被膜蛋白糖基化在HIV感染中的作用

在HIV感染过程中,病毒被膜蛋白糖基化起着重要作用。它使病毒粒子具有高度糖基化的表面,帮助HIV逃避人体免疫细胞识别和攻击。在病毒入侵时,被膜糖蛋白与宿主细胞表面的受体结合,并进行一系列构象变化,使病毒粒子顺利地与宿主细胞膜融合。介绍近年来对被膜蛋白糖基化过程与HIV成熟、感染和逃避免疫应答等方面分子水平作用机理的深入了解,这些作用机理将会有助于艾滋病疫苗的研制和以“糖链为靶”药物的开发。     

Comparison of Canavanyl-Envelope Proteins to Normal Envelope Proteins

Electrophoretic analysis showed arginine- and canavanine-containing envelope proteins to be qualitatively the same. Quantitative differences may be due to rapid degradation of some canavanine-containing envelope proteins.     

Redirecting Lentiviral Vectors Pseudotyped with Sindbis Virus-Derived Envelope Proteins to DC-SIGN by Modification of N-Linked Glycans of Envelope Proteins

Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.The ultimate goal of gene therapy is cell- and tissue-specific targeted delivery of therapeutic genes. A targeted system increases the therapeutic effects of transgenes at the site of action while reducing adverse effects in surrounding cells and tissues that commonly occur through nonspecific modes of gene delivery (5-8). Gene therapy vectors that can home to specific cells and tissues after intravenous administration, also known as targeting vectors, are ideal for targeted delivery (62). In the past, many attempts have been made to develop targeting viral vectors by using adenovirus, adeno-associated virus, oncoretrovirus, lentivirus, measles virus, and alphavirus (70, 89).To create targeting viral vectors, the natural tropisms of the viruses must first be eliminated and new binding specificities conferred (89). The binding of envelope viruses, such as oncoretrovirus, lentivirus, measles virus, and alphavirus, is mediated by envelope proteins. To redirect the tropisms of these viruses, the original receptor-binding regions of their envelope proteins must be eliminated. We have developed targeting oncoretroviral and lentiviral vectors by pseudotyping them with modified Sindbis virus envelope proteins and by mutating the receptor-binding regions of the envelope proteins, thereby reducing the nonspecific transduction of untargeted cells (61, 63-66). The mutated regions of the envelope protein originally interact directly with other receptors, including heparan sulfate, laminin receptor, and/or unknown molecules (10, 46, 67, 90). These mutations reduced the nonspecific transduction of the liver and spleen when the vectors were administered intravenously (66). By conjugating the virus with targeting ligands, including antibodies and peptides, the virus can transduce specific cells and tissues both in vitro and in vivo (53, 61, 63-66, 71, 72). These results demonstrated that we can eliminate the natural tropism of the Sindbis virus envelope protein while maintaining its fusion activity.However, the N-glycans of the envelope proteins are still intact and possibly interact with cell surface lectins. DC-SIGN is the best-known cell surface lectin expressed on dendritic cells, certain macrophages, and activated B cells (27, 29, 30).Structural and biochemical studies show flexible modes of DC-SIGN binding to cognate saccharides. The trimannose core unit of high-mannose N-glycans is the primary binding site for DC-SIGN (23), while nonreducing alpha1-2-linked terminal mannose moieties contribute to the high avidity seen when DC-SIGN binds the Man8 or Man9 structures common to many viral envelope glycoproteins (22). DC-SIGN traps a wide variety of viruses and viral vectors (HIV [29, 30], simian immunodeficiency virus [50], human T-cell leukemia virus type 1 [12], measles virus [17, 18], dengue virus [86], feline corona virus [77], herpes simplex virus type 1 [16], human cytomegalovirus [36], human herpesvirus type 8 [76], Ebola virus [1], West Nile virus [15], influenza virus [91], Marburg virus [57], and severe acute respiratory syndrome virus [93]) by binding to the N-glycans of the viruses and viral vectors. Binding of DC-SIGN with virus and viral vectors results in enhanced infection and/or transduction of DC-SIGN-positive cells (cis infection/transduction) and/or neighboring cells (trans infection/transduction).If any targeting vector can be trapped by DC-SIGN, it is necessary to eliminate its binding to DC-SIGN to increase the targeting specificity of the virus in vivo (28, 49, 73). In addition to enhanced infection/transduction, binding to DC-SIGN causes signaling that can activate DC-SIGN-expressing antigen-presenting cells (32, 38). Activation of antigen-presenting cells can lead to adverse effects, including systemic inflammation and immune reactions to viral vectors and their transgene products (7, 8, 32, 59, 