RESUMEN
Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.
Asunto(s)
Virus Chikungunya , Internalización del Virus , Virus Chikungunya/genética , Glicoproteínas/análisis , Humanos , Fusión de Membrana , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Virión/metabolismoRESUMEN
HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.
Asunto(s)
Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Virión/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructura , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/ultraestructura , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Secuencia de Aminoácidos , Disulfuros/farmacología , Epítopos/química , Células HEK293 , Proteína gp41 de Envoltorio del VIH/química , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Modelos Moleculares , Pruebas de Neutralización , Péptidos/química , Polisacáridos/química , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Rubella virus (RV) is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, RV infection leads to severe damage in fetuses. Despite its medical importance, compared with the related alphaviruses, very little is known about the structure of RV. The RV capsid protein is an essential structural component of virions as well as a key factor in virus-host interactions. Here we describe three crystal structures of the structural domain of the RV capsid protein. The polypeptide fold of the RV capsid protomer has not been observed previously. Combining the atomic structure of the RV capsid protein with the cryoelectron tomograms of RV particles established a low-resolution structure of the virion. Mutational studies based on this structure confirmed the role of amino acid residues in the capsid that function in the assembly of infectious virions.