RESUMO
Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the ß-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.
Assuntos
Antivirais , HIV-1 , Antivirais/farmacologia , Antivirais/química , Humanos , HIV-1/efeitos dos fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Relação Estrutura-Atividade , Tratamento Farmacológico da COVID-19 , Replicação Viral/efeitos dos fármacosRESUMO
The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Infecções por HIV , HIV-1 , Ubiquitina , Humanos , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologiaRESUMO
The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub1), here we show that it can also bind several diubiquitins (K48-Ub2, N-Ub2, and K63-Ub2), with a preference for K63-linked Ub2. The NMR structure of the Tsg101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lisina/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Humanos , Elementos da Série dos Lantanídeos/química , Lisina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Two decades ago, Tsg101, a component of the Endosomal Sorting Complexes Required for Transport (ESCRT) complex 1, was identified as a cellular factor recruited by the human immunodeficiency virus type 1 (HIV-1) to facilitate budding of viral particles assembled at the cell periphery. A highly conserved Pro-(Thr/Ser)-Ala-Pro [P(T/S)AP] motif in the HIV-1 structural polyprotein, Gag, engages a P(T/S)AP-binding pocket in the Tsg101 N-terminal domain. Since the same domain in Tsg101 that houses the pocket was found to bind mono-ubiquitin (Ub) non-covalently, Ub binding was speculated to enhance P(T/S)AP interaction. Within the past five years, we found that the Ub-binding site also accommodates di-Ub, with Lys63-linked di-Ub exhibiting the highest affinity. We also identified small molecules capable of disrupting Ub binding and inhibiting budding. The structural similarity of these molecules, prazoles, to nucleosides prompted testing for nucleic acid binding and led to identification of tRNA as a Tsg101 binding partner. Here, we discuss these recently identified interactions and their contribution to the viral assembly process. These new partners may provide additional insight into the control and function of Tsg101 as well as identify opportunities for anti-viral drug design.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vírion/metabolismo , Montagem de Vírus , Sítios de Ligação , Proteínas de Ligação a DNA/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , HIV-1/fisiologia , Humanos , Ligação Proteica , Fatores de Transcrição/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoAssuntos
Pessoal de Laboratório , Pesquisa , Retroviridae , Feminino , Humanos , Entrevistas como AssuntoRESUMO
BACKGROUND: A measure of episode spending, such as Medicare Spending Per Beneficiary (MSPB) is increasingly used to evaluate provider performance. Yet if the measure is unreliable, as is often true for low-volume providers, it cannot distinguish "good" from "poor" performance. OBJECTIVE: The objective of this study was to evaluate the reliability of a uniformly calculated MSPB measure for post-acute care (PAC) and the tradeoffs involved in setting a minimum case count threshold. DATA: Medicare claims for 15 million PAC episodes from April 2013 to March 2015. RESEARCH DESIGN: Given the overlap in patients treated in PAC settings, we developed a uniformly calculated MSPB measure for PAC providers that measures spending during the PAC stay and the following 30 days. We examine variation in the MSPB-PAC measure and characterize the measure's reliability and its relationship to provider case counts. RESULTS: Applied to our MSPB-PAC measure, a minimum threshold of 20 Medicare episodes as currently used by the Centers for Medicare & Medicaid Services (CMS) would not establish reasonably reliable measures and could result in drawing unduly erroneous conclusions about provider performance. The measures for home health agencies were considerably less stable and reliable than for institutional PAC providers. CONCLUSIONS: CMS should consider adopting a more stringent reliability standard for setting minimum case counts for MSPB-PAC and other measures. Its current threshold (R-statistic=0.4) reflects more random variation than differences in actual provider performance. To include as many providers as possible, CMS should consider pooling data over multiple years to avoid drawing incorrect conclusions about low-volume providers.
Assuntos
Medicare/economia , Cuidados Semi-Intensivos/economia , Agências de Assistência Domiciliar/economia , Humanos , Medicare/estatística & dados numéricos , Casas de Saúde/economia , Centros de Reabilitação/economia , Reprodutibilidade dos Testes , Cuidados Semi-Intensivos/estatística & dados numéricos , Estados UnidosRESUMO
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases, including cancers of lymphoid and epithelial cells. EBV cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase are essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation postreactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication, such as chronic active EBV infection and posttransplant lymphoproliferative disorders. IMPORTANCE Production of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans and can set the stage for development of EBV cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus and, by interfering with Tsg101, prevent EBV release from lytically active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely used prazoles as candidates to prevent development of posttransplant EBV lymphomas.
Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Antivirais/farmacologia , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Rabeprazol/farmacologia , Fatores de Transcrição/metabolismo , Liberação de Vírus/efeitos dos fármacos , Células A549 , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/prevenção & controle , Células HEK293 , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Inibidores da Bomba de Prótons/farmacologia , Carga Viral/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The ESCRT-I factor Tsg101 is essential for sorting endocytic cargo and is exploited by viral pathogens to facilitate egress from cells. Both the nucleocapsid (NC) domain and p6 domain in HIV-1 Gag contribute to recruitment of the protein. However, the role of NC is unclear when the P(S/T)AP motif in p6 is intact, as the motif recruits Tsg101 directly. The zinc fingers in NC bind RNA and membrane and are critical for budding. Tsg101 can substitute for the distal ZnF (ZnF2) and rescue budding of a mutant made defective by deletion of this element. Here, we report that the ubiquitin (Ub) E2 variant (UEV) domain in Tsg101 binds tRNA in vitro. We confirmed that Tsg101 can substitute for ZnF2 when provided at the viral assembly site as a chimeric Gag-Tsg101 protein (Gag-ΔZnF2-Tsg101) and rescue budding. The UEV was not required in this context; however, mutation of the RNA binding determinants in UEV prevented Tsg101 recruitment from the cell interior when Gag and Tsg101 were co-expressed. The same Tsg101 mutations increased recognition of Gag-Ub, suggesting that tRNA and Ub compete for binding sites. This study identifies a novel Tsg101 binding partner that may contribute to its function in recognition of Ub-modified cargo.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Interações Hospedeiro-Patógeno , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Relação Estrutura-Atividade , Fatores de Transcrição/químicaRESUMO
Two proton pump inhibitors, tenatoprazole and esomeprazole, were previously shown to inhibit HIV-1 egress by blocking the interaction between Tsg101, a member of the ESCRT-I complex, and ubiquitin. Here, we deepen our understanding of prazole budding inhibition by studying a range of viruses in the presence of tenatoprazole. Furthermore, we investigate the relationship between the chemistry of prodrug activation and HIV-1 inhibition for diverse prazoles currently on the market. We report that tenatoprazole is capable of inhibiting the replication of members of the enveloped filo, alpha, and herpes virus families but not the flavivirus group and not the non-enveloped poliovirus. Another key finding is that prazole prodrugs must be activated inside the cell, while their rate of activation in vitro correlated to their efficacy in cells. Our study lays the groundwork for future efforts to repurpose prazole-based compounds as antivirals that are both broad-spectrum and selective in nature.
Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , HIV-1/fisiologia , Inibidores da Bomba de Prótons/farmacologia , Replicação Viral/efeitos dos fármacos , Células HeLa , HumanosRESUMO
The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.
Assuntos
Vírus do Sarcoma Aviário/química , Membrana Celular/metabolismo , Produtos do Gene gag/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Galinhas , Chlorocebus aethiops , Produtos do Gene gag/química , Produtos do Gene gag/genética , Humanos , Proteínas de Membrana/metabolismo , Mutação , Fosfolipase C delta/metabolismo , Ligação Proteica , Domínios Proteicos , Liberação de Vírus/fisiologiaRESUMO
For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.
Assuntos
Vírus do Sarcoma Aviário/química , Membrana Celular/metabolismo , Produtos do Gene gag/metabolismo , Montagem de Vírus/fisiologia , Acilação , Sítios de Ligação , Membrana Celular/química , Produtos do Gene gag/química , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica , Domínios Proteicos , Eletricidade EstáticaRESUMO
HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety.
Assuntos
Precursores Enzimáticos/metabolismo , Protease de HIV/metabolismo , HIV-1/enzimologia , Sequência de Aminoácidos , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Células HEK293 , Protease de HIV/química , Protease de HIV/genética , Humanos , MutaçãoRESUMO
HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/metabolismo , Fatores de Transcrição/metabolismo , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , 2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Esomeprazol/farmacologia , Células HEK293 , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Liberação de Vírus/efeitos dos fármacos , Liberação de Vírus/genéticaRESUMO
BACKGROUND: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. RESULTS: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. CONCLUSIONS: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.
Assuntos
Infecções por HIV/virologia , Protease de HIV/genética , HIV-1/genética , Polimorfismo Genético , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Terapia Antirretroviral de Alta Atividade , Feminino , Células HEK293 , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Protease de HIV/metabolismo , Inibidores da Protease de HIV/uso terapêutico , HIV-1/enzimologia , Humanos , Mutação , Infecções do Sistema Genital/virologia , Fatores de Transcrição , Liberação de Vírus , Replicação ViralRESUMO
Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca(2+) from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca(2+) promotes viral particle production and our studies indicate that IP3R and the ER Ca(2+) store are the physiological providers of Ca(2+) for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca(2+) level originating from the ER store than control cells, suggesting that Gag induced release of store Ca(2+). This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca(2+) elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence (TIRF) and immunoelectron microscopy (IEM) or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an ability to modulate both ER store Ca(2+) release and ER store refilling.
