Quantification of the HIV-1 virus-like particle production process by super-resolution imaging: From VLP budding to nanoparticle analysis

Virus-like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super-resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293-transient gene expression (TGE), High Five-baculovirus expression vector system (BEVS) and Sf9-BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293-TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non-assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM-based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.     

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.     

IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production

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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.     

HIV-1 Gag pl7-p24在痘苗病毒中的表达及性质研究

研究了重组痘苗病毒表达的ＨＩＶ－１核心蛋白（Ｇａｇ）ｐ１７－ｐ２４蛋白的些生物学及免疫学特点。间接免疫荧光、Ｄｏｔ 及ＬＩＳＡ及Ｗｅｓｔｅｒｎ ｂｌｏｔ结果表明,构建的两株重组病毒分别表达了ＨＩＶ－１Ｇａｐ ｐ２４及ｐ１７－ｐ２４融合蛋白。电镜观察证实,Ｇａｇ ｐ２４及ｐ１７－２４重组蛋白均可形成病毒样粒子。重组病毒可诱导小鼠产生抗ＨＩＶ－１Ｇａｐ ｐ２４抗体。重组病毒感染ＢＨＫ２１细胞后,可见由     

HIV-1 Gag多表位嵌合基因的构建及表达蛋白的抗体制备

旨在通过构建Gag的抗原多表位融合基因及在原核系统的高表达,为HIV诊断及可能的疫苗制备提供试验基础。选定HIV-1 Gag基因中3个片段包含较多抗原表位的区域,设计带有酶切位点的引物,用PCR的方法从HIV-1HXB2全基因扩增编码这3个片段的基因序列,通过质粒提取、酶切、测序方法鉴定基因片段的正确性,SDS-PAGE和Western blotting测定融合蛋白的表达,并免疫动物制备相应抗体。结果显示,构建的HIV-1 Gag多表位嵌合基因的原核表达质粒,酶切和测序结果表明基因序列正确,基因全长576bp。在大肠杆菌BL21(DE3)中高效表达的重组蛋白分子量为27kD,以包涵体的形式存在。纯化目的蛋白免疫家兔,制备多克隆抗体IgG。ELISA和免疫荧光方法检测显示制备的多克隆抗体能具有特异性反应。成功构建和高表达了HIV-1 Gag多表位融合蛋白,纯化蛋白制备的抗体与HIV-1Gag有特异性结合。为进一步研究HIV-1奠定了试验基础。     

Covalent bonded Gag multimers in human immunodeficiency virus type-1 particles

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome.     

Flexibility in the P2 domain of the HIV-1 Gag polyprotein

The HIV-1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156-residue polypeptide that contains the C-terminal capsid subdomain (CA(CTD)) through the NC domain of Gag (CA(CTD)-p2-NC, Gag residues 276-431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CA(CTD) and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CA(CTD) and thirteen N-terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an alpha-helical conformation. The presence of a transient coil-to-helix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA-p2 junction. CA(CTD)-p2-NC forms dimers and self-associates with an equilibrium constant (Kd = 1.78 +/- 0.5 microM) similar to that observed for the intact capsid protein (Kd = 2.94 +/- 0.8 microM), suggesting that Gag self-association is not significantly influence by the P2 domain.     

The effect of HIV-1 Gag myristoylation on membrane binding

During the viral life cycle, an HIV protein, Gag, assembles at the host membrane, specifically at lipid raft regions, at very high concentrations leading to viral particle budding. Gag is post-translationally modified with an N-terminal myristate group which is thought to target Gag to lipid rafts thus aiding in assembly. Here we have analyzed the membrane binding of myristoylated HIV-1 Gag and a non-myristoylated form of HIV-1 Gag to various membrane models. After assessing the extent of myristoylation by HPLC and radiometric assays, we compared membrane binding using fluorescence methods. We found that myristoylated Gag shows a greater than twofold increase in binding affinity to model rafts. A structural model to explain these results is presented.     

Structure and Stoichiometry of Template-Directed Recombinant HIV-1 Gag Particles

Size polydispersity of immature human immunodeficiency virus type 1 (HIV-1) particles represents a challenge for traditional methods of biological ultrastructural analysis. An in vitro model for immature HIV-1 particles constructed from recombinant Gag proteins lacking residues 16-99 and the p6 domain assembled around spherical nanoparticles functionalized with DNA. This template-directed assembly approach led to a significant reduction in size polydispersity and revealed previously unknown structural features of immature-like HIV-1 particles. Electron microscopy and image reconstruction of these particles suggest that the Gag shell formed from different protein regions that are connected by a “scar”—an extended defect connecting the edges of two continuous, regularly packed protein layers. Thus, instead of a holey protein array, the experimental model presented here appears to consist of a continuous array of ∼ 5000 proteins enveloping the core, in which regular regions are separated by extended areas of disorder.     

Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag

We introduced mutations into the HIV-1 major homology region (MHR; capsids 153-172) and adjacent C-terminal region to analyze their effects on virus-like particle (VLP) assembly, membrane affinity, and the multimerization of the Gag structural protein. Results indicate that alanine substitutions at K158, F168 or E175 significantly diminished VLP production. All assembly-defective Gag mutants had markedly reduced membrane-binding capacities, but results from a velocity sedimentation analysis suggest that most of the membrane-bound Gag proteins were present, primarily in a higher-order multimerized form. The membrane-binding capacity of the K158A, F168A, and E175A Gag proteins increased sharply upon removal of the MA globular domain. While demonstrating improved multimerization capability, the two MA-deleted versions of F168A and E175A did not show marked improvement in VLP production, presumably due to a defect in association with the raft-like membrane domain. However, K158A bound to detergent-resistant raft-like membrane; this was accompanied by noticeably improved VLP production following MA removal. Our results suggest that the HIV-1 MHR and adjacent downstream region facilitate multimerization and tight Gag packing. Enhanced Gag multimerization may help expose the membrane-binding domain and thus improve Gag membrane binding, thereby promoting Gag multimerization into higher-order assembly products.     

Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 Gag

Suppressor of cytokine signaling 1 (SOCS1) is a recently identified host factor that positively regulates the intracellular trafficking and stability of HIV-1 Gag. We here examine the molecular mechanism by which SOCS1 regulates intercellular Gag trafficking and virus particle production. We find that SOCS1 colocalizes with Gag along the microtubule network and promotes microtubule stability. SOCS1 also increases the amount of Gag associated with microtubules. Both nocodazole treatment and the expression of the microtubule-destabilizing protein, stathmin, inhibit the enhancement of HIV-1 particle production by SOCS1. SOCS1 facilitates Gag ubiquitination and the co-expression of a dominant-negative ubiquitin significantly inhibits the association of Gag with microtubules. We thus propose that the microtubule network plays a role in SOCS1-mediated HIV-1 Gag transport and virus particle formation.

Structured summary

MINT-7014185: Gag (uniprotkb:P05888) and SOCS1 (uniprotkb:O15524) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7014239: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with RelA (uniprotkb:Q04206), RBX1 (uniprotkb:P62877), SOCS1 (uniprotkb:O15524), elongin B (uniprotkb:Q15369) and elongin C (uniprotkb:Q15370) by pull-down (MI:0096)MINT-7014046: gag (uniprotkb:P05888), SOCS1 (uniprotkb:O15524) and tubulin alpha (uniprotkb:Q13748) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7014269: tubulin alpha (uniprotkb:Q13748) physically interacts (MI:0218) with Gag (uniprotkb:P05888) by anti tag coimmunoprecipitation (MI:0007)MINT-7014036: tubulin alpha (uniprotkb:Q13748) and SOCS1 (uniprotkb:O15524) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7014201: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with RBX1 (uniprotkb:P62877), SOCS1 (uniprotkb:O15524), elongin B (uniprotkb:Q15369) and elongin C (uniprotkb:Q15370) by pull-down (MI:0096)MINT-7014257: Gag (uniprotkb:P05888) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7014221: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with Gag (uniprotkb:P05888), elongin C (uniprotkb:Q15370), elongin B (uniprotkb:Q15369), SOCS1 (uniprotkb:O15524) and RBX1 (uniprotkb:P62877) by pull-down (MI:0096)     

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.     

HIV-1 Env三聚体抗原改造研究进展

HIV-1疫苗研发是当前艾滋病研究的一大热点,其病毒表面包膜糖蛋白Env三聚体介导病毒与细胞融合,是HIV-1疫苗研究的重要靶点。因此,设计并在体外表达类天然Env三聚体对HIV-1疫苗的研发具有重要的意义。近年来,Env三聚体研究取得了显著的进展。SOSIP、NFL2P、UFO等抗原改造方法实现了类天然Env三聚体的体外表达,逐步解决了改造抗原产量低、结构不稳定等问题,且表达的Env三聚体抗原能在动物免疫中诱导机体产生较高的中和抗体水平。Env三聚体改造方法促进了HIV-1疫苗的研究。文中综述了SOSIP、NFL2P、UFO三种HIV-1 Env三聚体抗原改造方法,对比各个改造方法优缺点,并结合自身工作提出相关建议,为后续HIV-1抗原的相关设计提供指导。     

Conformation of the HIV-1 Gag protein in solution

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。     

Visualisation of phenotypically mixed HIV-1 and HTLV-I virus particles by electron microscopy

Virions produced after HIV-1 infection of HTLV-I transformed cells have an expanded tropism that has been attributed to the presence of HTLV-I glycoproteins in the envelope. This report now directly identifies these phenotypically mixed virions by immunogold labelling electron microscopy. Furthermore we estimate there are 2% of these in cell-free supernatant, which represents up to 1 x 10(7) particles/ml from an in vitro infection. HTLV-1 envelope labelling was localised to a single region, suggesting a defined event in packaging of foreign envelope proteins into HIV-1 virus particles.     

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。