A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.     

And the noise played on: stochastic gene expression and HIV-1 infection

Stochastic gene expression has been implicated in a variety of cellular processes, including cell differentiation and disease. In this issue of Cell, take an integrated computational-experimental approach to study the Tat transactivation feedback loop of HIV-1. They show that fluctuations in a key regulator, Tat, in an isogenic population of infected cells result in two distinct expression states corresponding to latent and productive HIV-1 infection. These findings demonstrate the importance of stochastic gene expression in molecular "decision-making."     

一个新的HIV-1治疗靶——Tat转录激活蛋白(英)

Tat是HIV-1病毒进行转录和复制的一个十分重要的蛋白质,同时,Tat也与HIV-1感染引起的严重病理学程度密切相关.Tat 的生物学性质和功能决定了其是一个理想的开发抗AIDS疫苗和药物的靶蛋白.基于Tat自身及其作用的TAR RNA,可以设计Tat疫苗、细胞外结合Tat的拮抗剂、抗Tat的反义核酸、抗TAR的反义核酸、抗Tat的细胞内抗体和细胞内Tat协同因子的抑制剂等.传统的抗病毒药物及蛋白酶抑制剂与新的细胞内和细胞外Tat拮抗剂联合使用,多靶点地抑制HIV-1的复制将是一个有效的抗AIDS的治疗方案.这一治疗方案能够防止HIV病毒耐药株的产生,减少单一作用靶点药物的用药剂量和降低相应的毒性,最终治愈AIDS相关的病理学变化.     

一个新的HIV-1治疗靶--Tat转录激活蛋白

Tat是HIV-1病毒进行转录和复制的一个十分重要的蛋白质,同时,Tat也与HIV-1感染引起的严重病理学程度密切相关.Tat的生物学性质和功能决定了其是一个理想的开发抗AIDS疫苗和药物的靶蛋白.基于Tat自身及其作用的TARRNA,可以设计Tat疫苗、细胞外结合Tat的拮抗剂、抗Tat的反义核酸、抗TAR的反义核酸、抗Tat的细胞内抗体和细胞内Tat协同因子的抑制剂等.传统的抗病毒药物及蛋白酶抑制剂与新的细胞内和细胞外Tat拮抗剂联合使用,多靶点地抑制HIV-1的复制将是一个有效的抗AIDS的治疗方案.这一治疗方案能够防止HIV病毒耐药株的产生,减少单一作用靶点药物的用药剂量和降低相应的毒性,最终治愈AIDS相关的病理学变化.     

A review of HIV-1 Tat protein biological effects

The authors have reviewed some biological properties of HIV-1 Tat protein, and have also reported some personal data. This viral regulatory protein is endowed with multifunctional activities, acting as an endogenous factor in the infected cells and exogenously, on those uninfected. In particular, Tat-induced proliferation and differentiation of HIV target cells which promotes viral infection, is discussed in this review. However, exogenous Tat protein can sometimes also produce, directly or indirectly, damaging effects in different organs and host systems, such as myocardium, kidney, liver and central nervous system (CNS). For example some data also demonstrate an increase in the apoptotic index induced by Tat at various levels, including the immune system. The effective role of HIV-1 Tat protein in promoting viral replication and its high immunogenicity suggest useful employment of this protein for therapeutic or preventive vaccine preparations.     

Furin cleavage of the HIV-1 Tat protein

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.     

The 5' UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed -1 ribosomal frameshift that generates HIV-1 enzymes

Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed -1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5' untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5' UTR of HIV-1 mRNA occupies the 5' end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5' UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5' end of the messenger. HIV-1 mRNA 5' UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5' UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5' UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.     

Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1

Specific spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specific associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodeficiency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains.     

Endocytosis and targeting of exogenous HIV-1 Tat protein.

The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.     

Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression

The HIV-1 Tat protein is a potent trans-activator essential for virus replication. We reported previously that HIV-1 Tat peptides containing residues 37-48 (mainly region II), a possible activating region, and residues 49-57 (region III), a nuclear targeting and putative nucleic acid binding region, possess minimal but distinct trans-activator activity. The presence of residues 58-72 (region IV) greatly enhances trans-activation. We postulate that Tat mutant peptides with an inactive region II and a functional region III can behave as dominant negative mutants. We synthesized minimal domain peptides containing single amino substitutions for amino acid residues within region II that are conserved among different HIV isolates. We identify four amino acid residues whose substitution within Tat minimal domain peptides leads to defects in transactivation. Some of these mutants are trans-dominant in several peptide backbones, since they strongly inhibit trans-activation by wild-type Tat protein added to cells or expressed from microinjected plasmid. Significantly, trans-activation of integrated HIV-LTRCAT is blocked by some trans-dominant mutant peptides. These results suggest an attractive approach for the development of an AIDS therapy.     

Epithelin/granulin growth factors: extracellular cofactors for HIV-1 and HIV-2 Tat proteins

Epithelin/granulin growth factor is synthesized as a 593 amino acid precursor protein that contains 7.5 imperfectly conserved repeats of approximately 57 amino acids. Processed epithelin/granulin peptides have been isolated from vertebrate/invertebrate species and are growth factors implicated in epithelial and haemic cell function. Here they are identified as Human Immunodeficiency Virus (HIV) Tat binding proteins using the yeast two-hybrid assay. Intracellularly in yeast, mutation of selected cysteines in an epithelin/granulin dimeric repeat caused loss of binding to Tat exon 1. In vitro binding of HIV-1 and HIV-2 Tat to epithelin/granulin dimeric and monomeric repeats was also observed by GST-glutathione bead "pulldown" assays. Because Tat is actively secreted from HIV-infected cells and has been shown to serve as a mitogenic factor for angiogenesis and for Kaposi-like cells, our observations suggest that epithelin/granulin growth factors may function as biologically important extracellular Tat co-factors.