Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.     

Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.     

禽流感病毒NS1蛋白对细胞的影响

NS1蛋白为流感病毒非结构蛋白,只在病毒侵入宿主细胞后产生.目前NS1蛋白对细胞整体水平上的作用仍不清楚,为了解NS1蛋白在病毒感染细胞中的作用,构建了重组质粒pCMV-myc-NS1并将其转染A549细胞,利用双向电泳技术检测了受NS1蛋白调控的宿主蛋白,以期从蛋白质组水平上研究禽流感病毒与宿主细胞间的相互作用.同时,还检测了转染NS1对细胞增殖和细胞周期的影响.结果显示,NS1在细胞中的表达,能够明显引起宿主细胞代谢的变化,并通过阻滞细胞周期的正常进行而减缓细胞的增殖.     

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

ＨＣＶ ＮＳ５Ｂ基因片段克隆入ＢＡＣ－ＴＯ－ＢＡＣ＾ＴＭ重组杆状病毒表达系统的ｐＦＡＳＴＨＴｃ载体质粒,转化ＤＨ１０ＢＡＣ＾ＴＭ感受态细菌获得重组的Ｂａｃｍｉｄ质粒,将重组Ｂａｃｍｉｄ质粒转染Ｓｆ细胞,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明,所表达蛋白为ＨＣＶ ＮＳ５Ｂ蛋白,具有多聚酶活性。     

Inhibition of dengue virus by targeting viral NS4B protein

We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC(50)], 1 to 4 μM; and 50% cytotoxic concentration [CC(50)], >40 μM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or nonflaviviruses (Western equine encephalomyelitis virus, Chikungunya virus, and vesicular stomatitis virus). A mode-of-action study suggested that the compound inhibits viral RNA synthesis. Replicons resistant to the inhibitor were selected in cell culture. Sequencing of the resistant replicons revealed two mutations (P104L and A119T) in the viral NS4B protein. Genetic analysis, using DENV-2 replicon and recombinant viruses, demonstrated that each of the two NS4B mutations alone confers partial resistance and double mutations confer additive resistance to the inhibitor in mammalian cells. In addition, we found that a replication defect caused by a lethal NS4B mutation could be partially rescued through trans complementation. The ability to complement NS4B in trans affected drug sensitivity when a single cell was coinfected with drug-sensitive and drug-resistant viruses. Mechanistically, NS4B was previously shown to interact with the viral NS3 helicase domain; one of the two NS4B mutations recovered in our resistance analysis-P104L-abolished the NS3-NS4B interaction (I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006). Collectively, the results suggest that the identified inhibitor targets the DENV NS4B protein, leading to a defect in viral RNA synthesis.     

登革2型病毒非结构蛋白NS4B的原核表达、纯化及多克隆抗体制备

目的：原核表达、纯化登革2型病毒非结构蛋白NS4B,并制备其多克隆抗体,以研究其结构与功能。方法：扩增编码登革2型病毒NS4B的24-238位氨基酸残基的基因序列,并将其克隆到原核表达载体pGEX-4T-1,转化大肠杆菌BL21（DE3）,IPTG诱导表达;采用蛋白浸提方法从SDS-PAGE胶中回收融合蛋白;用纯化后的融合蛋白免疫BALB／c鼠制备多克隆抗体,采用间接免疫荧光法检测抗体效价。结果：原核表达了NS4B-GST融合蛋白,并获得了其多克隆抗体,抗体效价为1：800。结论：登革2型病毒NS4B的24-238位氨基酸残基可诱导小鼠产生具有较高效价和特异性的多克隆抗体,这为研究NS4B的结构与功能奠定了基础。     

Hepatitis C virus NS5B protein delays s phase progression in human hepatocyte-derived cells by relocalizing cyclin-dependent kinase 2-interacting protein (CINP)

