Effect of tunicamycin on the biogenesis of hepatitis C virus glycoproteins

Hepatitis C virus (HCV) infects humans, with a prevalence around 3% of population, causing acute and chronic hepatitis and hepatocellular carcinoma. We studied the effect of inhibition of glycosylation on the assembly of the HCV particle. HCV possesses two envelope glycoproteins E1 and E2 that are highly modified by N-glycans. These glycan residues are crucial for viral entry and maturation of the progeny. Here, we examined the influence of inhibition of N-glycosylation on expression of E1 and E2. Since the propagation of HCV in cell culture is limited, we used a recombinant baculovirus producing viral-like particles in insect cells. Our data showed that blocking of N-glycan transfer to the nascent polypeptide chain with the antibiotic tunicamycin resulted in the loss of E1 and E2. We also found that a dose of tunicamycin that did not influence the cell viability significantly reduced the E2 level in infected cells. The results indicate that blocking of glycosylation at an early step efficiently reduces the assembly of HCV virions. Thus, we suggest that derivatives of tunicamycin that preferentially block glycosylation of viral proteins may become potential therapeutic agents against HCV.     

Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.     

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.     

Expression and functional characterization of classical swine fever virus E(rns) protein

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with unusual RNase activity. Recently, E(rns) was found to have a new function of counteracting the beta-interferon (IFN-beta) induction pathway. In this study, wildtype ErnsSM and two mutated E(rns) proteins ErnsH297k and ErnsH346k were expressed in insect cells and purified for RNase activity and function analysis. RNase activity assay in vitro demonstrated that only wildtype E(rns) protein had RNase activity. However, both wildtype ErnsSM and the two mutated E(rns)ErnsH297k and ErnsH346k as exogenous proteins had a block effect on Newcastle disease virus (NDV)-mediated IFN-beta promoter induction.     

Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns)

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.     

Role of N-glycan trimming in the folding and secretion of the pestivirus protein E(rns)

N-glycosylation inhibitors have antiviral effect against bovine viral diarrhea virus. This effect is associated with inhibition of the productive folding pathway of E1 and E2 envelope glycoproteins. E(rns) is the third pestivirus envelope protein, essential for virus infectivity. The protein is heavily glycosylated, its N-linked glycans counting for half of the apparent molecular weight. In this report we address the importance of N-glycan trimming in the biosynthesis, folding, and intracellular trafficking of E(rns). We show that E(rns) folding is not assisted by calnexin and calreticulin; however, the protein strongly interacts with BiP. Consistently, the N-glycan trimming is not a prerequisite for either the acquirement of the E(rns) native conformation, as it retains the RNase enzymatic activity in the presence of alpha-glucosidase inhibitors, or for dimerization. However, E(rns) secretion into the medium is severely impaired suggesting a role for N-glycosylation in the transport of the glycoprotein through the secretory pathway.     

Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines

An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein E(rns) of classical swine fever virus (CSFV) was used to rescue CSFV E(rns) deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of E(rns) from this cell line were indistinguishable from those of CSFV E(rns). Two E(rns) deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of E(rns) (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of E(rns) antibodies, due to a deletion of 66 amino acids in E(rns). The E(rns) deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by E(rns)-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that E(rns) from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) E(rns) in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.     

猪瘟病毒糖蛋白E^rns中和表位的鉴定和比较

猪瘟病毒 (CSFV)囊膜结构糖蛋白Erns(gp4 8)是诱导机体产生中和抗体及激发保护性免疫应答的第二抗原蛋白。E2和Erns与细胞表面受体的相互作用介导CSFV感染细胞的过程。Erns具有RNA酶活性 ,影响病毒自身复制并涉及对病毒的中和效应。采用抗CSFValfortT櫣ｂｉｎｇｅｎ毒株Ｅｒｎｓ糖蛋白的 1B5 ,b4_2 2和 2 4 16单克隆中和抗体 ,筛选噬菌体展示的 12肽随机肽库 ,进行Erns中和表位的鉴定和比较 ,获得分别针对 1B5、b4_2 2和 2 4 16单克隆抗体的 3个主要中和表 (拟 )位基序WxNxxP、DKNR (Q)G和A(T)CxYxKN ,分别定位于Erns的 35 1位～ 35 6位或 348位～ 35 0位、384位～ 386及 32 2位～ 32 3位、380位～ 386位氨基酸区域。分析表 (拟 )位基序与单克隆抗体的免疫反应性差异。b4_2 2和 2 4 16单克隆抗体识别基序存在共有序列KN ,识别Erns中的相似抗原区 ,但其侧翼序列及免疫印迹、免疫荧光抗体抑制试验结果均存在显著差异     

Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E(rns) (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553-9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser(476) and an HS-dependent recombinant virus (S-RT virus) with Arg(476) were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E(rns), E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg(476) was mutated back to Ser(476). Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E(rns) S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.     