88). Therefore, investigation of the interactions between viral vectors and DC-SIGN, identification of N-glycans that mediate binding to DC-SIGN, and elimination of interactions with DC-SIGN are important aspects of reducing adverse effects of vector administration and prolonging transgene expression.The envelope protein of our targeting lentiviral vectors, the Sindbis virus envelope protein, contains four N-linked glycans (9, 48). Sindbis virus can replicate in insect and mammalian cells, which have different types of enzymes to process N-glycans (3). Therefore, the structures of N-glycans differ between the virus produced in insect cells and that produced in mammalian cells (40, 58). The N-glycans of the virus produced in insect cells have either the high-mannose or the paucimannosidic structure. Paucimannosidic structure N-glycans, as well as high-mannose structure N-glycans, have terminal mannose residues, and all N-glycans produced in insect cells are predicted to be able to bind DC-SIGN (Fig. ​(Fig.11 a) (39, 47). On the other hand, two N-glycans of the virus produced in mammalian cells have the high-mannose structure, while two others have the complex structure (40, 58). The two complex structure N-glycans have been shown to be exposed on the surface of the envelope protein, while the two high-mannose structure N-glycans are buried within the center of the trimer of the envelope proteins (74, 94). Therefore, the virus produced in insect cells can access DC-SIGN as its receptor while the virus produced in mammalian cells cannot (47). Because our targeting vectors are produced in mammalian cells, they should not bind DC-SIGN efficiently. However, one group demonstrated that lentiviral vectors pseudotyped with a modified Sindbis virus envelope protein bind to DC-SIGN and target DC-SIGN-positive cells (92), in contrast to the results seen with replication-competent Sindbis virus. Both Sindbis virus and the pseudotyped lentiviral vectors were produced in mammalian cells; Sindbis virus was produced in baby hamster kidney (BHK) cells, chicken embryonic fibroblasts, and hamster fibroblast cells; and the pseudotyped vector was produced in human embryonic kidney fibroblast (293T) cells (69). Because it is known that the N-glycans of the HIV envelope protein produced in lymphocytes have structures different from those produced in macrophages, the different producer cells may account for the differences between the N-glycan structures of the virus and Sindbis virus envelope-pseudotyped lentivectors (54, 55). It is also known that the N-glycan structure of dengue virus can be altered by the presence of viral capsid (35). Thus, the capsid of Sindbis virus and HIV could also affect the structures of the N-glycans of envelope proteins differently.Open in a separate windowFIG. 1.(a) N-glycan structures and processing pathway. All N-glycans are first produced as the high-mannose structure in both mammalian cells and insect cells. In mammalian cells, certain N-glycans are further processed to the complex structure. In insect cells, certain N-glycans are further processed to the paucimannosidic structure. DMNJ inhibits mannosidase I, which is necessary for the formation of the complex structure; thus, all N-glycans have the high-mannose structure when generated in the presence of DMNJ. One representative structure of each N-glycan is shown. Man, mannose; GlcNAc, N-acetylglucosamine; SA, sialic acid; Gal, galactose. (b) Schematic representation of chimeric Sindbis virus envelope proteins. The Sindbis virus envelope protein is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate the E3, E2, 6K, and E1 proteins. E1 and E2 are incorporated into the viral envelope, and E3 and 6K are leader sequences for E2 and E1, respectively. The N-linked glycosylation sites of the envelope proteins are shown. 2.2 is a modified Sindbis virus envelope protein in which the IgG-binding domain of protein A (ZZ) was inserted into the E2 region at aa 70. 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) at aa 70 of the E2 protein. 2.2 ΔE2-196N does not have the N-glycan at E2 aa 196, 2.2 ΔE1-139N does not have the N-glycan at E1 aa 139, and 2.2 ΔE2-196N E1-139N does not have the N-glycans at either E2 aa 196 or E1 aa 139.In this study, we investigated whether our targeting vector binds DC-SIGN. We found that DC-SIGN does not mediate the transduction of our targeting vectors efficiently. The vectors can be redirected to DC-SIGN by modifying the structures of the N-glycans of the envelope proteins by using the mannosidase I inhibitor deoxymannojirimycin (DMNJ) (25, 47, 51).     