RESUMO
We previously demonstrated that ectodomain residue Asp286 in N2 neuraminidase (NA; Asp268 in N1 NA) present in budding-capable NA proteins contributes to productive NA plasma membrane transport partly by mediating escape from tetherin restriction [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Budding-incapable NA proteins contain a G at this position and either co-expression of human immunodeficiency virus type 1 vpu or siRNA-mediated depletion of tetherin rescued budding capabilities in these proteins [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Furthermore, replacement of D286 with G in budding-capable NA proteins caused loss of function, preventing release of NA virus-like particles (VLPs). Here, we show that mutation of this residue specifically modulates the ability of NA to escape tetherin restriction at the plasma membrane and results in virus attenuation in vivo. Based on immunogold electron microscopy and co-immunoprecipitation assays, both NAD286-containing and NAD286G-containing proteins associated with tetherin in the endoplasmic reticulum (ER). However, the NAD286G loss-of-function mutant also associated with the host factor outside the ER and in plasma-membrane-localized VLPs as visualized using immunogold electron microscopy. We conclude that the presence of aspartate at residue 286 liberates NA from tetherin-dependent restriction upon exit from the ER compartment thus preventing restriction at the plasma membrane. Underscoring the importance of these observations, knockdown of tetherin resulted in a 1-1.5 log increase in influenza virus growth. Additionally, the loss-of-function mutation conferred attenuation in a mouse model of influenza infection as evidenced by a 5-fold increase in LD50 and increases in either percent survival or time to death dependent on the administered dose in vivo.
Assuntos
Antígenos CD/metabolismo , Vírus da Influenza A/patogenicidade , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Antígenos CD/genética , Células COS , Chlorocebus aethiops , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Estrutura Terciária de Proteína , Proteínas Virais/genética , Vírion/fisiologiaRESUMO
BACKGROUND: HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. RESULTS: Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. CONCLUSIONS: The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Transcrição/metabolismo , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , HIV-1/genética , Microscopia Eletrônica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
A retroviral capsid (CA) protein consists of two helical domains, CA(N) and CA(C), which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a ß-hairpin motif upon processing from its precursor polyprotein Gag. The ß-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the ß-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the ß-hairpin structure demonstrated that folding of the ß-hairpin specifically extended the N terminus of helix α1 from Tyr(20) to Pro(17). This coil to helix transition involves the conserved sequence of Thr(16)-Pro(17)-Arg(18) (Ser(16)-Pro(17)-Arg(18) in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA(N) oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg(18), helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the ß-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA(N) oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA(N) domain oligomerization, independent to the faster EIAV-CA(C) domain dimerization.
Assuntos
Proteínas do Capsídeo/química , Vírus da Anemia Infecciosa Equina/química , Vírion/química , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Sequência Conservada , Escherichia coli/genética , Expressão Gênica , HIV-1/química , HIV-1/genética , Vírus da Anemia Infecciosa Equina/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Vírion/genética , Montagem de VírusRESUMO
More than a decade has elapsed since the link between the endosomal sorting complex required for transport (ESCRT) machinery and HIV-1 protein trafficking and budding was first identified. L domains in HIV-1 Gag mediate recruitment of ESCRT which function in bud abscission releasing the viral particle from the host cell. Beyond virus budding, the ESCRT machinery is also involved in the endocytic pathway, cytokinesis, and autophagy. In the past few years, the number of non-ESCRT host proteins shown to be required in the assembly process has also grown. In this paper, we highlight the role of recently identified cellular factors that link ESCRT machinery to calcium signaling machinery and we suggest that this liaison contributes to setting the stage for productive ESCRT recruitment and mediation of abscission. Parallel paradigms for non-ESCRT roles in virus budding and cytokinesis will be discussed.
RESUMO
Despite many changes made in 2010 and 2011 to Medicare's payment system for short-term stays in skilled nursing facilities, a flawed payment structure continues to underpay facilities for certain types of patients and overpay for others. The flaws in the payment structure create incentives to selectively admit or refuse patients based on the type and complexity of their conditions, while payments that vary with level of use encourage providers to furnish therapy services, such as rehabilitation care, that some patients might not need. We propose an alternative payment design and demonstrate that it would dampen such incentives by making payments that are more closely matched to costs and based on characteristics of the patients treated. We propose replacing the existing therapy component of payment with one that varies payments according to the expected care needs of the patient and adding a separate payment component that covers drugs and other nontherapy ancillary services, such as support for patients on ventilators. We also propose adding an outlier policy to provide additional reimbursement for patients requiring exceptionally high-cost care.