RESUMEN
The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , VIH-1/química , Imagen Individual de Molécula , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Animales , Anticuerpos Neutralizantes/inmunología , Bovinos , Disulfuros/química , Células HEK293 , VIH-1/genética , VIH-1/inmunología , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated "SOS" (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env.IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/inmunología , VIH-1/inmunología , Esparcimiento de Virus/inmunología , Esparcimiento de Virus/fisiología , Anticuerpos Neutralizantes/inmunología , Antígenos CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Disulfuros , Células HEK293 , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.
Asunto(s)
Técnicas Genéticas , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Artemisininas/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Prueba de Complementación Genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
During development in human erythrocytes, the malaria parasite Plasmodium falciparum internalizes a large part of the cellular content of the host cell. The internalized cytosol, consisting largely of hemoglobin, is transported to the parasite's food vacuole where it is degraded, providing nutrients and space for growth. This host cell cytosol uptake (HCCU) is crucial for parasite survival but the parasite proteins mediating this process remain obscure. Here, we identify P. falciparum VPS45 as an essential factor in HCCU. Conditional inactivation of PfVPS45 led to an accumulation of host cell cytosol-filled vesicles within the parasite and inhibited the delivery of hemoglobin to the parasite's digestive vacuole, resulting in arrested parasite growth. A proportion of these HCCU vesicle intermediates was positive for phosphatidylinositol 3-phosphate, suggesting endosomal characteristics. Thus PfVPS45 provides insight into the elusive machinery of the ingestion pathway in a parasite that contains an endolysosomal system heavily repurposed for protein secretion.