HIV-1 Tat蛋白神经毒作用的研究进展

HIV-1感染者常并发神经系统损伤,患者主要表现为运动功能障碍,认知和行为受损等。研究表明,HIV-1多种蛋白参与上述病理过程,其中Tat蛋白发挥了极为重要的作用。外周Tat蛋白可透过血脑屏障进入脑组织,与脑内感染细胞分泌或释放至胞外的Tat共同作用于NMDAR、mGluR1、多巴胺转运蛋白等,损伤神经系统。该文主要对Tat蛋白的神经毒作用作一综述。     

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.     

Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients

Recent in vitro studies have suggested that autophagy may play a role in both HIV-1 replication and disease progression. In this study we investigated whether autophagy protects the small proportion of HIV-1 infected individuals who remain clinically stable for years in the absence of antiretroviral therapy, these named long-term nonprogressors (LTNP) and elite controllers (EC). We found that peripheral blood mononuclear cells (PBMC) of the HIV-1 controllers present a significantly higher amount of autophagic vesicles associated with an increased expression of autophagic markers with respect to normal progressors. Of note, ex vivo treatment of PBMC from the HIV-1 controllers with the MTOR inhibitor rapamycin results in a more efficient autophagic response, leading to a reduced viral production. These data lead us to propose that autophagy contributes to limiting viral pathogenesis in HIV-1 controllers by targeting viral components for degradation.     

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还     

Analysis of HIV-1 Tat effects inXenopus laevis embryos

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in theXenopus embryo. We adopted theXenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function.Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that ofXbra andgsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a loss of function mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.     

JDV Tat反式激活LTR与HIV-1 Tat采用类似的细胞因子

为分析JDV Tat在反式激活JDV及HIV-1 LTR过程中是否采用与HIV-1 Tat类似的细胞因子,本文构建了包含完整激活域的jTat70和hTat47,同时构建了cyclin T1和CDK9真核表达及反义转译质粒.过量表达hTat47和jTat70对hTat反式激活HIV-1 LTR,jTat反式激活JDV和HIV-1 LTR均有明显的抑制作用推测jTat和hTat的反式激活作用可能涉及类似的细胞因子.通过cyclin T1和CDK9的反义转译质粒对jTat反式激活的抑制作用证实这两种细胞因子参与了jTat对JDV和HIV-1LTR的反式激活.     

HIV－1载体中存在着提高转录和翻译基因的元件

目的研究HIV-1载体中的一些元件如Rev和Tat蛋白对其骨架的转录及外源基因表达水平的影响。方法将HIV-1表达GFP载体（FUGW）单独或分别与Rev蛋白表达质粒（pLP2）、Tat蛋白表达质粒（pcDNA3.1-Tat）,及表达Rev和Tat蛋白的质粒（△8．9）等摩尔共转染人293T细胞后,经实时定量RT-PCR、FACS、荧光显微镜镜检等方法检测,比较其表达量。结果Rev与RRE结合后,载体骨架及外源基因的转录是单独转染FUGW时的3倍,Tat与TAR结合后,则提高其骨架及外源基因的转录近4倍,而Rev和Tat蛋白的协同作用,其转录本则可提高至6倍。FACS和荧光显微镜镜检也显示GFP蛋白表达量明显提高。F-TPO载体（HIV-1载体乳腺特异表达促血小板生成素）与△8．9在小鼠乳腺上皮细胞HC-11共转染和表达,则TPO蛋白的表达量接近pcDNA3．1-TPO载体的8倍。结论HIV-1载体中存在着提高转录和翻译基因的元件,可提高其骨架的转录和外源基因的表达,且该现象并不依赖于细胞类型和外源基因的种类。     

Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat

Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter.
In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

JDV Tat反式激活LTR与HIV-1 Tat采用类似的细胞因子

为分析JDV Tat在反式激活JDV及HIV-1 LTR过程中是否采用与H1V-1 Tat类似的细胞因子,本文构建了包含完整激活域的jTat70和hTat47,同时构建了cyclin T1和CDK9真核表达及反义转译质粒。过量表达hTat47和jTat70对hTat反式激活HIV-1 LTR,jTat反式激活JDV和HIV-1 LTR均有明显的抑制作用推测jTat和hTat的反式激活作用可能涉及类似的细胞因子。通过cyclin T1和CDK9的反义转译质粒对jTat反式激活的抑制作用证实这两种细胞因子参与了jTat对JDV和HIV-1 LTR的反式激活。     

DDX3 RNA helicase is required for HIV-1 Tat function

Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function.     

Endocytosis and autophagy: Shared machinery for degradation

Two key questions in the autophagy field are the mechanisms that underlie the signals for autophagy initiation and the source of membrane for expansion of the nascent membrane, the phagophore. In this review, we discuss recent findings highlighting the role of the classical endosomal pathway, from plasma membrane to lysosome, in the formation and expansion of the phagophore and subsequent degradation of the autophagosome contents. We also highlight the striking conservation of regulatory factors between the two pathways, including those regulating membrane budding and fusion, and the role of the lysosome in sensing the nutrient status of the cell, regulating mTORC1 activity, and ultimately the initiation of autophagy. Editor's suggested further reading in BioEssays The evolution of dynamin to regulate clathrin‐mediated endocytosis Abstract     

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.