RESUMO
With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.
Assuntos
COVID-19 , Inibidores da Fusão de HIV , Animais , Camundongos , Humanos , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/genética , Glicoproteína da Espícula de Coronavírus/genética , Camundongos Transgênicos , Peptídeos/farmacologiaRESUMO
Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.
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Cell therapy efforts for treating HIV+ patients are challenged by limited availability of donors with naturally occurring CCR5 mutations conferring resistance. Xu et al. (2019) report a CRISPR-based method for disrupting CCR5 in hematopoietic stem cells prior to transplant, providing a proof of concept for expanding the pool of potential donors.
Assuntos
Infecções por HIV , Leucemia-Linfoma Linfoblástico de Células Precursoras , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Humanos , Receptores CCR5 , Transplante de Células-Tronco , Doadores de TecidosRESUMO
Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem and progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 and AAVS1 loci in human HSPCs and cell lines. As a control, we used AAV6, which effectively edits HSPCs but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc-finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results, therefore, do not support that clade F AAVs can perform high-efficiency genome editing in the absence of a DSB.
Assuntos
Quebras de DNA de Cadeia Dupla , Dependovirus/fisiologia , Edição de Genes/métodos , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/citologia , Células Cultivadas , Dependovirus/classificação , Dependovirus/genética , Marcação de Genes , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Células-Tronco Hematopoéticas/metabolismo , Recombinação Homóloga , Humanos , Células K562 , Receptores CCR5/genética , Montagem de VírusRESUMO
Most studies of HIV latency focus on the peripheral population of resting memory T cells, but the brain also contains a distinct reservoir of HIV-infected cells in microglia, perivascular macrophages, and astrocytes. Studying HIV in the brain has been challenging, since live cells are difficult to recover from autopsy samples and primate models of SIV infection utilize viruses that are more myeloid-tropic than HIV due to the expression of Vpx. Development of a realistic small animal model would greatly advance studies of this important reservoir and permit definitive studies of HIV latency. When radiation or busulfan-conditioned, immune-deficient NSG mice are transplanted with human hematopoietic stem cells, human cells from the bone marrow enter the brain and differentiate to express microglia-specific markers. After infection with replication competent HIV, virus was detected in these bone marrow-derived human microglia. Studies of HIV latency in this model would be greatly enhanced by the development of compounds that can selectively reverse HIV latency in microglial cells. Our studies have identified members of the CoREST repression complex as key regulators of HIV latency in microglia in both rat and human microglial cell lines. The monoamine oxidase (MAO) and potential CoREST inhibitor, phenelzine, which is brain penetrant, was able to stimulate HIV production in human microglial cell lines and human glial cells recovered from the brains of HIV-infected humanized mice. The humanized mice we have developed therefore show great promise as a model system for the development of strategies aimed at defining and reducing the CNS reservoir.
Assuntos
Complexo AIDS Demência/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Microglia/efeitos dos fármacos , Inibidores da Monoaminoxidase/farmacologia , Proteínas do Tecido Nervoso/genética , Fenelzina/farmacologia , Proteínas Repressoras/genética , Complexo AIDS Demência/genética , Complexo AIDS Demência/fisiopatologia , Complexo AIDS Demência/virologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Encéfalo/virologia , Bussulfano/toxicidade , Diferenciação Celular , Proteínas Correpressoras , Modelos Animais de Doenças , Regulação da Expressão Gênica , HIV-1/patogenicidade , HIV-1/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/virologia , Proteínas do Tecido Nervoso/metabolismo , Ratos , Proteínas Repressoras/metabolismo , Transplante Heterólogo , Latência Viral/efeitos dos fármacos , Latência Viral/genética , Irradiação Corporal TotalRESUMO
Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Reparo de DNA por Recombinação/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Advances in gene therapy technologies, particularly in gene editing, are suggesting new avenues for the treatment of human immunodeficiency virus and other infectious diseases. This article outlines recent developments in antiviral gene therapies, including those based on the disruption of entry receptors or that target viral genomes using targeted nucleases, such as the CRISPR/Cas9 system. In addition, new ways to express circulating antiviral factors, such as antibodies, and approaches to harness and engineer the immune system to provide an antiviral effect that is not naturally achieved are described.
