RESUMEN
To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.
Asunto(s)
Productos del Gen gag , Virus de la Inmunodeficiencia Felina , Virus de la Inmunodeficiencia de los Simios , Ensamble de Virus , Dedos de Zinc , Animales , Virus de la Inmunodeficiencia Felina/genética , Virus de la Inmunodeficiencia Felina/metabolismo , Virus de la Inmunodeficiencia Felina/fisiología , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Productos del Gen gag/química , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Gatos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Línea Celular , Nucleocápside/metabolismo , Nucleocápside/genética , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , FenotipoRESUMEN
For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.
Asunto(s)
Virus del Mono Mason-Pfizer , Virus del Mono Mason-Pfizer/química , Virus del Mono Mason-Pfizer/fisiología , Proteínas , Productos del Gen gag/química , Endopeptidasas , Membrana Celular , Ensamble de VirusRESUMEN
During the HIV-1 assembly process, the Gag polyprotein multimerizes at the producer cell plasma membrane, resulting in the formation of spherical immature virus particles. Gag-genomic RNA (gRNA) interactions play a crucial role in the multimerization process, which is yet to be fully understood. We performed large-scale all-atom molecular dynamics simulations of membrane-bound full-length Gag dimer, hexamer, and 18-mer. The inter-domain dynamic correlation of Gag, quantified by the heterogeneous elastic network model applied to the simulated trajectories, is observed to be altered by implicit gRNA binding, as well as the multimerization state of the Gag. The lateral dynamics of our simulated membrane-bound Gag proteins, with and without gRNA binding, agree with prior experimental data and help to validate our simulation models and methods. The gRNA binding is observed to affect mainly the SP1 domain of the 18-mer and the matrix-capsid linker domain of the hexamer. In the absence of gRNA binding, the independent dynamical motion of the nucleocapsid domain results in a collapsed state of the dimeric Gag. Unlike stable SP1 helices in the six-helix bundle, without IP6 binding, the SP1 domain undergoes a spontaneous helix-to-coil transition in the dimeric Gag. Together, our findings reveal conformational switches of Gag at different stages of the multimerization process and predict that the gRNA binding reinforces an efficient binding surface of Gag for multimerization, and also regulates the dynamic organization of the local membrane region itself.
Asunto(s)
Productos del Gen gag , VIH-1 , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Genómica , VIH-1/metabolismo , ARN Viral/química , Ensamble de VirusRESUMEN
BACKGROUND: Activity-regulated cytoskeleton-associated (Arc) protein is predominantly expressed in excitatory glutamatergic neurons of vertebrates, where it plays a pivotal role in regulation of synaptic plasticity. Arc protein forms capsid-like particles, which can encapsulate and transfer mRNA in extracellular vesicles (EVs) between hippocampal neurons. Once glioma cell networks actively interact with neurons via paracrine signaling and formation of neurogliomal glutamatergic synapses, we predicted the involvement of Arc in a process of EV-mediated mRNA transfer between glioma cells. MATERIALS AND METHODS: Arc expression in three human glioma cell lines was evaluated by WB and immunocytochemistry. The properties of Arc protein/mRNA-containing EVs produced by glioma cells were analyzed by RT-PCR, TEM, and WB. Flow cytometry, RT-PCR, and fluorescent microscopy were used to show the involvement of Arc in EV-mediated mRNA transfer between glioma cells. RESULTS: It was found that human glioma cells can produce EVs containing Arc/Arg3.1 protein and Arc mRNA (or "Arc EVs"). Arc EVs from U87 glioma cells internalize and deliver Arc mRNA to recipient U87 cells, where it is translated into a protein. Arc overexpression significantly increases EV production, alters EV morphology, and enhances intercellular transfer of highly expressed mRNA in glioma cell culture. CONCLUSION: These findings indicate involvement of Arc EVs into mRNA transfer between glioma cells that could contribute to tumor progression and affect synaptic plasticity in cancer patients.
Asunto(s)
Vesículas Extracelulares , Glioma , Animales , Humanos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Productos del Gen gag/química , Productos del Gen gag/genética , Vesículas Extracelulares/metabolismo , Glioma/genéticaRESUMEN
For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use spatial stochastic computer simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol is highly likely and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<∆G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses.
Asunto(s)
Infecciones por VIH , Ensamble de Virus , Humanos , Ensamble de Virus/fisiología , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Péptido Hidrolasas/metabolismo , Endopeptidasas/metabolismo , Virión/metabolismo , Infecciones por VIH/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.
