RESUMEN
Newly synthesized membrane proteins are queried by ubiquitin ligase complexes and triaged between degradative and nondegradative fates. The mechanisms that convert modest differences in substrate-ligase interactions into decisive outcomes of ubiquitination are not well understood. Here, we reconstitute membrane protein recognition and ubiquitination in liposomes using purified components from a viral-mediated degradation pathway. We find that substrate-ligase interactions in the membrane directly influence processivity of ubiquitin attachment to modulate polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates in cultured cells of degraded and nondegraded clients. Both computational and experimental analyses identified continuous deubiquitination as a prerequisite for maximal substrate discrimination. Deubiquitinases reduce polyubiquitin dwell times preferentially on clients that dissociate more rapidly from the ligase. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in net degradation. These results provide a conceptual framework for substrate discrimination during membrane protein quality control.
Asunto(s)
Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Antígenos CD4/química , Antígenos CD4/metabolismo , Células HEK293 , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Liposomas/química , Liposomas/metabolismo , Proteínas de la Membrana/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Bone marrow stromal antigen 2 (BST2)/tetherin is a restriction factor that reduces HIV-1 dissemination by tethering virus at the cell surface. BST2 also acts as a sensor of HIV-1 budding, establishing a cellular antiviral state. The HIV-1 Vpu protein antagonizes BST2 antiviral functions via multiple mechanisms, including the subversion of an LC3C-associated pathway, a key cell intrinsic antimicrobial mechanism. Here, we describe the first step of this viral-induced LC3C-associated process. This process is initiated at the plasma membrane through the recognition and internalization of virus-tethered BST2 by ATG5, an autophagy protein. ATG5 and BST2 assemble as a complex, independently of the viral protein Vpu and ahead of the recruitment of the ATG protein LC3C. The conjugation of ATG5 with ATG12 is dispensable for this interaction. ATG5 recognizes cysteine-linked homodimerized BST2 and specifically engages phosphorylated BST2 tethering viruses at the plasma membrane, in an LC3C-associated pathway. We also found that this LC3C-associated pathway is used by Vpu to attenuate the inflammatory responses mediated by virion retention. Overall, we highlight that by targeting BST2 tethering viruses, ATG5 acts as a signaling scaffold to trigger an LC3C-associated pathway induced by HIV-1 infection.
Asunto(s)
Antígeno 2 del Estroma de la Médula Ósea , Virus , Antivirales/metabolismo , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Virus/metabolismo , HumanosRESUMEN
Mammalian cells are equipped with so-called "restriction factors" that suppress virus replication and help to prevent virus transmission from one species to another. This Essay discusses the host restriction factor tetherin, which blocks the release of enveloped viruses like HIV-1, and the factors evolved by primate lentiviruses, such as Vpu and Nef, that antagonize tetherin's action.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Proteínas Ligadas a GPI , Productos del Gen nef/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Lentivirus/genética , Infecciones por Lentivirus/tratamiento farmacológico , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.
Asunto(s)
COVID-19 , Infecciones por VIH , Poliovirus , Humanos , SARS-CoV-2/metabolismo , Proteínas Viroporinas , Proteínas Reguladoras y Accesorias Virales , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Tetherin is an interferon-induced protein whose expression blocks the release of HIV-1 and other enveloped viral particles. The underlying mechanism by which tetherin functions and whether it directly or indirectly causes virion retention are unknown. Here, we elucidate the mechanism by which tetherin exerts its antiviral activity. We demonstrate, through mutational analyses and domain replacement experiments, that tetherin configuration rather than primary sequence is critical for antiviral activity. These findings allowed the design of a completely artificial protein, lacking sequence homology with native tetherin, that nevertheless mimicked its antiviral activity. We further show that tetherin is incorporated into HIV-1 particles as a parallel homodimer using either of its two membrane anchors. These results indicate that tetherin functions autonomously and directly and that infiltration of virion envelopes by one or both of tetherin's membrane anchors is necessary, and likely sufficient, to tether enveloped virus particles that bud through the plasma membrane.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Virión/metabolismo , Animales , Antígenos CD/química , Antígenos CD/genética , Línea Celular , Membrana Celular/metabolismo , Ebolavirus/metabolismo , Proteínas Ligadas a GPI , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutagénesis , Estructura Terciaria de Proteína , Ratas , Proteínas de la Matriz Viral/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
Human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) and HIV type 1 (HIV-1), encode an antisense gene in the negative strand of the provirus. Besides coding for proteins, the messenger RNAs (mRNAs) of retroviral antisense genes have also been found to regulate transcription directly. Thus, it has been proposed that retroviruses likely localize their antisense mRNAs to the nucleus in order to regulate nuclear events; however, this opposes the coding function of retroviral antisense mRNAs that requires a cytoplasmic localization for protein translation. Here, we provide direct evidence that retroviral antisense mRNAs are localized predominantly in the nuclei of infected cells. The retroviral 3' LTR induces inefficient polyadenylation and nuclear retention of antisense mRNA. We further reveal that retroviral antisense RNAs retained in the nucleus associate with chromatin and have transcriptional regulatory function. While HTLV-1 antisense mRNA is recruited to the promoter of C-C chemokine receptor type 4 (CCR4) and enhances transcription from it to support the proliferation of HTLV-1-infected cells, HIV-1 antisense mRNA is recruited to the viral LTR and inhibits sense mRNA expression to maintain the latency of HIV-1 infection. In summary, retroviral antisense mRNAs are retained in nucleus, act like long noncoding RNAs instead of mRNAs, and contribute to viral persistence.
