Egy átlagostól eltérő fiziko-kémiai tulajdonságokkal rendelkező fehérje, a Hepatitis B vírus X antigén, immun-biotechnológiai modellvizsgálata

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Phylogenetic Studies of the United States Bluetongue Viruses and Characterization of the Viral VP4 Protein

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4. The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region of the M1 gene, which encodes the VP4 protein, possesses an open reading frame with an initiation codon (ATG) at nucleotides #9-11 and a stop codon (TAA) at nucleotides #1941-1943. This open reading frame encodes a protein of 644 amino acid residues with a predicted molecular weight of about 75 kDa. A potential leucine zipper motif was detected near the carboxyl terminus of the deduced VP4 amino acid sequence. The phylogenetic analysis of bluetongue viruses using the sequences of these five cognate M1 genes is consistent with the results of previous phylogenetic studies. Serotypes-10, -11, -13, and -17 are closely related and serotype-2 is the most distantly related among the five US BTV serotypes. Heterologously expressed bluetongue virus VP4 protein was purified to near homogeneity. Six linear epitopes of VP4 were mapped at both termini and in the middle of the protein. By using enzyme-linked immunosorbent assay and peptide competition assay, six linear epitopes were found to be surface accessible. The VP4 protein was shown to be an oligomer by chemical cross-linking. VP4 protein was identified as a ssRNA-binding protein. The VP4 protein has binding activity towards both capped and non-capped ssRNA. RNA-binding activity was not specific to BTV ssRNA. A leucine-zipper motif of VP4 is not required for RNA-binding activity. One RNA-binding domain was mapped between amino acid residues #112-158 by a Northwestern assay and by deletion mutant analysis. Using sequence-specific synthetic peptides corresponding to VP4 in the arginine-and lysine-rich regions, four potential ssRNA-binding domains of VP4 protein were mapped

Expression of hepatitis B virus chimeric proteins in prokaryotic and eukaryotic systems

The particulate form of the core antigen of hepatitis B virus (HBcAg), is highly immunogenic. It has been used as a carrier molecule, for expression and presentation of heterologous viral epitopes on the surface of hybrid core particles, in immunogenicity studies. The aim of this project was to produce a hybrid antigen comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions, by investigating the influence of position of the foreign epitope on antigenicity, immunogenicity and hybrid core particle formation. A part of the HCMV genome, encoding a neutralizing glycoprotein epitope gp58, was inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg in separate constructs and expressed in a prokaryotic system (E.coli). At the same time, in order to express the same fusion proteins in a eukaryotic system, the baculovirus expression vector system (BEVS) was selected and as an initial control two recombinant baculoviruses, containing genes encoding HBcAg and hepatitis B surface antigen were isolated by dot blot hybridization. It was realized that work in BEVS would require more time than previously expected therefore further work was only carried out on the prokaryotic system. The HBcAg carboxyl terminal fusion (HBC 3 - 1 4 4 -HCMV) was expressed in high yields in E.coli and assembled into core like particles resembling native HBcAg. A similar fusion in the amino terminus of HBcAg (HCMV-HBC1-183) could not be purified or characterized immunologically, although it formed core like particles. HBc3-144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected. Following immunization of rabbits with HBC3-144-HCMV, a high level of anti-HBc specific antibody was produced along with HCMV/gp58 specific antibody. The data presented here provide evidence that the HCMV/gp58 region can be used as a candidate immunogen for an HCMV subunit vaccine and that HBcAg can effectively present this foreign epitope joined to its carboxyl terminus, to the immune system

Tubules composed of non-structural protein NS1 of african horsesickness virus as system for the immune display of foreign peptides

