登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用

根据登革 2型病毒衣壳蛋白C基因和葡萄球菌核酸酶SN基因序列设计引物 ,从构建的原核表达载体pLEX D2C SN中扩增获得编码登革病毒衣壳蛋白和葡萄球菌核酸酶的融合基因D2C SN ,将其插入到真核表达载体pcDNA6 V5 His中 ,筛选获得重组质粒pcDNA D2C SN .电穿孔转染BHK细胞后 ,5mg Lblasticidin压力筛选 ,通过RT PCR、间接免疫荧光和免疫印迹鉴定表达的蛋白 ,体外DNA消化试验检测核酸酶活性 .结果表明 ,融合蛋白D2C SN在BHK细胞中获得了稳定表达 ,表达的融合蛋白能够被抗登革病毒衣壳蛋白的单克隆抗体特异识别 ,并具有良好的核酸酶活性 ,能够对DNA进行切割 .同时 ,BHK细胞中稳定表达的融合蛋白D2C SN能够有效抑制登革病毒的增殖 ,使其感染性降低 10 3 ～ 10 4倍 .这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于人类抗登革病毒感染奠定了基础     

猪瘟病毒衣壳蛋白靶向核酸酶表达系统的建立及鉴定

根据猪瘟病毒衣壳蛋白(C)基因序列设计一对引物,RT-PCR扩增获得编码猪瘟病毒衣壳蛋白的C基因,将其插入到含有葡萄球菌核酸酶(SN)基因的真核表达载体pcDNA-SN中,筛选获得重组质粒pcDNA-C-SN。脂质体转染猪肾细胞(PK-15),并经G418稳定筛选,通过RT-PCR、免疫印迹和间接免疫荧光鉴定表达的融合蛋白,体外DNA消化试验检测核酸酶活性。结果表明融合蛋白C-SN在PK-15细胞中获得了稳定表达,能够被兔抗猪瘟病毒衣壳蛋白多抗所识别,并具有良好的核酸酶活性,能够对DNA进行切割。同时,稳定表达融合蛋白C-SN的PK-15细胞系中能够有效抑制猪瘟野毒的增殖,使其感染性降低102～103倍。这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于抵抗猪瘟病毒感染奠定了基础。     

An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.     

The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry

Dengue virus (DV) is a flavivirus and its urban transmission is maintained largely by its mosquito vectors and vertebrate host, often human. In this study, investigation was carried out on the involvement of domain III of the envelope (E) glycosylated protein of dengue virus serotypes 1 and 2 (DV-1 and DV-2 DIII) in binding to host cell surfaces, thus mediating virus entry. Domain III protein of flavivirus can also serve as an attractive target in inhibiting virus entry. The respective DV DIII proteins were expressed as soluble recombinant fusion proteins before purification through enzymatic cleavage and affinity purification. The purified recombinant DV-1 and DV-2 DIII proteins both demonstrated the ability to inhibit the entry of DV-1 and DV-2 into HepG2 cells and C6/36 mosquito cells. As such, the DV DIII protein is indeed important for the interaction with cellular receptors in both human and mosquito cells. In addition, this protein induced antibodies that completely neutralized homologous dengue serotypes although not with the same efficiency among the heterologous serotypes. This observation may be of importance when formulating a generic vaccine that is effective against all dengue virus serotypes.     

登革2型病毒衣壳蛋白在哺乳动物细胞中的表达与鉴定

从构建的重组质粒pLEX—C中高保真PCR获得编码登革2型病毒43株C基／E／（D2C）DNA片段,通过基因重组的方法将其克隆入真核表达载体pcDNA6／V5-His获得了重组真核表达载体pc／D2C。经电穿孔的方法转染BHK21细胞后,分别通过RT—PCR、免疫荧光和western印迹鉴定表达的蛋白。结果重组蛋白在BHK21细胞中获得表达,表达的蛋自主要存在于胞浆中,并具有较好的抗原性,能够被抗登革病毒衣壳蛋白单克隆抗体特异识别。此研究为深入了解登革病毒衣壳蛋白在病毒复制及组装过程中的生物学功能奠定了基础。     

Differential cholesterol binding by class II fusion proteins determines membrane fusion properties

The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.     

登革2型病毒非结构蛋白NS4B及其突变体的真核表达与鉴定

目的：构建登革2型病毒非结构蛋白NS4B及其突变体Δ2K-NS4B基因的真核载体,并观察二者在哺乳动物细胞内的定位情况。方法：从登革2型病毒43株的全长cDNA克隆载体上扩增获得编码NS4B及缺失2K片段的NS4B突变体Δ2K-NS4B的基因;通过基因重组的方法分别将2段基因克隆入真核表达载体pcDNA6/V5-HisA,获得重组真核表达载体pc/D2-NS4B和pc/D2-Δ2K-NS4B;经脂质体法转染BHK-21细胞后,用RT-PCR、间接免疫荧光和Western印迹鉴定表达的蛋白。结果：重组蛋白D2-NS4B和D2-Δ2K-NS4B可在BHK-21细胞中表达,二者均定位于细胞质中,并具有较好的抗原性,能够被抗登革2型病毒NS4B的多克隆抗体特异识别。结论：重组蛋白D2-NS4B和D2-Δ2K-NS4B在哺乳动物细胞胞质中的正确表达,为深入了解NS4B在登革病毒致病过程中的生物学功能奠定了基础。     

Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1alpha, and MIP-1beta, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-kappaB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.     

Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits virus spread.

The antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly. Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro. A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold. A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins. The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold. An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.     

Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI

Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Clinical studies have shown that significant decrease in the level of lipoproteins is correlated with severe illness in DHF/DSS patients. Available evidence also indicates that lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are able to facilitate cell entry of HCV or other flaviviruses via corresponding lipoprotein receptors. In this study, we found that pre-incubation of DV with human serum leads to an enhanced DV infectivity in various types of cells. Such enhancement could be due to interactions between serum components and DV particles. Through co-immunoprecipitation we revealed that apolipoprotein A-I (ApoA-I), the major protein component in HDL, is associated with DV particles and is able to promote DV infection. Based on that observation, we further found that siRNA knockdown of the scavenger receptor class B type I (SR-BI), the cell receptor of ApoA-I, abolished the activity of ApoA-I in enhancement of DV infection. This suggests that ApoA-I bridges DV particles and cell receptor SR-BI and facilitates entry of DV into cells. FACS analysis of cell surface dengue antigen after virus absorption further confirmed that ApoA-I enhances DV infection via promoting initial attachment of the virus to cells. These findings illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.     

Passive Protection Assay of Monoclonal Antibodies Against Dengue Virus in Suckling Mice

Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are highly infectious diseases caused by dengue virus (DV). Specific monoclonal antibodies (mAbs) against DV are vital for diagnosis, pathological studies, and passive immune therapy. In this study, purified DV serotype 2 (DV2) was used as antigen and BALB/c mice were immunized to induce specific antibodies. We established five hybridoma cell lines, called 78#, 1E7, 7F7, 8F12, and 8H1, respectively, and evaluated them by enzyme-linked immunosorbent assay, indirect immunofluorescence assay, Western blot, plaque reduction neutralization test, and suckling mice protection assay. Lines 78#, 1E7, 7F7, and 8F12 showed a neutralizing effect, and lines 78#, 1E7, 8F12, and 8H1 recognized envelope glycoprotein of DV2. Among them, lines 78# and 8F12 had stronger neutralizing ability in vitro and could protect some suckling mice from virus challenge. Our results demonstrate that immunization with purified virion is efficient for the production of specific neutralizing mAbs against DV2, and these mAbs could be useful tools for studying or treating DV infection.     

Therapeutic Effect of a Gag-Nuclease Fusion Protein against Retroviral Infection In Vivo

Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.     

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

Background

Increased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.

Methods and Findings

Here, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.

Conclusions

These results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus

The pathogenesis of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), both serious complications of dengue virus (DV) infection, remains unclear. In this study, we found that anti-DV NS1 (nonstructural protein 1) polyclonal antibodies cross-reacted with human umbilical vein endothelial cells (HUVECs). We further identified a complex-specific mAb, DB16-1, which could recognize DV NS1 and cross-react with HUVECs and human blood vessels. The target protein of DB16-1 was further purified by immunoaffinity chromatography. LC-MS/MS analysis and co-immunoprecipitation revealed that the target protein of DB16-1 was human LYRIC (lysine-rich CEACAM1 co-isolated). Our newly generated anti-LYRIC mAbs bound to HUVECs in a pattern similar to that of DB16-1. The B-cell epitope of DB16-1 displayed a consensus motif, Lys-X-Trp-Gly (KXWG), which corresponded to amino acid residues 116-119 of DV NS1 and mimicked amino acid residues 334-337 in LYRIC. Moreover, the binding activity of DB16-1 in NS1 of DV-2 and in LYRIC disappeared after the KXWG epitope was deleted in each. In conclusion, DB16-1 targeted the same epitope in DV NS1 and LYRIC protein on human endothelial cells, suggesting that it might play a role in the pathogenesis of DHF/DSS. Future studies on the role of the anti-NS1 antibody in causing vascular permeability will undoubtedly be performed on sera collected from individuals before, during, and after the endothelial cell malfunction phase of a dengue illness.     

Peptide inhibitors of flavivirus entry derived from the E protein stem

Peptides derived from the "stem" of dengue virus (DV) type 2 (DV2) envelope (E) protein inhibit DV2 infectivity, targeting a late-stage fusion intermediate. We show here that stem peptides from all DV serotypes cross-inhibit DV1 to DV4 but that corresponding peptides derived from related flaviviruses do not. This failure to inhibit infection is not due to poor interaction with the E protein but rather to loss of association with the virion membrane. Residues 442 to 444 of the stem are determinants of inhibition; increasing hydrophobicity in this region increases inhibitory strength. These results support a two-step model of how stem-derived peptides inhibit viral entry.     

衣壳蛋白靶向灭活 一种新型抗病毒策略

衣壳蛋白靶向灭活（CTVI）是一种新型抗病毒策略,它是通过将病毒衣壳蛋白与核酸酶（金黄色葡萄球菌和酸梅、核糖核酸酶Barnase、大肠杆菌RNase HI等）的融合蛋白装配到病毒粒子中,使核酸酶接触并降解病毒核酸,从而达到抑制病毒复制的目的。该策略已经在人免疫缺陷病毒、鼠白血病病毒、乙肝病毒、登革病毒等病毒的抗病毒研究中取得良好效果,展示了广阔的应用前景。     

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.