按痘苗病毒优势密码子改造HIV-1 gag基因提高表达水平的研究

为确定痘苗病毒密码子偏向性与基因表达的关系及其在痘苗病毒与宿主细胞相互作用过程中的作用,按痘苗病毒的优势密码子对HIV-1 gag基因进行改造,并对合成基因与野生型HIV-1 gag基因在痘苗病毒载体系统的表达水平进行了研究。结果显示：①各目的基因分别正向插入了痘苗病毒TK区7．5k启动子下游;②免疫荧光检测显示,改造前后的gag基因均能够很好地在痘苗病毒中表达;③Western blot检测显示,在相同感染量时,改造后的gag基因具有更高的表达水平;④流式细胞术检测显示,密码子改造后的gag基因较野生型gag基因表达水平提高约17％。上述结果表明：按照痘苗病毒优势密码子进行外源基因改造,可作为提高外源基因在痘苗病毒中表达的策略,同时提示,密码子偏向性是痘苗病毒与宿主细胞相互作用的重要调控因素。     

HIV-1 B亚型gag基因密码子优化及其免疫原性的研究

从河南HIV-1流行区感染者中克隆HIV-1 B亚型gag基因,通过序列比对获得其一致性共有序列,对该共有序列按照哺乳动物优势密码子的使用原则进行优化,以Western blot方法比较优化前后gag基因体外表达量.发现对gag基因进行密码子优化可显著提高其表达水平.将优化后的mod.gag基因插入重组腺病毒载体,构建了重组病毒rAdV-mod.gag.在BALB/c小鼠体内分别以108PFIJ及108PFU rAdV-mod.gag疫苗单独免疫两次均可产生较高水平的gag特异性细胞免疫反应.由此得出结论,对gag基因的密码子优化是成功的;表达优化后gag基因的重组腺病毒疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.     

Codon engineering for improved antibody expression in mammalian cells

While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.     

Mycobacterial induction of autophagy varies by species and occurs independently of mammalian target of rapamycin inhibition

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to latent infection. Although many factors are involved, the mammalian autophagy pathway is recognized as a determinant that can influence the course of infection. Intervention aimed at utilizing autophagy to clear infection requires an examination of the autophagy and signal transduction induced by mycobacteria under native conditions. With both pathogenic and non-pathogenic mycobacteria, we show that infection correlates with an increase in the mammalian target of rapamycin (mTOR) activity indicating that autophagy induction by mycobacteria occurs in an mTOR-independent manner. Analysis of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guérin (BCG), which respectively induce high and low autophagy responses, indicates that lipid material is capable of inducing both autophagy and mTOR signaling. Although mycobacterial infection potently induces mTOR activity, we confirm that bacterial viability can be reduced by rapamycin treatment. In addition, our work demonstrates that BCG can reduce autophagy responses to M. smegmatis suggesting that specific mechanisms are used by BCG to minimize host cell autophagy. We conclude that autophagy induction and mTOR signaling take place concurrently during mycobacterial infection and that host autophagy responses to any given mycobacterium stem from multiple factors, including the presence of activating macromolecules and inhibitory mechanisms.     

Codon optimization of bacterial luciferase (lux) for expression in mammalian cells

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5×10⁵ vs 2.9×10⁶ relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7×10⁷ RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells     

Ovariectomy-induced bone loss occurs independently of B cells

Estrogen withdrawal is associated with a significant expansion in B cell precursor and mature B cell populations. However, despite significant circumstantial evidence the role of B lineage cells in ovariectomy-induced bone loss in vivo is unclear. In vitro studies have demonstrated that mature B cells have the potential to both positively and negatively impact osteoclastogenesis by virtue of their capacity to secrete pro-osteoclastogenic cytokines including receptor activator of NFkappaB ligand (RANKL), as well as anti-osteoclastogenic cytokines such as osteoprotegerin (OPG) and transforming growth factor beta (TGFbeta). Although several studies have suggested that expansion of the B lineage following ovariectomy may play a key role in the etiology of ovariectomy-induced bone loss, in vivo studies to directly test this notion have yet to be conducted. In this study, we performed ovariectomy on microMT(-/-) mice which are specifically deficient in mature B cells. Analysis of bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) and micro-computed tomography (CT) demonstrate that mature B cell-deficient mice undergo an identical loss of bone mass relative to wild-type (WT) control mice. Our data demonstrate that mature B cells are not central mediators of ovariectomy-induced bone loss in vivo.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330     

Effects of signal peptide exchange on HIV-1 glycoprotein expression and viral infectivity in mammalian cells