Non-structural protein, NS1 of African horse sickness virus is a hydrophobic protein of 63 kDa that spontaneously assembles into highly distinct tubular structures when expressed in mammalian or insect cells. The spontaneous assembly of these proteins into a predictable multimeric structure, high levels of expression and ease of purification make this protein an ideal candidate for the immune display of foreign peptides. The potential of such a display system has been investigated for BTV NS1 that is able to successfully elicit both a humoral and a cellular immune response against inserted peptides. The aims of this study were to investigate both the stability of the AHSV NS1 particulate structure after insertion of peptides as well as the antigenicity and immunogenicity of the peptides presented in this system. Two overlapping regions consisting of 40 and 150 amino acids, and which correspond to a neutralising region identified within the AHSV major neutralising protein VP2, were inserted into an internal site in NS1. This site offered the best surface display of inserted peptides on the tubular structures. An enhanced green fluorescent protein, 240 amino acids long, was also inserted into the NS1 protein. Sucrose gradient analysis of the recombinant proteins indicated that the majority of the baculovirus expressed chimeric proteins formed particulate structures with a sedimentation value similar to that of the native NS1 protein. This was confirmed by transmission electron microscopic analysis, which clearly showed that all the chimeric proteins assembled into tubular structures similar to those observed for AHSV NS1 proteins. Furthermore, fluorescence analysis of sucrose gradients of NS1/eGFP also showed high levels of fluorescence that corresponded directly to particle formation. Not only do the inserts remain functional but are also presented successfully on the surface of the intact NS1 tubule structure. The potential of the NS1 vector to efficiently present peptides to the immune system was subsequently investigated. The serums generated against these chimeric proteins in guinea pigs were tested against chimeric constructs, the baculovirus expressed inserts (for eGFP) and the inserts presented on other presentation vectors. Western blot analysis showed that most of the serums generated against the chimeric proteins contained antibodies not only against the chimeric proteins but antibodies that reacted specifically with the inserted peptides on their own or on another presentation system. Preliminary immune studies seem to indicate that the humoral immune response elicited by the chimeric NS1 proteins is predominantly against the inserts. The inserts are successfully presented to the immune system on the surface of the NS1 vector and are able to elicit the production of antibodies with the potential to provide a protective immune response.Dissertation (MSc (Genetics))--University of Pretoria, 2010.Geneticsunrestricte

Production and evaluation of pseudotyped viruses for Hepacivirus and Flavivirus studies

Flaviviridae is a diverse family of RNA viruses that includes many important human pathogens such as dengue virus, West Nile virus, Japanese encephalitis virus (JEV), and hepatitis C virus (HCV). Handling many of these pathogens requires high containment level biosafety laboratories and some cannot replicate in cell culture. The use of replication deficient pseudotyped viruses (PV) circumvents these issues. This thesis shows the development of two Flaviviridae PVs, GB Virus B (GBV-B) and JEV, for serological assay development and virus-host interaction studies. HCV is a major global concern with over 70 million people chronically infected. Until the recent discovery of related animal hepaciviruses, GBV-B was the only known homolog of HCV and is used in new world monkeys as an animal model. However, there is still little known about the viral-host interactions. This thesis first describes the production and optimization of GBV-B PV to develop a neutralization assay for screening sera from experimentally infected tamarins. Viral entry mechanisms were investigated using the GBV-B PV. Cells without Claudin 1 (CLDN1), a known HCV receptor, had no detectible GBV-B infection. CLDN1 was subsequently confirmed to be an entry factor when restoration of expression conferred susceptibility to GBV-B PV. Chimeric claudin proteins consisting of the permissive CLDN1 and non-permissive CLDN9 were created to investigate the region of importance for GBV-B and CLDN1 interaction. The important region was identified as extracellular loop 2, or downstream, which is different from the dependence of CLDN1-restricted HCV strains on regions in extracellular loop 1. Research with JEV requires high containment level biosafety labs which are often unavailable in resource limited areas. Described here is the production of JEV PV using two alternative pseudotyping methods, in vitro assembly of subviral particles with envelope-less lentiviral particles and dengue virus core, after failed attempts at using traditional systems

Hepatitis C Virus NS5A Targets the Nucleosome Assembly Protein NAP1L1 to Control the Interferon Response

