RESUMEN
The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but detailed structural information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryoelectron tomography and single particle analysis (SPA) cryoelectron microscopy to determine structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a method of liposome scaffold-based in vitro lattice assembly ("lattice templating") that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (lenacapavir). Our work reveals key structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and SPA as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds.
Asunto(s)
Proteínas de la Cápside , VIH-1 , Proteínas de la Cápside/química , Cápside/metabolismo , VIH-1/metabolismo , Microscopía por Crioelectrón/métodos , Ensamble de VirusRESUMEN
A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.
Asunto(s)
VIH-1/metabolismo , Fosfatos de Inositol/metabolismo , Virión/metabolismo , Ensamble de Virus , Arginina/metabolismo , Cápside/química , Cápside/metabolismo , Cristalografía por Rayos X , VIH-1/química , VIH-1/genética , Técnicas In Vitro , Lisina/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Virión/química , Virión/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
In this Letter, the Protein Data Bank (PDB) accessions were incorrectly listed as '6BH5, 6BHT and 6BHS' instead of '6BHR, 6BHT and 6BHS'; this has been corrected online.
RESUMEN
During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. "Complex" retroviruses, such as human immunodeficiency virus (HIV), use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. "Simple" retroviruses, such as Mason-Pfizer monkey virus (MPMV) and murine leukemia virus (MLV), exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous sarcoma virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling-deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to wild-type (WT) virus and surprisingly is replication competent, albeit with a slower rate of spread than observed in WT virus. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. IMPORTANCE While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. Rous sarcoma virus (RSV) Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Productos del Gen gag/genética , Virus del Sarcoma de Rous/genética , Transporte Activo de Núcleo Celular/genética , Animales , Línea Celular , Núcleo Celular/virología , Productos del Gen gag/metabolismo , Genoma Viral/genética , Humanos , Ratones , ARN Viral/genética , Retroviridae/genética , Virus del Sarcoma de Rous/metabolismo , Virión/metabolismo , Ensamble de VirusRESUMEN
Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.
Asunto(s)
Vectores Genéticos/administración & dosificación , Infecciones por VIH/virología , Fosfatos de Inositol/metabolismo , Lentivirus de los Primates/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ácido Fítico/metabolismo , Virión/fisiología , Animales , Vectores Genéticos/genética , VIH/fisiología , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Humanos , Fosforilación , PrimatesRESUMEN
Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.
Asunto(s)
Anemia Infecciosa Equina/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus de la Anemia Infecciosa Equina/fisiología , Ácido Fítico/metabolismo , Virión/fisiología , Secuencia de Aminoácidos , Animales , Tomografía con Microscopio Electrónico , Anemia Infecciosa Equina/virología , Productos del Gen gag/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , VIH-1/ultraestructura , Caballos , Interacciones Huésped-Patógeno , Virus de la Anemia Infecciosa Equina/química , Virus de la Anemia Infecciosa Equina/genética , Virus de la Anemia Infecciosa Equina/ultraestructura , Alineación de Secuencia , Virión/genética , Virión/ultraestructura , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Understanding the molecular mechanisms of retroviral assembly has been a decades-long endeavor. With the recent discovery of inositol hexakisphosphate (IP6) acting as an assembly co-factor for human immunodeficiency virus (HIV), great strides have been made in retroviral research. In this review, the enzymatic pathways to synthesize and metabolize inositol phosphates (IPs) relevant to retroviral assembly are discussed. The functions of these enzymes and IPs are outlined in the context of the cellular biology important for retroviruses. Lastly, the recent advances in understanding the role of IPs in retroviral biology are surveyed.
Asunto(s)
Fosfatos de Inositol/metabolismo , Retroviridae/fisiología , Ensamble de Virus/fisiología , Vías Biosintéticas , Infecciones por VIH/virología , Humanos , Ácido Fítico/metabolismo , Virión/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.