaces in infectious capsids. These assemblies reveal unique capsid-targeting mechanisms for each of the anti-HIV factors, TRIMCyp, MxB, and TRIM5α, linked to inhibition of virus uncoating and nuclear entry, as well as the HIV-1 cofactor FEZ1 that facilitates virus intracellular trafficking. This capsid assembly repertoire enables elucidation of capsid recognition modes by known capsid-interacting factors, identification of new capsid-interacting factors, and potentially, development of capsid-targeting therapeutics.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/ultraestrutura , Capsídeo/química , Capsídeo/ultraestrutura , HIV-1/fisiologia , HIV-1/ultraestrutura , Animais , Fármacos Anti-HIV/farmacologia , Fatores de Restrição Antivirais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , HIV-1/genética , Humanos , Macaca fascicularis , Macaca mulatta , Proteínas de Resistência a Myxovirus , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Modular type I polyketide synthases (PKSs) are versatile biosynthetic systems that initiate, successively elongate, and modify acyl chains. Intermediate transfer between modules is mediated via docking domains, which are attractive targets for PKS pathway engineering to produce natural product analogs. We identified a class 2 docking domain in cyanobacterial PKSs and determined crystal structures for two docking domain pairs, revealing a distinct class 2 docking strategy for promoting intermediate transfer. The selectivity of class 2 docking interactions, demonstrated in binding and biochemical assays, could be altered by mutagenesis. We determined the ideal fusion location for exchanging class 1 and class 2 docking domains and demonstrated effective polyketide chain transfer in heterologous modules. Thus, class 2 docking domains are tools for rational bioengineering of a broad range of PKSs containing either class 1 or 2 docking domains.