Hepatitis C virus (HCV) is a hepatotropic virus affecting more than 150 million people worldwide. HCV establishes a chronic infection in the majority of cases, which leads to severe liver complications such as cirrhosis and hepatocellular carcinoma. Fortunately, recent potent direct acting antivirals can now cure the infection. However, such treatments can induce resistance, are extremely costly limiting their use to wealthier countries and are ineffective for the complications of the infection. Therefore, a better understanding of the interaction of HCV with the host cell remains a priority both to increase the armamentarium of antiviral drugs and to define the relationship between infection and malignant transformation. My study focuses on the mechanisms governing the evasion of the innate immune system, which are required to establish a chronic infection. The non-structural viral protein NS5A has the capacity to interact with a large number of cellular factors involved in promoting viral replication/assembly and in the cell antiviral response to HCV. Interaction of NS5A with the nucleosome assembly protein NAP1L1 has been recently characterized in my laboratory. NAP1L1 is a histone chaperone protein with various functions related to nuclear chromatin remodelling that impact on the regulation of cell cycle, on cell differentiation and on transcription. I have confirmed the interaction of NS5A with NAP1L1 in the cytoplasm and demonstrated the NS5A-dependent impairment of NAP1L1 nuclear translocation. Whole genome transcription analysis performed in NAP1L1 depleted hepatocytes indicated that its nuclear function might be essential for the transcriptional control of several interferon stimulated genes and the function of key innate immunity pathways. Indeed, I was able to demonstrate that NAP1L1 is a novel factor involved in the interferon response and specifically modulates TBK1/IKKε mediated IRF-3 phosphorylation and NF-κB levels. Hence, both the TLR3 and RIG-I/MDA5 pathways are affected by NAP1L1 depletion. I could further demonstrate that NAP1L1 controls the basal transcription of genes involved in the immune pathway and that it interacts with the adaptor protein MAVS, which is required for RIG-I/MDA5 signalling. In conclusion, by studying the interaction of the viral protein NS5A with the cellular factor NAP1L1 I could discover a novel mechanism of regulation of the innate response mediated by NAP1L1. These findings have wider implications for HCV and beyond, further highlighting the importance of studying viruses to uncover cellular functions

Visualization of the HIV-1 Nuclear Preintegration Complex Structure by High Precision Correlative Light - and Electron Microscopy and - Tomography

Upon fusion of the viral envelope with the host cell membrane, the capsid of the human immunodeficiency virus 1 (HIV-1) is released into the host cell cytoplasm. To productively infect a cell, the viral RNA genome needs to be reverse transcribed into viral DNA. This in turn needs to become integrated into the host cell genome. Integration can, however, only happen, after the viral genome is released from its capsid-container, in a process called uncoating. This is a vital process and needs to be regulated and orchestrated in certain ways – which are still elusive and controversially discussed. Some studies suggest that uncoating takes place soon after -, or concomitant with viral entry. Other researchers came to the result that the capsid needs to retain its structure to shield the viral components from being sensed by the innate cellular immune system. Both hypotheses, early uncoating and prolonged structural retention, are solidly supported by experimental data. Therefore, the timing and kinetics of uncoating remain unresolved. Based on previous results from our group, we had reason to believe that the capsid might indeed be retained, possibly even within the nucleus. A method was developed, that allows the detection of viral DNA. The presence of viral DNA was used as a criterion to discriminate between productive and nonproductive subviral particles in infected cells. Surprisingly, productive subviral particles displayed an intense, stable signal for capsid protein in immunofluorescence experiments, throughout the cytoplasm and even within the nuclei of infected cells. A strong signal is can be understood as a high concentration of labeled protein, which in turn might indicate the presence of a retained structure. However, intense immunofluorescence signals can also mean more efficient binding of antibodies due to structural rearrangements (such as uncoating), and a high spatial concentration of proteins cannot be directly interpreted as structure retention. In this study, we present a unique way to address and solve this important question. We specifically focused on the small fraction of productive particles. Light Microscopy allows specific labeling but has low resolution. Electron Microscopy yields much higher resolution, but specific (immuno)labeling is difficult and often detrimental to ultrastructural retention. We overcame both limitations by correlative light – and electron microscopy: Regions of interest were identified by specific nuclear subviral particle surrogate markers in light microscopy. On these regions, tilt series electron tomography was performed, to visualize the subviral particles’ structure, as well as the subcellular environment, around the region of interest. Performing high resolution tilt series electron tomography, we could repeatedly and convincingly visualize a capsid-reminiscent structure that underlies HIV-1 nuclear preintegration complexes. This apparent structure is very similar in shape, but smaller in size compared to capsids of virus particles of mostly identical preparations. The discovery of a retained capsid structure in the nucleus of an infected cell will advance on our understanding of nuclear entry and provides whole new insights into the overall understanding of HIV-1 in early steps of infection