白斑综合症病毒囊膜蛋白VP19、VP28的融合表达及中和抗体的制备

根据GenBank上WSSV囊膜蛋白基因vp19和vp28的序列,设计并合成两对引物,PCR扩增得到vp19和vp28两基因,大小分别为370bp和630bp.通过EcoRI位点连接两基因,再按正确的阅读框插入表达载体pET-22b(+)中,构建出重组表达载体pET-vp(19+28)并转化大肠杆菌BL21(DE3).基因工程菌株35℃IPTG诱导,表达产物经SDS-PAGE检测显示有与预期大小41kDa相吻合的融合蛋白带.用Ni2+-柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清,进行螯虾活体中和病毒实验,结果表明抗血清对WSSV的中和效率达到了100%.     

白斑综合症病毒囊膜蛋白VP19、VP28的融合表达及中和抗体的制备

根据GenBank上WSSV囊膜蛋白基因vp19和vp28的序列,设计并合成两对引物,PCR扩增得到vp19和vp28两基因,大小分别为370bp和630bp。通过EcoRI位点连接两基因,再按正确的阅读框插入表达载体pET-22b( )中,构建出重组表达载体pET-vp(19 28)并转化大肠杆菌BL21(DE3)。基因工程菌株35℃IPTG诱导,表达产物经SDS-PAGE检测显示有与预期大小41kDa相吻合的融合蛋白带。用Ni^2 -柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清,进行螯虾活体中和病毒实验,结果表明抗血清对WSSV的中和效率达到了100％。     

Human Immunodeficiency Virus (HIV) Envelope Binds to CXCR4 Independently of CD4, and Binding Can Be Enhanced by Interaction with Soluble CD4 or by HIV Envelope Deglycosylation

Chemokine receptor CXCR4 (also known as LESTR and fusin) has been shown to function as a coreceptor for T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have developed a binding assay to show that HIV envelope (Env) can interact with CXCR4 independently of CD4 but that this binding is markedly enhanced by the previous interaction of Env with soluble CD4. We also show that nonglycosylated HIV-1_SF-2 gp120 or sodium metaperiodate-treated oligomeric gp160 from HIV-1₄₅₁ bound much more readily to CXCR4 than their counterparts with intact carbohydrate residues did.In the recent past, several members of the family of chemokine receptors have been identified as cofactors for human immunodeficiency virus type 1 (HIV-1) entry (1, 6, 8, 10). Specifically, CCR5 (as well as CCR3 and CCR2b in some instances) has been shown to mediate entry of viruses characterized as macrophage tropic or dual tropic (1, 5–8), while CXCR4 has been shown to mediate entry of T-cell-tropic or dual-tropic strains (7, 10). While several ligands have been found for CCR5, CXC chemokine stromal derivative factor (SDF1) remains the only known ligand for CXCR4 (4, 24). Coimmunoprecipitation studies have shown that HIV-1 Env from T-cell-tropic strains forms a complex with CD4 and CXCR4 (18), but the nature of the binding events leading to the formation of this complex and the possibility of a direct interaction between HIV Env and CXCR4 remained speculative. Data from Hesselgesser et al. (15) have more recently shown that gp120 from the T-cell-tropic strains IIIB or BRU was able to compete with SDF1 for binding to CXCR4 in hNT cells (a neuronal CD4-negative cell line), indicating the possibility of a direct interaction between CXCR4 and gp120, but no information was presented on the relevance of the interaction with CD4. Other data have shown that gp120 from macrophage-tropic strains of HIV might be able to bind directly to CCR5 and that the affinity for binding between the two molecules can be increased significantly by the presence of soluble CD4 (sCD4) (34), although this effect could not be reproduced by a different group (32).We have performed the following studies to determine if HIV Env binds to CXCR4 independently of CD4 and, if so, what would be the effect of previous binding of HIV Env to sCD4.

