RESUMO
The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.
Assuntos
Fármacos Anti-HIV , Capsídeo , HIV-1 , Humanos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Capsídeo/química , Capsídeo/efeitos dos fármacos , Microscopia Crioeletrônica , Cristalografia por Raios X , Medição da Troca de Deutério , Células HEK293 , Células HeLa , HIV-1/química , HIV-1/efeitos dos fármacos , Fatores de Poliadenilação e Clivagem de mRNA/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Domínios Proteicos , Integração ViralRESUMO
While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Edição de Genes , Humanos , Masculino , Complexo Mediador/química , Complexo Mediador/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Estrutura Quaternária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Molecular solids and polymers can form low-symmetry crystal structures that exhibit anisotropic electron and ion mobility in engineered devices or biological systems. The distribution of molecular orientation and disorder then controls the macroscopic material response, yet it is difficult to image with conventional techniques on the nanoscale. We demonstrated a new form of optical nanocrystallography that combines scattering-type scanning near-field optical microscopy with both optical antenna and tip-selective infrared vibrational spectroscopy. From the symmetry-selective probing of molecular bond orientation with nanometer spatial resolution, we determined crystalline phases and orientation in aggregates and films of the organic electronic material perylenetetracarboxylic dianhydride. Mapping disorder within and between individual nanoscale domains, the correlative hybrid imaging of nanoscale heterogeneity provides insight into defect formation and propagation during growth in functional molecular solids.