Asunto(s)
VIH-1/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Latencia del Virus/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , Provirus/genética , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/metabolismo , ARN Viral/genética , Proteínas de los Retroviridae/genética , Proteínas de los Retroviridae/metabolismo , Secuencias Repetidas Terminales/genética , Transcripción Genética/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.
Asunto(s)
Linfocitos T CD4-Positivos , Infecciones por VIH , VIH-1 , Proteínas del Virus de la Inmunodeficiencia Humana , Proteínas Reguladoras y Accesorias Virales , Proteínas Viroporinas , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/virología , Citometría de Flujo , Infecciones por VIH/fisiopatología , VIH-1/genética , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/aislamiento & purificación , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/genética , Proteínas Viroporinas/aislamiento & purificación , Proteínas Viroporinas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Infecciones por VIH/virología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteoma/metabolismo , Nexinas de Clasificación/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , VIH-1/fisiología , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Microscopía Electrónica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteoma/análisis , Nexinas de Clasificación/química , Nexinas de Clasificación/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Viroporinas/genéticaRESUMEN
The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.
Asunto(s)
VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Integración Viral , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virología , Cromatina/genética , Cromatina/metabolismo , Citoplasma/metabolismo , Citoplasma/virología , Proteínas del Citoesqueleto/metabolismo , Transcriptasa Inversa del VIH/fisiología , VIH-1/enzimología , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Interfase , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Conformación Proteica , Dominios Proteicos , Factores de Transcripción/deficiencia , Factores de Transcripción/fisiología , Integración Viral/genética , Integración Viral/fisiología , Latencia del Virus , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/deficiencia , Factores de Escisión y Poliadenilación de ARNm/fisiologíaRESUMEN
Ethanol (EtOH) exerts its effects through various protein targets, including transient receptor potential melastatin 7 (TRPM7) channels, which play an essential role in cellular homeostasis. We demonstrated that TRPM7 is expressed in rat brain microvascular endothelial cells (rBMVECs), the major cellular component of the blood-brain barrier (BBB). Heavy alcohol drinking is often associated with HIV infection, however mechanisms underlying alcohol-induced BBB damage and HIV proteins, are not fully understood. We utilized the HIV-1 transgenic (HIV-1Tg) rat to mimic HIV-1 patients on combination anti-retroviral therapy (cART) and demonstrated TRPM7 expression in rBMVECs wass lower in adolescent HIV-1Tg rats compared to control animals, however control and HIV-1Tg rats expressed similar levels at 9 weeks, indicating persistent presence of HIV-1 proteins delayed TRPM7 expression. Binge exposure to EtOH (binge EtOH) decreased TRPM7 expression in control rBMVECs in a concentration-dependent manner, and abolished TRPM7 expression in HIV-1Tg rats. In human BMVECs (hBMVECs), TRPM7 expression was downregulated after treatment with EtOH, HIV-1 proteins, and in combination. Next, we constructed in vitro BBB models using BMVECs and found TRPM7 antagonists enhanced EtOH-mediated BBB integrity changes. Our study demonstrated alcohol decreased TRPM7 expression, whereby TRPM7 could be involved in the mechanisms underlying BBB alcohol-induced damage in HIV-1 patients on cART.