CD4-independent binding of HIV Env to CXCR4.

The phenotypes of the T-cell lines CEM-SS and Jurkat 25 (J25) were evaluated with respect to surface expression of both CD4 and CXCR4. J25 clone 22F6 cells (3, 21) were grown in complete medium (RPMI 1640, 2% penicillin-streptomycin, 2% l-glutamine; BioWhittaker, Walkersville, Md.) containing heat-inactivated 10% fetal calf serum at 37°C in a 5% CO₂ atmosphere. CEM-SS is a T-cell line that was obtained from the AIDS Research and Reference Reagent Program and maintained in complete medium. CEM-SS cells were derived from a human lymphoblastoid tumor (22, 23). Commercial monoclonal antibody (MAb) to CD4 (mouse immunoglobulin G2a [IgG2a], clone S3.5), fluorescein isothiocyanate (FITC) labeled, and the necessary isotypic controls were obtained from Caltag Laboratories (San Francisco, Calif.). Mouse MAb 12G5 against CXCR4 was raised in BALB/c mice and has been described previously (9). Goat anti-mouse IgG–FITC was purchased from Becton Dickinson (San Jose, Calif.). Flow cytometric analysis was performed on a Becton Dickinson FACScan cytometer equipped with a 15-mW argon laser emitting at 488 nm. Dead cells were detected on the basis of their scatter and eliminated from the analysis. Live cells (10,000) were analyzed for each marker. CXCR4 surface expression was determined by washing the cells taken in logarithmic growth phase with phosphate-buffered saline (PBS) containing 1% horse serum and incubating them with 10 μl of 12G5 antibody/100 μl (0.16 mg/ml) at 4°C for 30 min. The cells were then washed again in PBS, and a secondary goat anti-mouse IgG–FITC (Becton Dickinson) was incubated with the cells for another 30 min at 4°C. Finally, the cells were washed with PBS and fixed with 2% paraformaldehyde. As a control, equal amounts of mouse IgG2a (the same isotype as 12G5) were used. Both cell lines expressed significant levels of CXCR4 on their surfaces (Fig. ​(Fig.1),1), but only CEM-SS had measurable levels of surface CD4. This characteristic of the phenotype of J25 cells, with respect to CD4 expression, has been reported before (3). To assess binding of HIV Env to CXCR4, the following binding assay was developed. Oligomeric gp160 (ogp160) was purified from cell cultures (obtained from T. C. Van Cott (Henry M. Jackson Foundation, Rockville, Md.) infected with HIV₄₅₁ (17). The cells were washed once with PBS and then incubated with ogp160 for 1 h at 37°C in RPMI medium. The cells were washed again in PBS and incubated with 10 μg of human MAb 1331A [IgG3(λ)]/ml, which is specific for the C terminus of gp120 (i.e., amino acids 510 to 516 of HIV_LAI), or with a human MAb against p24 (MAb 71-31) as a control (12) for 30 min at 4°C. The secondary antibody was a goat anti-human IgG phycoerythrin labeled (Caltag). The cells were fixed in 2% paraformaldehyde, and the fluorescence intensity was determined by flow cytometry. Background was obtained by adding MAb 1331 and goat anti-human IgG, phycoerythrin labeled, to the cells in the absence of ogp160. The results of the binding assay with ogp160 from HIV₄₅₁ and both cell lines are shown in Fig. ​Fig.2A.2A. By using the high-affinity human MAb 1331A against the C-terminal region of gp120, our assay was able to detect significant binding of the ogp160 molecule to the surfaces of both cell lines even at concentrations of only 88 nM. The very high relative affinity of MAb 1331A