Asunto(s)
Condensados Biomoleculares , Productos del Gen gag , Proteínas Intrínsecamente Desordenadas , Virus del Sarcoma de Rous , Humanos , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/virología , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus del Sarcoma de Rous/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Transición de FaseRESUMEN
During the late stages of the HIV-1 lifecycle, immature virions are produced by the concerted activity of Gag polyproteins, primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains, which assemble into a spherical lattice, package viral genomic RNA, and deform the plasma membrane. Recently, inositol hexakisphosphate (IP6) has been identified as an essential assembly cofactor that efficiently produces both immature virions in vivo and immature virus-like particles in vitro. To date, however, several distinct mechanistic roles for IP6 have been proposed on the basis of independent functional, structural, and kinetic studies. In this work, we investigate the molecular influence of IP6 on the structural outcomes and dynamics of CA/SP1 assembly using coarse-grained (CG) molecular dynamics (MD) simulations and free energy calculations. Here, we derive a bottom-up, low-resolution, and implicit-solvent CG model of CA/SP1 and IP6, and simulate their assembly under conditions that emulate both in vitro and in vivo systems. Our analysis identifies IP6 as an assembly accelerant that promotes curvature generation and fissure-like defects throughout the lattice. Our findings suggest that IP6 induces kinetically trapped immature morphologies, which may be physiologically important for later stages of viral morphogenesis and potentially useful for virus-like particle technologies.
Asunto(s)
VIH-1 , Proteínas de la Cápside/metabolismo , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , VIH-1/metabolismo , Cinética , Ácido Fítico/metabolismo , ARN Viral/metabolismo , Virión , Ensamble de Virus/fisiologíaRESUMEN
The Gag proteins of retroviruses play an essential role in virus particle assembly by forming a protein shell or capsid and thus generating the virion compartment. A variety of human proteins have now been identified with structural similarity to one or more of the major Gag domains. These human proteins are thought to have been evolved or "domesticated" from ancient integrations due to retroviral infections or retrotransposons. Here, we report that X-ray crystal structures of stably folded domains of MOAP1 (modulator of apoptosis 1) and PEG10 (paternally expressed gene 10) are highly similar to the C-terminal capsid (CA) domains of cognate Gag proteins. The structures confirm classification of MOAP1 and PEG10 as domesticated Gags, and suggest that these proteins may have preserved some of the key interactions that facilitated assembly of their ancestral Gags into capsids.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Proteínas de Unión al ADN , Productos del Gen gag , Proteínas de Unión al ARN , Retroelementos/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Secuencia Conservada/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Productos del Gen gag/química , Productos del Gen gag/genética , Humanos , Modelos Moleculares , Dominios Proteicos/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Retroviridae/genética , Infecciones por RetroviridaeRESUMEN
Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism.
Asunto(s)
Variaciones en el Número de Copia de ADN , Productos del Gen gag/química , Retroelementos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas Reguladoras de la Apoptosis/química , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Cristalografía por Rayos X , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Mutación , Dominios Proteicos , Multimerización de Proteína , Estabilidad Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology.
Asunto(s)
Síndrome de Angelman/metabolismo , Síndrome de Angelman/fisiopatología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Productos del Gen gag/química , Proteínas de Unión al ARN/metabolismo , Retroviridae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Movimiento Celular , Preescolar , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios Proteicos , Retroelementos/genética , Gránulos de Estrés/metabolismo , Gránulos de Estrés/ultraestructura , Transcriptoma/genéticaRESUMEN
The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid (CA) lattice is located downstream of the CA protein in many retroviral Gags. The HIV-1 Gag protein contains a stretch of 5 amino acid residues termed the "clasp motif," important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of HIV-1 and Mason-Pfizer monkey virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide a comparable function. The importance of the sequences spanning the CA-nucleocapsid (NC) cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study, we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant proteins in vitro and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant viruses in vivo. The mutants revealed major defects in virion assembly and release in HEK 293T and HeLa cells and even larger defects in infectivity. Our data identify the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient retroviral infection. IMPORTANCE The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short "clasp" motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.
Asunto(s)
Proteínas de la Cápside/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus del Mono Mason-Pfizer/fisiología , Virión/metabolismo , Ensamble de Virus , Secuencias de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Productos del Gen gag/genética , Genoma Viral , Células HEK293 , Células HeLa , Humanos , Virus del Mono Mason-Pfizer/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dominios Proteicos , ARN Viral/genética , ARN Viral/metabolismo , Empaquetamiento del Genoma ViralRESUMEN
Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag-PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P2 than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(-)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(-)MA binds to PI(4,5)P2 via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA-IP binding is governed by charge-charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P2and phosphatidylserine enhance myr(-)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(-)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly.