Asunto(s)
Infecciones por VIH , Canales Catiónicos TRPM , Canales de Potencial de Receptor Transitorio , Ratas , Animales , Humanos , Adolescente , Barrera Hematoencefálica/metabolismo , Canales Catiónicos TRPM/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Células Endoteliales/metabolismo , Etanol/toxicidad , Etanol/metabolismo , Ratas Transgénicas , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Host proteins with antiviral activity have evolved as first-line defenses to suppress viral replication. The HIV-1 accessory protein viral protein U (Vpu) enhances release of the virus from host cells by down-regulating the cell-surface expression of the host restriction factor tetherin. However, the exact mechanism of Vpu-mediated suppression of antiviral host responses is unclear. To further understand the role of host proteins in Vpu's function, here we carried out yeast two-hybrid screening and identified the V0 subunit C of vacuolar ATPase (ATP6V0C) as a Vpu-binding protein. To examine the role of ATP6V0C in Vpu-mediated tetherin degradation and HIV-1 release, we knocked down ATP6V0C expression in HeLa cells and observed that ATP6V0C depletion impairs Vpu-mediated tetherin degradation, resulting in defective HIV-1 release. We also observed that ATP6V0C overexpression stabilizes tetherin expression. This stabilization effect was specific to ATP6V0C, as overexpression of another subunit of the vacuolar ATPase, ATP6V0Câ³, had no effect on tetherin expression. ATP6V0C overexpression did not stabilize CD4, another target of Vpu-mediated degradation. Immunofluorescence localization experiments revealed that the ATP6V0C-stabilized tetherin is sequestered in a CD63- and lysosome-associated membrane protein 1 (LAMP1)-positive intracellular compartment. These results indicate that the Vpu-interacting protein ATP6V0C plays a role in down-regulating cell-surface expression of tetherin and thereby contributes to HIV-1 assembly and release.
Asunto(s)
Antígenos CD/biosíntesis , Regulación hacia Abajo , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Liberación del Virus , Antígenos CD/genética , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Células HEK293 , VIH-1/genética , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , ATPasas de Translocación de Protón Vacuolares/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4+ and CD8+ T cells and is upregulated on the surface of these cells upon infection by human immunodeficiency virus type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Here, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5-positive (Rab 5+) vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4+ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates the surface expression of Tim-3, but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Regulación hacia Abajo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Células HEK293 , VIH-1/metabolismo , Células HeLa , Humanos , Interferón beta/metabolismo , ARN Interferente Pequeño/metabolismo , Red trans-Golgi/metabolismoRESUMEN
BST-2/CD317/tetherin is a host transmembrane protein that potently inhibits human immunodeficiency virus type 1 (HIV-1) virion release by tethering the nascent virions to the plasma membrane. Viral protein U (Vpu) is an accessory protein encoded by HIV-1 as well as by some simian immunodeficiency viruses (SIVs) infecting wild chimpanzees, gorillas, or monkeys (SIVcpz, SIVgor, or SIVgsn/SIVmon/SIVmus, respectively). HIV-1 Vpu directly binds to and downregulates human BST-2. The antagonism is highly species specific because the amino acid sequences of BST-2 are different among animal species. Here, we show that Vpu proteins from several SIVcpz, SIVgsn, SIVmon, or SIVmus isolates fail to antagonize human BST-2. Only Vpu from an SIVgsn isolate (SIVgsn-99CM71 [SIVgsn71]) was able to antagonize human BST-2 as well as BST-2 of its natural host, greater spot-nosed monkey (GSN). This SIVgsn Vpu interacted with human BST-2, downregulated cell surface human BST-2 expression, and facilitated HIV-1 virion release in the presence of human BST-2. While the unique 14AxxxxxxxW22 motif in the transmembrane domain of HIV-1NL4-3Vpu was reported to be important for antagonizing human BST-2, we show here that two AxxxxxxxW motifs (A22W30 and A25W33) exist in SIVgsn71 Vpu. Only the A22W30 motif was needed for SIVgsn71 Vpu to antagonize GSN BST-2, suggesting that the mechanism of this antagonism resembles that of HIV-1NL4-3 Vpu against human BST-2. Interestingly, SIVgsn71 Vpu requires two AxxxxxxxW (A22W30 and A25W33) motifs to antagonize human BST-2, suggesting an as-yet-undefined way that SIVgsn71 Vpu works against human BST-2. These results imply an evolutionary impact of primate BST-2 on lentiviral Vpu.IMPORTANCE Genetic alterations conferring a selective advantage in protecting from life-threating pathogens are maintained during evolution. In fact, the amino acid sequences of BST-2 differ among primate animals and their susceptibility to viral proteins is species specific, suggesting that such genetic diversity has arisen through the evolutionarily controlled balance between the host and pathogens. The M (main) group of HIV-1 is thought to be derived from SIVcpz, which utilizes Nef, but not Vpu, to antagonize chimpanzee BST-2. SIVcpz Nef is, however, unable to antagonize human BST-2, and Vpu was consequently chosen again as an antagonist against human BST-2 in the context of HIV-1. Studies on how Vpu lost and acquired this ability, together with the distinct mechanisms by which SIVgsn71 Vpu binds to and downregulates human or GSN BST-2, may help to explain the evolution of this lentiviral protein as a result of host-pathogen interactions.