for the gp120 molecule appears to be critical to demonstrate this interaction, as other antibodies with lower relative affinities for gp120 were incapable of detecting this low-level binding (data not shown). The binding of ogp160 to the CD4-expressing CEM-SS cells was several orders of magnitude higher than that to the J25 cells. To prove the specificity of the binding assay for CXCR4, a synthetic form of SDF1 was produced and tested for its ability to block infection by the HIV-1 strain NL_4-3 in HeLa CD4-positive long terminal repeat (LTR)-LacZ cells. These data have been published elsewhere (2). SDF1 synthesis and composition have been described previously (24). Exposure of J25 cells to SDF1 was shown to produce a dose-dependent blockage of the binding of ogp160 to the surfaces of the J25 cells (Fig. ​(Fig.2B),2B), indicating the specific nature of the assay. Open in a separate windowFIG. 1Phenotype analysis of CEM-SS and J25 cell lines. Thin solid line, background; thick solid line, CD4; dashed line, CXCR4.Open in a separate windowFIG. 2(A) Binding of ogp160 from HIV₄₅₁ to the surfaces of CEM-SS or J25 cells. Fluorescence intensity is expressed on a logarithmic scale on the x axis, with each line representing one-half log. Concentrations of ogp160 are shown at the right of each graph. The experiments were done in duplicate to ensure consistency of results. (B) Effect of RANTES (250 nM) or increasing amounts of SDF1 (up to 250 nM) on binding of ogp160 (355 nM) to J25 cells. The results are expressed as mean channel fluorescence. Experiments were repeated twice to ensure consistency of results.To further test the fact that HIV Env binding to CXCR4 could occur independently of CD4, and to evaluate the effect of prior binding of Env to sCD4, the following experiments were performed. We preexposed CEM-SS as well as J25 cells to either the anti-CD4 antibody Leu3a (Becton Dickinson), which blocks the CD4 binding domain of HIV Env, or OKT4 (Ortho Diagnostics, Costa Mesa, Calif.), which does not block binding of HIV Env to CD4. The cells were then tested for their ability to bind ogp160 to their surfaces. As shown in Fig. ​Fig.3,3, OKT4 had no significant effect on the binding of ogp160 to either CEM-SS or J25 cells while Leu3a readily inhibited binding of ogp160 to CEM-SS cells but had no such effect on J25 cells. Furthermore, when ogp160 was allowed to react in advance with recombinant sCD4 produced in CHO cells (Intracel, Issaquah, Wash.) for 30 min at 4°C at a concentration of 1 μg/ml, we were able to show a clear decrease in the surface binding of ogp160 to CEM-SS cells while the opposite, an obvious enhancement in surface binding, was demonstrated for J25 cells (Fig. ​(Fig.3).3). Open in a separate windowFIG. 3Binding of ogp160 to CEM-SS or J25 cells after exposure of the cells to the anti-CD4 antibodies Leu3a (thin solid lines), OKT4 (dotted lines), or a combination of ogp160 with sCD4 (dashed lines). The shaded areas represent background. The thick solid lines represent binding in the absence of antibodies or sCD4. The experiments were performed in quadruplicate with similar results. Mean channel fluorescence is represented on the x axis.Taken together, these data indicate that HIV Env can bind to CXCR4 independently of CD4. On the other hand, prior interaction of HIV Env with CD4 results in a clear increase in the binding of HIV Env to CXCR4.