Cell cycle dysregulation is a critical event in virus infection-associated tumorigenesis. Previous studies have suggested that hepatitis C virus NS5B modulates cell cycle progression in addition to participating in RNA synthesis as an RNA-dependent RNA polymerase. However, the molecular mechanisms have thus far remained unclear. In this study, a HepG2 Tet-On NS5B stable cell line was generated to confirm the effect of NS5B on the cell cycle. To better understand the role of NS5B in cell cycle regulation, yeast two-hybrid assays were performed using a human liver cDNA library. The cyclin-dependent kinase 2-interacting protein (CINP) was identified. The interaction between NS5B and CINP was further demonstrated by in vivo and in vitro assays, and their association was found to be indispensable for S phase delay and cell proliferation suppression. Further experiments indicated that NS5B relocalized CINP from the nucleus to the cytoplasm. Directly knocking down CINP by specific siRNA resulted in a significant alteration in the DNA damage response and expression of cell cycle checkpoint proteins, including an increase in p21 and a decrease in phosphorylated Retinoblastoma and Chk1. Similar results were observed in cells expressing NS5B, and the effects were partially reversed upon ectopic overexpression of CINP. These studies suggest that the DNA damage response might be exploited by NS5B to hinder cell cycle progression. Taken together, our data demonstrate that NS5B delays cells in S phase through interaction with CINP and relocalization of the protein from the nucleus to the cytoplasm. Such effects might contribute to hepatitis C virus persistence and pathogenesis.     

Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.     

In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3.

We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.     

Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.     

Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus

Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENV) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.     

The Hepatitis C Virus Non-structural NS5A Protein Impairs Both the Innate and Adaptive Hepatic Immune Response in Vivo

The role of hepatitis C virus (HCV) protein non-structural (NS) 5A in HCV-associated pathogenesis is still enigmatic. To investigate the in vivo role of NS5A for viral persistence and virus-associated pathogenesis a transgenic (Tg) mouse model was established. Mice with liver-targeted NS5A transgene expression were generated using the albumin promoter. Alterations in the hepatic immune response were determined by Western blot, infection by lymphocytic choriomeningitis virus (LCMV), and using transient NS3/4A Tg mice generated by hydrodynamic injection. Cytotoxic T lymphocyte (CTL) activity was investigated by the Cr-release assay. The stable NS5A Tg mice did not reveal signs of spontaneous liver disease. The intrahepatic immunity was disrupted in the NS5A Tg mice as determined by clearance of LCMV infection or transiently NS3/4A Tg hepatocytes in vivo. This impaired immunity was explained by a reduced induction of interferon β, 2′,5′-OAS, and PKR after LCMV infection and an impairment of the CTL-mediated elimination of NS3-expressing hepatocytes. In conclusion, these data indicate that in the present transgenic mouse model, NS5A does not cause spontaneous liver disease. However, we discovered that NS5A could impair both the innate and the adaptive immune response to promote chronic HCV infection.Chronic hepatitis C virus (HCV)⁴ infection is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). The HCV genome is a single-stranded positive-sense RNA molecule of ∼9600 bp (1). The viral RNA codes for one large polyprotein of ∼3100 amino acids that is post-translationally processed by cellular and viral proteases, leading to the structural proteins core, E1 and E2, the p7 protein, and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The mature NS5A protein is generated by the action of the NS3/NS4A serine protease. NS5A is a phosphoprotein that exists in a basal or in a hyperphosphorylated state (p56 and p58) (3). Through an amphipathic α-helix, NS5A is associated with the cytoplasmic face of the ER (4) and is an integral part of the replication complex (5). Mutations in NS5A affect the rate of HCV replication suggesting a role of NS5A in modulating viral expression and replication (6). Moreover, NS5A is able to interfere with a variety of cellular proteins. Some of these interaction partners, such as Grb2, PI3K, p53, or Raf-1 are important key players in host cell signal transduction, enabling NS5A to deregulate important cellular check points (7–10). Recent reports even suggest that NS5A may deregulate cell cycle progression by modulating the expression of cell cycle regulatory genes (11). In light of these observations and that it has been suggested to transform murine fibroblasts (12), it is speculated that NS5A could represent an important factor for the development of HCV-associated HCC (13).Infection of transgenic mice expressing the complete HCV polyprotein with lymphocytic choriomeningitis virus (LCMV) showed a reduced IFN response and a delayed viral elimination (14). Cell culture-based experiments have shown that NS5A interacts directly with the interferon-dependent induced protein kinase R (PKR), a key player in the cellular antiviral response and that this interaction results in an inhibition of PKR function (15). Therefore, a role of NS5A for the establishment of a chronic HCV infection by inhibiting the innate immunity is conceivable.To enable in vivo studies of NS5A-specific effects transgenic mice were generated with a liver-specific expression of NS5A. We used these mice to show that NS5A affects both the innate and the adaptive hepatic immunity.     

Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.

