Expression of Structural Protein E2 of Hepatitis C Virus

Introduction HepatitisCvirus(HCV)isanRNAvirusthatcausesacuteor chronichepatitis,cirrhosis,andhepatocellularcarcinoma(HCC)[1,2].DespiterecentadvancesinthetherapyofHCV,eventhemostrecent combinationofpegylatedalpha-interferonandribavirinfailstoelimi nateinfectioninnearly50%ofthoseinfected[3,4].Nowadays,itis wellknownthatvaccineisstillthemostefficientwaystopreventvirus infection[5].Thestudyingofviralvaccinehasbeenhamperedbythe lackofanefficientcellculturesystem.Asaenvelopeglycoprotein,E2prot…     

Purification and application of bacterially expressed chimeric protein E1E2 of hepatitis C virus

E1 and E2 glycoproteins are structural components of hepatitis C virus (HCV) virion. They are involved in cellular receptors interaction, neutralising antibodies elicitation, and viral morphogenesis. They are considered as major candidates for anti-HCV vaccine. In this report, we first expressed tandem E1E2 as well as C-terminally truncated E1 fragment and C-terminally truncated E2 fragment, respectively, in Escherichia coli cells and the proteins were purified to homogenesis. All the purified proteins can react specifically with patient sera. Both purified chimeric protein E1E2 and protein E2 can interact with a putative cellular receptor CD81, while purified protein E1 cannot interact with CD81. The sera of rabbit immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81 and reacted with both proteins E1 and E2, respectively. Anti-E1 and E2 antibodies can be generated simultaneously in the rabbit immunized with the E1E2, and the titers of antibodies were 63 or 56% higher than the titers induced by E1 or E2 alone, respectively. The results suggest that E1 and E2 can enhance their immunogenicity each other in chimeric protein E1E2 and the E. coli-derived chimeric protein E1E2 and corresponding antisera can be used as an useful tools in anti-HCV vaccine research.     

Multimeric HCV E2 protein obtained from <Emphasis Type="Italic">pichia pastoris</Emphasis> cells induces a strong immune response in mice

Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2₆₈₀) was produced in Pichia pastoris cells. E2₆₈₀, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulide-linked multimers. Both monomeric and oligomeric forms of E2₆₈₀ were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCV-positive patients were reactive against E2₆₈₀. By immunizing E2₆₈₀ in BALB/c mice, both a specific cellular immune response and anti-E2₆₈₀ IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2₆₈₀ could be useful to successfully induce strong anti-HCV immunity.     

Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.     

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.     

Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions

Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.     

Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is continuously evolving, and this poses a major threat to antibody therapies and currently authorized Coronavirus Disease 2019 (COVID-19) vaccines. It is therefore of utmost importance to investigate and predict the putative mutations on the spike protein that confer immune evasion. Antibodies are key components of the human immune system’s response to SARS-CoV-2, and the spike protein is a prime target of neutralizing antibodies (nAbs) as it plays critical roles in host cell recognition, fusion, and virus entry. The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations. In this review, we discuss (1) the emerging SARS-CoV-2 variants; (2) the structural basis for antibody-mediated neutralization of SARS-CoV-2 and nAb classification; and (3) identification of the RBD escape mutations for several antibodies that resist antibody binding and neutralization. These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines.     

Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly

Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.     

Molecular and functional aspects of latent transforming growth factor-β binding protein: just a masking protein?

Latent transforming growth factor-beta (TGFbeta) binding protein (LTBP), a component of the high-molecular-weight latent TGFbeta complex, is found in various cell and tissue types. Originally described as a TGFbeta-masking protein, recent detections of four isoforms and numerous splice variants provide new aspects of its putative functional role. Regulation and sequestration of TGFbeta activity and structural remodeling of the extracellular matrix (ECM) seem to be the main tasks, but other possible functions might exist. The mechanism by which LTBP interacts with cell surface molecules or cellular receptors and ECM components remains unclear. Cellular, molecular and functional aspects will be discussed.     

Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.     

The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.     

Scavenger receptor class B type I and hepatitis C virus infection of primary tupaia hepatocytes

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.     

Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli

Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene. We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus. 1) 14 kDa is a trimer of identical subunits. 2) A monomer binds to itself more strongly than to a dimer or a trimer. 3) Oligomerization does not require cellular factors. 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity. 5) 14-kDa binds with specificity to the cell surface of cultured cells.     

K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function

The Kaposi's sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, approximately 50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman's disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.     

The human papillomavirus-16 (HPV-16) oncoprotein E7 conjugates with and mediates the role of the transforming growth factor-beta inducible early gene 1 (TIEG1) in apoptosis

The human papillomavirus (HPV) oncoprotein E7 is a major transforming protein. The E7 protein does not possess intrinsic enzymatic activity, but rather functions through direct and indirect interactions with cellular proteins, several of which are well known cellular tumor suppressors. Using the yeast two-hybrid system, we found that transforming growth factor-beta inducible early gene 1 (TIEG1), a member of the Krüppel-like family (KLF) that has been implicated as a putative tumor suppressor, interacts and forms a specific complex with HPV-16 E7. TIEG1 has been shown to mimic the effects of TGF-beta in various carcinoma cells and plays a critical role in the apoptotic cascade. Our results indicate that E7 binds to the C-terminus of TIEG1 and induces its degradation via the ubiquitin pathway. E7 not only increased the ubiquitination of TIEG1 but also influenced the ability of TIEG1 to affect apoptosis. Our results suggest that suppression of TIEG1-mediated signaling by E7 may contribute to HPV-associated carcinogenesis.     

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.     

Expression in Pichia pastoris and immunological evaluation of a truncated dengue envelope protein

Among the Dengue virus structural proteins, the Envelope glycoprotein is the most important because of its antigenic characteristics. In this work, the E protein from Dengue-2 virus truncated at the C-terminus region was successfully expressed in Pichia pastoris. The E2trunc gene was cloned under the AOX1 promoter from P. pastoris and the signal peptide of the sucrose invertase gene from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression revealed the presence of a protein with the expected size, which was completely associated to the insoluble fraction after cellular disruption. The recombinant N-glycosylated protein reacted with two conformational antibodies against Dengue-2, indicating a proper folding of it. In addition, it was able to induce antiviral antibodies after mice immunization.