Asunto(s)
Membrana Celular/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Sitios de Unión , Línea Celular , Membrana Celular/virología , Productos del Gen gag/genética , Virus Linfotrópico T Tipo 1 Humano/química , Humanos , Células Jurkat , Microscopía Confocal , Modelos Moleculares , Mutación , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.
Asunto(s)
Productos del Gen gag/química , Guanina/química , Virus del Mono Mason-Pfizer/química , ARN Viral/química , Uracilo/química , Animales , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Regulación Viral de la Expresión Génica , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Guanina/metabolismo , Interacciones Huésped-Patógeno , Virus del Mono Mason-Pfizer/genética , Virus del Mono Mason-Pfizer/metabolismo , Conformación de Ácido Nucleico , Papio , Unión Proteica , Conformación Proteica , Huella de Proteína , ARN Viral/genética , ARN Viral/metabolismo , Transducción de Señal , Uracilo/metabolismoRESUMEN
The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Interacciones Huésped-Patógeno , Polielectrolitos/química , Retroviridae/fisiología , Ensamble de Virus , Alpharetrovirus/fisiología , Animales , Betaretrovirus/fisiología , Células Cultivadas , Gammaretrovirus/fisiología , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Polielectrolitos/metabolismo , Retroviridae/ultraestructura , ViriónRESUMEN
The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.
Asunto(s)
Moléculas de Adhesión Celular , Adhesiones Focales , Productos del Gen gag , Virus Linfotrópico T Tipo 1 Humano , Proteínas de Microfilamentos , Fosfoproteínas , Linfocitos T , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Adhesiones Focales/química , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Adhesiones Focales/virología , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Virus Linfotrópico T Tipo 1 Humano/química , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , Linfocitos T/química , Linfocitos T/metabolismo , Linfocitos T/virologíaRESUMEN
Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.
Asunto(s)
Productos del Gen env/química , Productos del Gen gag/química , Virus del Mono Mason-Pfizer/química , Productos del Gen env/genética , Productos del Gen gag/genética , Virus del Mono Mason-Pfizer/genética , Dominios ProteicosRESUMEN
During the maturation of HIV-1 particle, the Gag polyprotein is cleaved into several proteins by the HIV-1 protease. These proteins rearrange to form infectious virus particles. In this study, the solution structure and dynamics of a monomeric mutated domain encompassing the C-terminal of capsid, the spacer peptide SP1 and the nucleocapsid from Gag was characterized by Nuclear Magnetic Resonance in the presence of maturation inhibitor EP39, a more hydro-soluble derivative of BVM. We show that the binding of EP39 decreases the dynamics of CA-SP1 junction, especially the QVT motif in SP1, and perturbs the natural coil-helix equilibrium on both sides of the SP1 domain by stabilizing the transient alpha helical structure. Our results provide new insight into the structure and dynamics of the SP1 domain and how HIV-1 maturation inhibitors interfere with this domain. They offer additional clues for the development of new second generation inhibitors targeting HIV-1 maturation.
Asunto(s)
Fármacos Anti-VIH/farmacología , Productos del Gen gag/metabolismo , VIH-1/efectos de los fármacos , Secuencia de Aminoácidos , Sitios de Unión , Dimerización , Productos del Gen gag/química , VIH-1/fisiología , Humanos , Resonancia Magnética Nuclear Biomolecular , Factor de Transcripción Sp1/química , Factor de Transcripción Sp1/metabolismoRESUMEN
The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions.
Asunto(s)
Sustitución de Aminoácidos , Productos del Gen gag/química , Productos del Gen gag/genética , Motivos de Unión al ARN , Virus del Sarcoma de Rous/fisiología , Proteínas Virales/química , Proteínas Virales/genética , Productos del Gen gag/metabolismo , Genoma Viral , Humanos , Unión Proteica , Transporte de Proteínas , Proteínas Virales/metabolismo , Ensamble de Virus , Liberación del VirusRESUMEN
We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Fragmentos de Inmunoglobulinas/química , Nanopartículas/química , Anticuerpos de Dominio Único/química , Cristalografía por Rayos X , ADN/química , Bases de Datos de Proteínas , Escherichia coli , Transferencia Resonante de Energía de Fluorescencia , Productos del Gen gag/química , Células HEK293 , VIH-1/química , Células HeLa , Humanos , Cinética , Ligandos , Microscopía Fluorescente , Mitosis , Dominios Proteicos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
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.