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1.
J Virol ; 96(6): e0192921, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35080425

RESUMO

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.


Assuntos
Linfócitos T CD4-Positivos , Infecções por HIV , HIV-1 , Proteínas do Vírus da Imunodeficiência Humana , Proteínas Virais Reguladoras e Acessórias , Proteínas Viroporinas , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/virologia , Citometria de Fluxo , Infecções por HIV/fisiopatologia , HIV-1/genética , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/isolamento & purificação , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Viroporinas/genética , Proteínas Viroporinas/isolamento & purificação , Proteínas Viroporinas/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/isolamento & purificação , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo
2.
BMC Biotechnol ; 9: 96, 2009 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-19930574

RESUMO

BACKGROUND: In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. RESULTS: The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. CONCLUSION: We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.


Assuntos
Nicotiana/genética , Interferência de RNA , Tombusvirus/genética , Proteínas do Core Viral/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/biossíntese , Cromatografia de Afinidade , Homólogo 5 da Proteína Cromobox , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Espectrometria de Massas , RNA Interferente Pequeno/genética , Nicotiana/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/isolamento & purificação
3.
Transgenic Res ; 18(4): 499-512, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19169897

RESUMO

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.


Assuntos
Anticorpos Anti-HIV/biossíntese , Antígenos HIV/biossíntese , Plantas Geneticamente Modificadas/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/biossíntese , Vetores Genéticos , Anticorpos Anti-HIV/genética , Antígenos HIV/genética , Humanos , Testes de Neutralização , Plantas Geneticamente Modificadas/classificação , Plantas Geneticamente Modificadas/genética , Estabilidade Proteica , Rhizobium/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/isolamento & purificação
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