Asunto(s)
Antígenos CD/biosíntesis , Regulación hacia Abajo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos CD/genética , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Células HEK293 , VIH-1/genética , VIH-1/metabolismo , Haplorrinos , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Virus de la Inmunodeficiencia de los Simios/genética , Especificidad de la Especie , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread.IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD4/biosíntesis , Regulación hacia Abajo , Infecciones por VIH , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Antígenos CD/genética , Antígenos CD4/genética , Línea Celular Transformada , Enfermedad Crónica , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.
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Antígenos CD/genética , VIH-1/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/metabolismo , Secuencia de Aminoácidos , Antígenos CD/biosíntesis , Antígenos CD/aislamiento & purificación , Antígenos CD/farmacología , Secuencia de Bases , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/aislamiento & purificación , Proteínas Ligadas a GPI/farmacología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , VIH-1/patogenicidad , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Cuerpos de Inclusión/química , Unión Proteica , Replegamiento Proteico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Viroporinas/genéticaRESUMEN
HIV-1 can incite activation of chemokine receptors, inflammatory mediators, and glutamate receptor-mediated excitotoxicity. The mechanisms associated with such immune activation can disrupt neuronal and glial functions. HIV-associated neurocognitive disorder (HAND) is being observed since the beginning of the AIDS epidemic due to a change in the functional integrity of cells from the central nervous system (CNS). Even with the presence of antiretroviral therapy, there is a decline in the functioning of the brain especially movement skills, noticeable swings in mood, and routine performance activities. Under the umbrella of HAND, various symptomatic and asymptomatic conditions are categorized and are on a rise despite the use of newer antiretroviral agents. Due to the use of long-lasting antiretroviral agents, this deadly disease is becoming a manageable chronic condition with the occurrence of asymptomatic neurocognitive impairment (ANI), symptomatic mild neurocognitive disorder, or HIV-associated dementia. In-depth research in the pathogenesis of HIV has focused on various mechanisms involved in neuronal dysfunction and associated toxicities ultimately showcasing the involvement of various pathways. Increasing evidence-based studies have emphasized a need to focus and explore the specific pathways in inflammation-associated neurodegenerative disorders. In the current review, we have highlighted the association of various HIV proteins and neuronal cells with their involvement in various pathways responsible for the development of neurotoxicity.
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Complejo SIDA Demencia/inmunología , Complejo SIDA Demencia/virología , Sistema Nervioso Central/virología , VIH-1/metabolismo , Proteínas Virales/metabolismo , Complejo SIDA Demencia/fisiopatología , Antirretrovirales/uso terapéutico , Astrocitos/virología , Sistema Nervioso Central/fisiopatología , Genoma , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Inflamación , Quinurenina/metabolismo , Macrófagos/virología , Microglía/virología , Neuronas/virología , Oligodendroglía/virología , Receptores de N-Metil-D-Aspartato/metabolismo , Carga Viral , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
OBJECTIVES: Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection. METHODS: Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting. RESULTS: The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56-60% as the bands of ~ 63 and ~ 72 kDa confirmed in western blotting, respectively. CONCLUSION: The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.
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Vacunas contra el SIDA , Epítopos de Linfocito T/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Vacunas de ADN , Animales , Simulación por Computador , Epítopos de Linfocito T/metabolismo , Células HEK293 , VIH-1/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Modelos Moleculares , TransfecciónRESUMEN
Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.
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Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Antígenos CD/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Nanotecnología/métodos , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
The endoplasmic reticulum-associated degradation (ERAD) pathway mediates the endoplasmic reticulum-to-cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K-resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.
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Degradación Asociada con el Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Células HEK293 , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Desplegamiento Proteico , Receptores del Factor Autocrino de Motilidad/genética , Receptores del Factor Autocrino de Motilidad/metabolismo , Especificidad por Sustrato , Trimetrexato/farmacología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target.IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.