The proteolytic cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions of hepatitis C virus (HCV) polyprotein are effected by the virus-encoded serine protease contained within NS3. Using transient expression in HeLa cells of cDNA fragments that code for regions of the HCV polyprotein, we studied whether viral functions other than NS3 are required for proteolytic processing at these sites. We found that, in addition to NS3, a C-terminal 33-amino-acid sequence of the NS4A protein is required for cleavage at the NS3-NS4A and NS4B-NS5A sites and that it accelerates the rate of cleavage at the NS5A-NS5B junction. In addition, we show that NS4A can activate the NS3 protease when supplied in trans. Our data suggest that HCV NS4A may be the functional analog of flavivirus NS2B and pestivirus p10 proteins.     

Human parvovirus B19 nonstructural protein (NS1) induces cell cycle arrest at G(1) phase

Human parvovirus B19 infects predominantly erythroid precursor cells, leading to inhibition of erythropoiesis. This erythroid cell damage is mediated by the viral nonstructural protein 1 (NS1) through an apoptotic mechanism. We previously demonstrated that B19 virus infection induces G(2) arrest in erythroid UT7/Epo-S1 cells; however, the role of NS1 in regulating cell cycle arrest is unknown. In this report, by using paclitaxel, a mitotic inhibitor, we show that B19 virus infection induces not only G(2) arrest but also G(1) arrest. Interestingly, UV-irradiated B19 virus, which has inactivated the expression of NS1, still harbors the ability to induce G(2) arrest but not G(1) arrest. Furthermore, treatment with caffeine, a G(2) checkpoint inhibitor, abrogated the B19 virus-induced G(2) arrest despite expression of NS1. These results suggest that the B19 virus-induced G(2) arrest is not mediated by NS1 expression. We also found that NS1-transfected UT7/Epo-S1 and 293T cells induced cell cycle arrest at the G(1) phase. These results indicate that NS1 expression plays a critical role in G(1) arrest induced by B19 virus. Furthermore, NS1 expression significantly increased p21/WAF1 expression, a cyclin-dependent kinase inhibitor that induces G(1) arrest. Thus, G(1) arrest mediated by NS1 may be a prerequisite for the apoptotic damage of erythroid progenitor cells upon B19 virus infection.     

Hepatitis C virus proteins activate NRF2/ARE pathway by distinct ROS-dependent and independent mechanisms in HUH7 cells

Hepatitis C virus (HCV) is a highly pathogenic human virus associated with liver fibrosis, steatosis, and cancer. In infected cells HCV induces oxidative stress. Here, we show that HCV proteins core, E1, E2, NS4B, and NS5A activate antioxidant defense Nrf2/ARE pathway via several independent mechanisms. This was demonstrated by the analysis of transient co-expression in Huh7 cells of HCV proteins and luciferase reporters. Expression, controlled by the promoters of stress-response genes or their minimal Nrf2-responsive elements, was studied using luminescence assay, RT-qPCR and/or Western-blot analysis. All five proteins induced Nrf2 activation by protein kinase C in response to accumulation of reactive oxygen species (ROS). In addition, expression of core, E1, E2, NS4B, and NS5A proteins resulted in the activation of Nrf2 in a ROS-independent manner. The effect of core and NS5A was mediated through casein kinase 2 and phosphoinositide-3 kinase, whereas those of NS4B, E1, and E2, were not mediated by either PKC, CK2, PI3K, p38, or ERK. Altogether, on the earliest stage of expression HCV proteins induced a strong up-regulation of the antioxidant defense system. These events may underlie the harmful effects of HCV-induced oxidative stress during acute stage of hepatitis C.     

丙型肝炎病毒基因组部份NS5区蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组ＮＳ５区Ａ段和部份Ｂ段蛋白。ＮＳ５Ａ蛋白溶于水,可经过ＧＳＴ亲和层析柱纯化;ＮＳ５Ｂ蛋白不溶于水,经ＰＢＳ－Ｔｒｉｔｏｎ Ｘ－１００洗涤,尿素溶解后,通过离子交换柱纯化。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｍｂｌｏｔ分析,ＮＳ５Ａ蛋白除在５７ｋＤ左右有条带外,还有不同程度的降解产物;ＮＳ５Ｂ蛋白主要在５８ｋＤ左右有条带出现。为查明表达蛋白抗体在病人血清中的分布,取９     

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。