In certain cell systems, exchange of the human immunodeficiency virus (HIV) Env signal peptide (SP) sequence with that of heterologous SPs has been shown to increase gp120 transport and secretion. Here we demonstrate that exchange of the HIV-Env-SP with those from erythropoietin or tissue plasminogen activator in the proviral context does not increase wild-type membrane-bound Env expression or incorporation into released virions. In fact, virion infectivity was decreased. These infectivity decreases were largely due to effects on Env transport and/or function and only to a minor extent to cis effects as a result of the sequence exchanges themselves. Thus, in fact, it is not advantageous to employ heterologous SPs to achieve high-level expression of functional cell surface membrane- or virion-associated HIV-Env.     

Cell-dependent replication potentials of HIV-1 gag mutants

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.     

Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells.

Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.     

Endocytosis occurs independently of annexin VI in human A431 cells

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild- type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.     

Bacterial expression, purification, and in vitro N-myristoylation of HIV-1 p17gag.

The coding region of the N-terminal 17-kDa portion of HIV-1 Pr55gag (p17gag) was cloned into the pET-3c expression vector and was used to overexpress HIV-1 p17gag in Escherichia coli. Induction of the transformed bacteria caused the accumulation of a 17-kDa polypeptide in the soluble cell fraction which was released by sonication in hypotonic nondetergent buffer. The 17-kDa polypeptide was purified by ammonium sulfate precipitation and successive chromatography on G-75 Sephadex, DEAE-Sephacel, and S-Sephadex. The final product was purified 12-fold with about a 16% recovery from the original soluble cell lysate and was judged to be 97+% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting with two different antibodies confirmed the identify of the purified 17-kDa polypeptide as authentic p17gag. In the presence of myristoyl-CoA and bovine brain N-myristoyl-transferase, p17gag was quantitatively N-myristoylated in vitro with a pseudo-first-order rate constant of 4.7 +/- 1.0 x 10(-3) min-1, but with only about 3% of the catalytic efficiency of N-myristoylation of a 16-residue peptide homologous to the N-terminus of p17gag. The myristate group in the N-myristoylated p17gag was stable to treatment with detergent and hydroxylamine consistent with a covalent N-acyl-amide linkage. The N-myristoylglycyl linkage was confirmed by partial acid hydrolysis and identification of the p-nitrobenzylazlactone derivative of the resulting N-myristoylglycine by high-performance liquid chromatography.     

Solution properties of murine leukemia virus gag protein: differences from HIV-1 gag

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ～7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.     

Ribosomal RACK1 promotes proliferation of neuroblastoma cells independently of global translation upregulation

Neuroblastoma is the most frequent solid tumor among those diagnosed during infancy and like most tumors, it is characterized by elevated rates of cell proliferation, migration and invasion. RACK1 is among the top 10 genes identified for unfavorable prognosis at 5 years in neuroblastoma cases and its depletion negatively affects proliferation, invasion and migration. Here, we show that the ribosomal localization of RACK1 modulates the proliferation of SH-SY5Y neuroblastoma cells by regulating the expression of cell cycle genes, such as Cyclin D1, D3 and B1 independently of global translation increase. Ribosomal RACK1 is not involved in general protein synthesis, which is instead dependent on total RACK1 and PKC but independent from mTOR. Thus, ribosomal RACK1 may represent a new target to develop more efficient therapies for neuroblastoma treatment.     

Regulated expression of HIV-1 Rev function in mammalian cell lines.

In order to facilitate further investigation of Rev function, we have generated two systems for the inducible expression of Rev in mammalian cell lines (HeLa and U937) using either a tetracycline-regulated promoter or fusion of Rev to a modified form of the hormone binding domain of the estrogen receptor. In the case of the fusion of Rev to the modified hormone binding domain of the estrogen receptor, we demonstrated induction of Rev function in response to tamoxifen administration to levels comparable to that of the unmodified Rev protein. Subsequently, U937 lines were generated that retained the observed pattern of hormone-dependent function of the Rev fusion protein. In the case of the tetracycline-regulated system, cell lines (both HeLa and U937) were generated that displayed tight regulation of Rev. In the case of the HeLa cell lines, they were used for the subsequent generation of stable cell lines expressing either HIV-1 env or chloramphenicol acetyl transferase (CAT) in a Rev-dependent fashion. Using the latter cell lines, we demonstrate the ability to control Rev expression over a broad concentration range and find that, as soon as Rev expression is detectable, induction of Rev-dependent gene expression is also observed.