Synthesis and antiviral activity of a novel glycosyl sulfoxide against classical swine fever virus

A novel compound—2″,3″,4″,6″-tetra-O-acetyl-β-d-galactopyranosyl-(1→4)-2′,3′,6′-tri-O-acetyl-1-thio-β-d-glucopyranosyl-(5-nitro-2-pyridyl) sulfoxide—designated GP6 was synthesized and assayed for cytotoxicity and in vitro antiviral properties against classical swine fever virus (CSFV) in this study. We showed that the examined compound effectively arrested CSFV growth in swine kidney cells (SK6) at a 50% inhibitory concentration (IC₅₀) of 5 ± 0.12 μg/ml without significant toxicity for mammalian cells. Moreover, GP6 reduced the viral E2 and E^rns glycoproteins expression in a dose-dependent manner. We have excluded the possibility that the inhibitor acts at the replication step of virus life cycle as assessed by monitoring of RNA level in cells and culture medium of SK6 cells after single round of infection as a function of GP6 treatment. Using recombinant E^rns and E2 proteins of classical swine fever virus produced in baculovirus expression system we have demonstrated that GP6 did not influence glycoprotein production and maturation in insect cells. In contrast to mammalian glycosylation pathway, insect cells support only the ER-dependent early steps of this process. Therefore, we concluded that the late steps of glycosylation process are probably the main targets of GP6. Due to the observed antiviral effect accompanied by low cytotoxicity, this inhibitor represents potential candidate for the development of antiviral agents for anti-flavivirus therapy. Further experiments are needed for investigating whether this compound can be used as a safe antiviral agent against other viruses from unrelated groups.     

猪瘟病毒石门强毒株和兔化弱毒疫苗株E2蛋白糖基化位点差异分析

猪瘟病毒强毒株和兔化弱毒疫苗株E2糖蛋白分别含有5个和6个潜在的糖基化位点,其中986N是兔化弱毒疫苗株所特有的。为了分析二者糖基化位点差异及其影响,将去掉信号肽和跨膜区的猪瘟病毒石门强毒株(Shi-men)和兔化弱毒疫苗株(HCLV)E2基因置于蜂素信号肽序列下游,使其在Sf9细胞内表达重组Shimen-E2和HCLV-E2蛋白。结果显示,重组E2蛋白以二聚体的形式分泌表达于细胞培养液中,但二者分子量存在差异。用endo H和PNGase F对纯化后的重组E2蛋白进行去糖基化处理后,二者分子量大小变成一致,证实石门强毒株和兔化弱毒株E2蛋白分子量大小的差异可能是由于糖基化程度的差异所致。对986N糖基化位点进行定点突变后发现,突变后的Shimen-E2与野生型HCLV-E2分子量大小一致,而突变后的HCLV-E2与野生型Shimen-E2分子量大小一致,表明Shimen-E2和HCLV-E2分子量大小的差异的确是由于986N糖基化位点的差异引起的。     

A novel role of classical swine fever virus E(rns) glycoprotein in counteracting the newcastle disease virus (NDV)-mediated IFN-beta Induction

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that E(rns) counteracts Newcastle disease virus (NDV)-mediated induction of IFN-beta. For this purpose, E(rns) fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, E(rns)-EGFP was found to prevent IFN-beta promoter-driven luciferase expression and block the induction of IFN-beta promoter mediated by NDV in a dosedependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-beta mRNA in NDV-infected PK15 cells was observed in the presence of E(rns)-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, E(rns)-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-beta. These evidences establish a novel function for CSFV E(rns) glycoprotein in counteraction of the IFN-beta induction pathway.     

Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

Three proteins, namely, the core protein C and envelope glycoproteins E1 and E2, are main structural proteins forming a hepatitis C virus (HCV) virion. The virus structure and assembly and the role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Highly efficient heterologous expression systems make it possible to obtain self-assembled, nonreplicating, genome-lacking particles that are morphologically similar to intact virions. Using recombinant baculoviruses expressing the HCV structural protein genes in insect cells, the individual HCV structural proteins were expressed to 25–35% of the total cell protein, and the CE1 and E1E2 heterodimers and HCV-like particles were obtained. It was demonstrated that the recombinant C, E1, and E2 proteins underwent posttranslational modification, the glycoproteins formed a noncovalent heterodimer, and HCV- like particles were located in endoplasmic reticulum membranes of infected cells. The formation of E1E2 dimers and HCV-like particles was used to study the effect of E1 glycosylation on the expression and processing of the coat proteins.     