Relevance of the glycosylation state of HIV Env in binding to CXCR4.

The binding of HIV Env to CD4 is dependent on the appropriate conformation of the Env molecule (27), which can be significantly altered by changes in its carbohydrate content. We next tested the hypothesis that alterations in the carbohydrate moieties of Env would affect its binding to CXCR4. To do so, we used the gp120 molecule from HIV_SF2, produced in CHO cells, and its counterpart, nonglycosylated HIV_SF2 Env 2-3, produced in yeast strain 2150, and tested both in the binding assay with CEM-SS or J25 cells. HIV_SF-2 gp120 and its nonglycosylated counterpart, Env 2-3, were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Kathelyn Steimer, Chiron Corp. (13, 14, 19, 26, 29–31). The results are shown in Fig. ​Fig.4.4. As expected, nonglycosylated HIV_SF2 Env 2-3 bound to the surfaces of the CEM-SS cells to a lesser extent than did HIV_SF2 gp120. On the other hand, and unexpectedly, nonglycosylated HIV_SF2 Env 2-3 bound much more readily to the surfaces of the J25 cells than its glycosylated counterpart, HIV_SF-2 gp120, even when used at equal molar concentrations. To determine whether these findings could be generalized to other Env molecules that lacked intact carbohydrate molecules, we treated ogp160 with sodium metaperiodate. ogp160 from HIV₄₅₁ at 1.25 μg/ml was treated with sodium metaperiodate (Sigma, St. Louis, Mo.) in acetate buffer for 2 h at 4°C in the dark (33). The cells to be tested had been treated previously with 1% glycine (Sigma) for 30 min at 37°C. Such treatment results in the oxidation and cleavage of the carbohydrate hydroxyl groups without affecting the structure of the polypeptide chains (33). Nonspecific binding by the resulting aldehyde groups was prevented by blocking the target cells beforehand with 1% glycine. The results are shown in Fig. ​Fig.4.4. Sodium metaperiodate treatment of ogp160 resulted in a marked inhibition of the binding of ogp160 to the surfaces of the CEM-SS cells. In contrast, sodium metaperiodate treatment of ogp160 resulted in a very clear increase in the binding of HIV Env to the surfaces of the J25 cells. The preexposure of CEM-SS cells to SDF1 did not significantly affect the binding of ogp160 or sodium metaperiodate-treated ogp160. On the other hand, preexposure of J25 cells to 250 nM SDF1 resulted in a marked decrease in binding of both ogp160 and sodium metaperiodate-treated ogp160. These data indicate the specificity of the interaction of the deglycosylated form of ogp160 with CXCR4. The results of these experiments suggest that the alteration in the carbohydrate content of the HIV Env molecules resulted in a better exposure of the epitopes involved in gp120 binding to CXCR4. Open in a separate windowFIG. 4Binding of HIV_SF-2 gp120 or the nonglycosylated form, HIV_SF-2 Env 2-3 (Non-glyc SF-2 gp120), to CEM-SS or J25 cells. The concentration was 355 nM for both. The binding of ogp160 and sodium metaperiodate-treated ogp160 (De-glyc ogp160), each at a concentration of 355 nM, to CEM-SS or J25 cells is also shown. The two right-hand bars in each graph show results for cells preexposed to SDF1 at 150 nM. The results are expressed as mean channel fluorescence. The experiments were performed in duplicate with similar results.The understanding of the underlying mechanisms by which HIV Env, CD4, and the newly discovered HIV coreceptors interact to mediate viral entry remains a very significant issue. The way that HIV Env and CD4 interact is well established (28), and some information exists about the interaction between HIV Env, CCR5, and CD4 (34). In this paper we have shown that HIV Env is able to interact in a CD4-independent manner with CXCR4. Still, the extent of such interaction was clearly lower than that of the sCD4-HIV Env complex and CXCR4. This effect of sCD4 seems to be consistent with the observation that the complexing of this molecule with HIV Env from the strains JRFL or BAL resulted in a significant increase in the affinity of HIV Env for CCR5 (34). We speculate that this interaction between sCD4 and HIV Env results in a conformational change that exposes the binding epitopes in HIV Env relevant for binding to CXCR4, as it does with other gp120 epitopes (16). A different scenario would involve a change in both molecules, resulting in a newly formed common binding epitope. This second alternative seems less likely given our data showing CD4-independent binding of HIV Env to CXCR4, as well as previous data showing the existence of HIV strains capable of CD4-independent entry into target cells (9, 15).The gp120 molecule from HIV contains 20 potential N-linked glycosylation sites, with N-linked glycans representing at least 50% of the molecular mass. Their role in CD4 binding has been studied extensively, although some of the results remain somewhat controversial. Most of the available data seem to indicate that complete lack of glycosylation completely (20), or at least partially (25), inhibits HIV Env binding to CD4. Also, enzymatic manipulation of the carbohydrate residues results in a significant decrease but not in complete abrogation of the binding of HIV Env to CD4 (11, 20, 25). It was therefore somewhat unexpected to find that the nonglycosylated form, as well as the sodium metaperiodate-treated form, of HIV Env was able to bind in such an enhanced way to CXCR4. This would appear to reinforce the concept of the existence of a binding epitope for CXCR4 within HIV Env which is different from the one for CD4. It also suggests that the changes occurring as a consequence of the manipulation of the carbohydrate residues likely result in a better exposure of the CXCR4 binding epitope(s) within the HIV Env molecule.In summary, we have shown that HIV Env can interact with CXCR4 in a CD4-independent manner. We have also shown how the interaction of CD4 with HIV Env results in a significant increase in the binding of the latter to CXCR4 and how the alterations in the carbohydrate composition of the HIV Env molecule affect its binding to CXCR4. The complete definition of these interactions may result in novel approaches to protect against cell infection by HIV.     

