RESUMO
The extensive diversity of human immunodeficiency virus type 1 (HIV-1) and its capacity to mutate and escape host immune responses are major challenges for AIDS vaccine development. Ancestral sequences, which minimize the genetic distance to circulating strains, provide an opportunity to design immunogens with the potential to elicit broad recognition of HIV epitopes. We developed a phylogenetics-informed algorithm to reconstruct ancestral HIV sequences, called Center of Tree (COT). COT sequences have potentially significant benefits over isolate-based strategies, as they minimize the evolutionary distances to circulating strains. COT sequences are designed to surmount the potential pitfalls stemming from sampling bias with the consensus method and outlier bias with the most-recent-common-ancestor approach. We computationally derived COT sequences from circulating HIV-1 subtype B sequences for the genes encoding the major viral structural protein (Gag) and two regulatory proteins, Tat and Nef. COT genes were synthesized de novo and expressed in mammalian cells, and the proteins were characterized. COT Gag was shown to generate virus-like particles, while COT Tat transactivated gene expression from the HIV-1 long terminal repeat and COT Nef mediated downregulation of cell surface major histocompatibility complex class I. Thus, retrodicted ancestral COT proteins can retain the biological functions of extant HIV-1 proteins. Additionally, COT proteins were immunogenic, as they elicited antigen-specific cytotoxic T-lymphocyte responses in mice. These data support the utility of the COT approach to create novel and biologically active ancestral proteins as a starting point for studies of the structure, function, and biological fitness of highly variable genes, as well as for the rational design of globally relevant vaccine candidates.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Evolução Molecular Direcionada/métodos , HIV-1/genética , HIV-1/imunologia , Vacinas contra a AIDS/genética , Algoritmos , Sequência de Aminoácidos , Animais , Antígenos Virais/classificação , Sequência de Bases , Epitopos/genética , Epitopos/imunologia , Feminino , Produtos do Gene gag/classificação , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene nef/classificação , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene tat/classificação , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Using nucleotide sequences from the first exon of the tat gene of the human immunodeficiency virus 1 (HIV-1), we tested the hypothesis that a Florida dentist (a common source) infected five of his patients in the course of dental procedures against the null hypothesis that the dentist and each individual of the dental group independently acquired the virus within the local community. This novel approach of analyzing the tat gene region was used because it may, in some circumstances, be more informative for phylogenetic epidemiology than the more commonly used C2-V3 envelope gene region. The first exon of the tat gene was polymerase chain reaction (PCR)-amplified and directly sequenced from uncultured peripheral blood mononuclear cells. Patient's sequences were compared with sequences from six HIV-1 infected heterosexual couples unrelated to the dentist or the five patients, but from the same general geographic area. In addition, a sixth infected dental patient, previously inferred to have acquired HIV-1 from a source other than the dentist, was included. Multiple phylogenetic analyses demonstrated that the sequences of the five patients were significantly more closely related to each other than to sequences of the controls. Our results using tat sequences, combined with envelope sequence data, strongly support a common phylogenetic epidemiological relationship among these five patients, and the HIV-1 infected dentist who treated them. Correct recovery of known epidemiological relationships among couples included in the analysis further strengthens this conclusion.
Assuntos
Produtos do Gene tat/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Sequência de Bases , DNA Viral , Estudos Epidemiológicos , Produtos do Gene tat/classificação , Proteína gp120 do Envelope de HIV/classificação , HIV-1/classificação , Humanos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência do Ácido Nucleico , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Jembrana disease virus (JDV) is a bovine lentivirus genetically similar to bovine immunodeficiency virus; it causes an acute and sometimes fatal disease in infected animals. This virus carries a very potent Tat that can strongly activate not only its own long terminal repeat (LTR) but also the human immunodeficiency virus (HIV) LTR. In contrast, HIV Tat cannot reciprocally activate the JDV LTR (H. Chen, G. E. Wilcox, G. Kertayadnya, and C. Wood, J. Virol. 73:658-666, 1999). This indicates that in transactivation JDV Tat may utilize a mechanism similar to but not the same as that of the HIV Tat. To further study the similarity of JDV and HIV tat in transactivation, we first tested the responses of a series of HIV LTR mutants to the JDV Tat. Cross-transactivation of HIV LTR by JDV Tat was impaired by mutations that disrupted the HIV type 1 transactivation response element (TAR) RNA stem-loop structure. Our results demonstrated that JDV Tat, like HIV Tat, transactivated the HIV LTR at least partially in a TAR-dependent manner. However, the sequence in the loop region of TAR was not as critical for the function of JDV Tat as it was for HIV Tat. The competitive inhibition of Tat-induced transactivation by the truncated JDV or HIV Tat, which consisted only of the activation domain, suggested that similar cellular factors were involved in both JDV and HIV Tat-induced transactivation. Based on the one-round transfection assay with HIV tat mutant proviruses, the cotransfected JDV tat plasmid can functionally complement the HIV tat defect. To further characterize the effect of JDV Tat on HIV, a stable chimeric HIV carrying the JDV tat gene was generated. This chimeric HIV replicated in a T-cell line, C8166, and in peripheral blood mononuclear cells, which suggested that JDV Tat can functionally substitute for HIV Tat. Further characterization of this chimeric virus will help to elucidate how JDV Tat functions and to explain the differences between HIV and JDV Tat transactivation.
Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV , HIV-1/genética , Lentivirus Bovinos/genética , Replicação Viral/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Bovinos , Células Cultivadas , Expressão Gênica , Produtos do Gene tat/classificação , Produtos do Gene tat/genética , Teste de Complementação Genética , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/citologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Provírus/genética , RNA Viral , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
This article looks at the history, development, progress and research of the Human Immunodeficiency Virus (HIV) which causes AIDS. The author reports of the ongoing research into a vaccine for HIV, he examines the viral life cycle and indicates the points at which the virus can be attacked, and classifies antiviral strategies