Assuntos
Doenças Transmissíveis/terapia , Terapia Genética , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV/genética , Sistemas CRISPR-Cas , Resistência à Doença/genética , Edição de Genes , Expressão Gênica , Marcação de Genes , Engenharia Genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/genética , Genoma Viral , Humanos , Imunomodulação/genética , Receptores de HIV/genética , Receptores de HIV/metabolismo , TransgenesRESUMO
Immune-mediated effector molecules can limit cancer growth, but lack of sustained immune activation in the tumour microenvironment restricts antitumour immunity. New therapeutic approaches that induce a strong and prolonged immune activation would represent a major immunotherapeutic advance. Here we show that the arenaviruses lymphocytic choriomeningitis virus (LCMV) and the clinically used Junin virus vaccine (Candid#1) preferentially replicate in tumour cells in a variety of murine and human cancer models. Viral replication leads to prolonged local immune activation, rapid regression of localized and metastatic cancers, and long-term disease control. Mechanistically, LCMV induces antitumour immunity, which depends on the recruitment of interferon-producing Ly6C+ monocytes and additionally enhances tumour-specific CD8+ T cells. In comparison with other clinically evaluated oncolytic viruses and to PD-1 blockade, LCMV treatment shows promising antitumoural benefits. In conclusion, therapeutically administered arenavirus replicates in cancer cells and induces tumour regression by enhancing local immune responses.
Assuntos
Arenavirus/fisiologia , Vigilância Imunológica , Interferon Tipo I/metabolismo , Neoplasias/imunologia , Neoplasias/virologia , Replicação Viral/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neoplasias/irrigação sanguínea , Vírus Oncolíticos/metabolismo , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).
RESUMO
Despite significant advances in HIV drug treatment regimens, which grant near-normal life expectancies to infected individuals who have good virological control, HIV infection itself remains incurable. In recent years, novel gene- and cell-based therapies have gained increasing attention due to their potential to provide a functional or even sterilizing cure for HIV infection with a one-shot treatment. A functional cure would keep the infection in check and prevent progression to AIDS, while a sterilizing cure would eradicate all HIV viruses from the patient. Genome editing is the most precise form of gene therapy, able to achieve permanent genetic disruption, modification, or insertion at a predesignated genetic locus. The most well-studied candidate for anti-HIV genome editing is CCR5, an essential coreceptor for the majority of HIV strains, and the lack of which confers HIV resistance in naturally occurring homozygous individuals. Genetic disruption of CCR5 to treat HIV has undergone clinical testing, with seven completed or ongoing trials in T cells and hematopoietic stem and progenitor cells, and has shown promising safety and potential efficacy profiles. Here we summarize clinical findings of CCR5 editing for HIV therapy, as well as other genome editing-based approaches under pre-clinical development. The anticipated development of more sophisticated genome editing technologies should continue to benefit HIV cure efforts.
Assuntos
Linfócitos T CD4-Positivos/virologia , Edição de Genes , Terapia Genética/métodos , Infecções por HIV/terapia , Receptores CCR5/genética , Ensaios Clínicos Controlados como Assunto , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Humanos , Terapia de Alvo Molecular/métodos , Edição de RNA , RNA Guia de Cinetoplastídeos/genética , Receptores CCR5/metabolismoRESUMO
HIV/AIDS has long been at the forefront of the development of gene- and cell-based therapies. Although conventional gene therapy approaches typically involve the addition of anti-HIV genes to cells using semirandomly integrating viral vectors, newer genome editing technologies based on engineered nucleases are now allowing more precise genetic manipulations. The possible outcomes of genome editing include gene disruption, which has been most notably applied to the CCR5 coreceptor gene, or the introduction of small mutations or larger whole gene cassette insertions at a targeted locus. Disruption of CCR5 using zinc finger nucleases was the first-in-human application of genome editing and remains the most clinically advanced platform, with 7 completed or ongoing clinical trials in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we review the laboratory and clinical findings of CCR5 editing in T cells and HSPCs for HIV therapy and summarize other promising genome editing approaches for future clinical development. In particular, recent advances in the delivery of genome editing reagents and the demonstration of highly efficient homology-directed editing in both T cells and HSPCs are expected to spur the development of even more sophisticated applications of this technology for HIV therapy.
Assuntos
Síndrome da Imunodeficiência Adquirida , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Mutagênese Insercional , Receptores CCR5 , Linfócitos T/metabolismo , Reparo Gênico Alvo-Dirigido/métodos , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/metabolismo , Síndrome da Imunodeficiência Adquirida/terapia , Desoxirribonucleases/genética , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismoRESUMO
Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.
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Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.