Hepatitis C virus: The role of N-glycosylation sites of viral genotype 1b proteins for formation of viral particles in insect and mammalian cells

Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1], [2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants.     

Identification of antigenic regions of the Erns protein for pig antibodies elicited during classical swine fever virus infection

The structural glycoprotein E(rns) of classical swine fever virus (CSFV) is one of the major antibody targets upon infection of pigs with the virus. Molecular dissection of the structure of E(rns) would define the minimal immunodominant regions that induce antibody responses after infection and may thus help design an effective diagnostic reagent or vaccine. In this study, deletion analysis was made within amino acids (aa) 297 to 776 of the CSFV Alfort/187 polyprotein containing the large C-terminal portion of the E(rns) protein (aa 27 to 227), the entire E1 protein (aa 1 to 195), and the N-terminal portion of the E2 protein (aa 1 to 87). Various protein fragments with target deletions from N- or/and C-terminal ends were constructed with pET30, expressed in Escherichia coli and probed on Western blots with antisera from pigs infected with CSFV. This has resulted in the identification within E(rns) of three overlapping antigenic regions: AR1(E(rns)aa 65-145), AR2 (E(rns)aa 84-160) and AR3 (E(rns)aa 109-220). N- or C-terminal deletions as small as 3 residues introduced into these regions disrupt their reactivity with antibodies, indicating that they are the minimum requirements for recognition by pig antibodies. The three minimal antigenic regions correlated well with the hydropathy profiles and the 3D structural model of E(rns). Each individual region and a protein fragment containing AR1, AR2 and AR3 reacted equally well with pig anti-CSFV sera. Since variable and conserved sequences are present within the three overlapping antigenic regions of E(rns) of different pestiviruses, specific serological detection of CSFV infection or broad detection of pestivirus infections may be achieved with the use of a single E(rns) region or a combination of two or three E(rns) regions.     

Determinants of virulence of classical swine fever virus strain Brescia

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB (476)R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB (476)R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB (476)S variant into a His residue did not affect virulence. However, the (710)His in the C1.1.1/CoBrB virus, together with adaptive mutations (276)R, (476)R, and (477)I in E(rns), resulted in reduced virulence in pigs. These results indicated that mutations in E(rns) and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E(rns) may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the (710)L-->H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.     

Misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum

As a part of our studies on the folding of glycoproteins in the ER, we analyzed the fate of viral glycoproteins that have misfolded either spontaneously or through inhibition of N-linked glycosylation. Newly synthesized Semliki Forest virus spike glycoproteins E1 and p62 and influenza hemagglutinin were studied in infected and transfected tissue culture cells. Misfolded proteins aggregated in less than 1 min after release from polysomes and aberrant interchain disulfide bonds were formed immediately. When more than one protein was misfolded, mixed aggregates were generated. This indicated that the formation of complexes was nonspecific, random, and not restricted to products from single polysomes. The size of the aggregates varied from small oligomers to complexes of several million daltons. BiP was associated noncovalently with the aggregates and with some of the nonaggregated products. We conclude that aggregation reflects the poor solubility of incompletely folded polypeptide chains.     

针对猪瘟病毒E2蛋白的嵌合猪源化单克隆抗体的表达及抗病毒活性鉴定

猪瘟(Classical swine fever,CSF)是严重危害养猪业的一种烈性传染病,常造成巨大的经济损失,是世界动物卫生组织要求必须申报的动物疫病之一。猪瘟的病原是猪瘟病毒(Classical swine fever virus,CSFV),CSFV的结构蛋白由衣壳蛋白(C)和囊膜糖蛋白(E~(rns)、E1、E2)构成。E2蛋白是CSFV主要的保护性抗原,可以诱导机体产生中和抗体,从而抵抗CSFV的感染。此前,本团队制备了一株针对CSFV E2蛋白的鼠源单克隆抗体HQ06。文中将HQ06抗体重链和轻链可变区基因与猪源恒定区基因嵌合后克隆至真核表达载体,利用中国仓鼠卵巢(CHO)细胞制备一株针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06。应用ELISA、Western blotting试验证实了c HQ06与CSFV E2蛋白具有良好的反应性;中和试验结果表明c HQ06可以中和CSFV。综上所述,本研究应用CHO细胞稳定表达了具有良好反应性和中和活性的针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06,为研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂奠定基础。     

Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.     

Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System

E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.