ZAP融合蛋白抑制HIV病毒模型的构建及功能分析

ZAP是一种抗病毒因子,能够特异性结合病毒RNA并招募细胞中的RNA酶降解所结合的靶RNA,从而抑制某些病毒的复制,如鼠白血病病毒(MLV)、辛德比斯病毒(SIN).ZAP对HIV病毒抑制作用并不明显.Tat和Rev是HIV编码的两种可以特异性结合HIVRNA的蛋白质,将它们与ZAP构建成融合蛋白,使得融合蛋白通过Tat或Rev结合HIVRNA并通过ZAP降解HIVRNA,从而抑制HIV假病毒载体携带基因的表达.这一结果为抑制HIV病毒提供了一个新思路,也支持了ZAP招募mRNA降解机器降解靶RNA的模型.     

Contributions of Herpes Simplex Virus 1 Envelope Proteins to Entry by Endocytosis

Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the required HSV glycoproteins, gB, gD, and gH-gL, may be sufficient for entry regardless of entry route taken. This may be distinct from entry mechanisms employed by other human herpesviruses.     

白斑综合征病毒囊膜蛋白在对虾免疫保护应用中的研究进展

白斑综合征自上世纪90年代初在水产养殖业中爆发以来,其病原体白斑综合征病毒的研究一直在深入开展,特别是WSSV结构蛋白的功能学研究尤为广泛,其主要方向集中在病毒囊膜蛋白对虾体的免疫保护上,并取得了显著的保护效果。从利用病毒囊膜蛋白作为亚单位疫苗免疫虾体、利用囊膜蛋白对应抗体保护虾体、构建囊膜蛋白基因核酸疫苗和利用RNAi干扰技术保护虾体等四个方面,对当前WSSV囊膜蛋白在对虾免疫保护中的应用进行了概述,并对其应用前景作一展望,旨在为及早开发出有效防治白斑综合征疾病的技术途径提供借鉴参考。     

Enhanced Immunogenicity of HIV-1 Envelope gp140 Proteins Fused to APRIL

Current HIV-1 vaccines based on the HIV-1 envelope glycoprotein spike (Env), the only relevant target for broadly neutralizing antibodies, are unable to induce protective immunity. Env immunogenicity can be enhanced by fusion to costimulatory molecules involved in B cell activation, such as APRIL and CD40L. Here, we found that Env-APRIL signaled through the two receptors, BCMA and TACI. In rabbits, Env-APRIL induced significantly higher antibody responses against Env compared to unconjugated Env, while the antibody responses against the APRIL component were negligible. To extend this finding, we tested Env-APRIL in mice and found minimal antibody responses against APRIL. Furthermore, Env-CD40L did not induce significant anti-CD40L responses. Thus, in contrast to the 4-helix cytokines IL-21 and GM-CSF, the TNF-superfamily members CD40L and APRIL induced negligible autoantibodies. This study confirms and extends previous work and shows that fusion of Env-based immunogens to APRIL can improve Env immunogenicity and might help in designing HIV vaccines that induce protective humoral immunity.     

重组包涵体蛋白质的折叠复性

综述了减少包涵体形成、包涵体分离和溶解以及包涵体折叠复性的策略及其最新进展 .详细讨论了包涵体蛋白质折叠复性的基本原则、包涵体折叠复性促进剂和包涵体折叠复性方法     

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.     

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background

Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.

Methodology/Principal Findings

Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.

Conclusions/Significance

Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.     

Two-Dimensional Polyacrylamide Gel Electrophoresis of Envelope Proteins of Escherichia coli

A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight.     

利用植物系统表达重组蛋白的的研究进展

植物是可以生产不同生物药剂品的成本低廉的生物反应器,综述了稳定转化系统、瞬时表达系统以及叶绿体基因组转化方法,这三种不同的植物表达系统的特点和研究现状,并对其存在的问题及未来的前景进行了分析。     

杆状病毒出芽病毒粒子膜融合蛋白的研究进展

杆状病毒是一类感染节肢动物的病原微生物,其基因组为双链环状DNA,大小为80～180kb.