Assuntos
Antígenos CD4/imunologia , Dependovirus/genética , Imunoglobulinas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Internalização do Vírus , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Antagonistas dos Receptores CCR5/imunologia , Antígenos CD4/genética , Feminino , Terapia Genética , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , HIV-2/imunologia , Imunoglobulinas/genética , Macaca mulatta , Masculino , Testes de Neutralização , Receptores CCR5/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaAssuntos
Infecções por HIV , Soronegatividade para HIV , Antirretrovirais/uso terapêutico , Berlim , Boston , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia/complicações , Leucemia/terapia , Linfoma/complicações , Linfoma/terapiaRESUMO
Genetic strategies to block expression of CCR5, the major co-receptor of human immunodeficiency virus type 1 (HIV-1), are being developed as anti-HIV therapies. For example, human hematopoietic stem/precursor cells (HSPC) can be modified by the transient expression of CCR5-targeted zinc finger nucleases (ZFNs) to generate CCR5-negative cells, which could then give rise to HIV-resistant mature CD4(+) T cells following transplantation into patients. The safety and anti-HIV effects of such treatments can be evaluated by transplanting ZFN-treated HSPC into immunodeficient mice, where the extent of human cell engraftment, lineage differentiation and anti-HIV activity arising from the engineered HSPC can be examined. In this way, humanized mice are providing a powerful small animal model for pre-clinical studies of novel anti-HIV therapies.
Assuntos
Terapia Biológica/métodos , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/fisiologia , Células-Tronco Hematopoéticas/virologia , Receptores CCR5/genética , Receptores de HIV/genética , Animais , Terapia Biológica/efeitos adversos , Humanos , Camundongos , Camundongos SCID , Receptores CCR5/deficiência , Receptores de HIV/deficiência , Dedos de ZincoRESUMO
The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.
Assuntos
Endonucleases/genética , Genômica/métodos , Células-Tronco Hematopoéticas/citologia , Receptores CCR5/genética , Dedos de Zinco/genética , Síndrome da Imunodeficiência Adquirida/terapia , Adenoviridae/genética , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Apoptose , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Endonucleases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Deleção de Genes , Marcação de Genes , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Receptores CCR5/metabolismoRESUMO
BST-2/tetherin is an interferon-inducible host restriction factor that blocks the release of newly formed enveloped viruses. It is enriched in lipid raft membrane microdomains, which are also the sites of assembly of several enveloped viruses. Viral anti-tetherin factors, such as the HIV-1 Vpu protein, typically act by removing tetherin from the cell surface. In contrast, the Ebola virus glycoprotein (GP) is unusual in that it blocks tetherin restriction without apparently altering its cell surface localization. We explored the possibility that GP acts to exclude tetherin from the specific sites of virus assembly without overtly removing it from the cell surface and that lipid raft exclusion is the mechanism involved. However, we found that neither GP nor Vpu had any effect on tetherin's distribution within lipid raft domains. Furthermore, GP did not prevent the colocalization of tetherin and budding viral particles. Contrary to previous reports, we also found no evidence that GP is itself a raft protein. Together, our data indicate that the exclusion of tetherin from lipid rafts is not the mechanism used by either HIV-1 Vpu or Ebola virus GP to counteract tetherin restriction.
Assuntos
Antígenos CD/metabolismo , Ebolavirus/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Doença pelo Vírus Ebola/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/genética , Linhagem Celular , Ebolavirus/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Doença pelo Vírus Ebola/virologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/virologia , Ligação Proteica , Transporte Proteico , Proteínas do Envelope Viral/genética , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Candid#1 (Cd1) is an attenuated vaccine strain of Junin virus, the causative agent of Argentine hemorrhagic fever. Although several substitutions are present in Cd1, their importance for attenuation has not been established. We functionally characterized the substitutions present in the Cd1 glycoprotein (GP) and identified F427I in the transmembrane domain of the GP2 subunit as reducing infectivity in a reconstituted viral system. We further showed that this phenotype derives from the destabilization of the GP metastable conformation. Lastly, we identified an increased dependence of Cd1 GP on human transferrin receptor type 1 (hTfR-1) for entry, which may affect the tropism of the attenuated strain in vivo.
Assuntos
Antígenos CD/metabolismo , Vírus Junin/patogenicidade , Glicoproteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Fatores de Virulência/metabolismo , Internalização do Vírus , Substituição de Aminoácidos , Animais , Linhagem Celular , Humanos , Vírus Junin/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Conformação Proteica , Vacinas Atenuadas/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34(+) hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rgamma(null) mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4(+) T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5(-/-) cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5(-/-) HSPCs can populate an infected animal with HIV-1-resistant, CCR5(-/-) progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.