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The role of Hepatitis B virus capsid protein in the host ubiquitin proteasome pathway
Eliáš, Vratislav ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...
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The role of capsid protein in hepatitis B virus life cycle
Eliáš, Vratislav ; Weber, Jan (advisor) ; Drda Morávková, Alena (referee)
Hepatitis B virus (HBV) can cause either an acute or a chronic infection of hepatocytes, often leading to a hepatocellular carcinoma or a liver cirrhosis. HBV encodes seven proteins in its 3.2kb genome. Two of these proteins are transcribed from the same ORF (HBcAg and HBeAg). HBeAg, as a soluble variant of the core protein, plays a crucial role in virus immunogenicity. HBcAg, apart from building viral capsid, represents a pleiotropic protein engaging in multiple viral stages. Its primary structure is divided into two domains: an assembly domain and a C-terminal domain (CTD). The latter, containing four arginine clusters, is an important life cycle regulation element. By interaction with multiple viral and cellular factors, it directs the virus through a successful replication. In this thesis, we have collected the available data about the various roles of HBcAg in viral life cycle. We believe that further investigation of this field could lead to therapeutic advances resulting in the decrease of HBV infections worldwide, subsequently to a lower lethality connected with the diseases caused by HBV. Powered by TCPDF (www.tcpdf.org)
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Contribution of gag region to overall HIV replicative fitness in patients with different disease progression
Suchý, Tomáš ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Human immunodeficiency virus (HIV) is globally spread virus without available cure. Since its life-long presence, virus is carefully monitored as well as patient's immunological status. Replicative fitness of the virus is one of important aspects which can be taken into account, when monitoring HIV. Here, we are measuring HIV replicative fitness of gag recombinant viruses and comparing the results with replicative fitness of primary isolates. Further, we are comparing our findings of replicative fitness change over time with disease progression in the patient. We found that gag can be major contributor to overall fitness, although not in all cases. Additionally, we observed a correlation of replicative fitness development and slope of patient's CD4+ T cells. Moreover, this relation was even more noticeable in patients with slow disease progression or in carriers of protective alleles. In summary, our results extend the understanding of replicative fitness and its role in disease progression; and pave the way to use the recombinant HIV for replicative fitness measurement in clinical practice. Keywords: HIV, replicative fitness, recombinant virus, HIV disease progression, gag
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Interactions of polyomavirus structures in the endoplasmic reticulum and on the path to the nucleus
Svobodová, Terezie ; Huerfano Meneses, Sandra (advisor) ; Weber, Jan (referee)
Mouse polyomavirus is a member and model virus of Polyomaviridae family. In order to infect cells and produce viral progeny, the viral chromosome must be transported to the nucleus. Several studies suggest that virions are transporeted to the endoplasmic reticulum, from which they are transferred to the cytosol with assistace of host proteins. Two of these proteins are the chaperon, BiP (binding immunoglobulin protein) and the cochaperone, DNAJ B14. Polyomaviruses probably enter the nucleus through nuclear pores with the assistence of importins. These processes were mainly studied with SV40. In this work, we show that MPyV infection induces a change in distribution of the DNAJ B14 protein, which became clustered into foci, where it co-localizes with the viral capsid protein, VP1. The occurrence of foci varies during infection. With use of proximity ligation assay, we have shown that during an early fase of MPyV infection, DNAJ B14 and BiP get in the close proximity with VP1. It is suggested that negatively charged amino acids at the N-terminus of the minor capsid protein, VP2, are required for targeting virions to translocon and proteins associated with ERAD. We created MPyV with VP2 mutated in these amino acids. The negatively charged amino acid at position 17 is not necessary for successful...
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The role of Hepatitis B virus capsid protein in the host ubiquitin proteasome pathway
Eliáš, Vratislav ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...
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Rous sarcoma virus replication blocks in mammalian cells
Koslová, Anna ; Hejnar, Jiří (advisor) ; Ruml, Tomáš (referee) ; Weber, Jan (referee)
One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...
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Interactions of polyomavirus structures in the endoplasmic reticulum and on the path to the nucleus
Svobodová, Terezie ; Huerfano Meneses, Sandra (advisor) ; Weber, Jan (referee)
Mouse polyomavirus is a member and model virus of Polyomaviridae family. In order to infect cells and produce viral progeny, the viral chromosome must be transported to the nucleus. Several studies suggest that virions are transporeted to the endoplasmic reticulum, from which they are transferred to the cytosol with assistace of host proteins. Two of these proteins are the chaperon, BiP (binding immunoglobulin protein) and the cochaperone, DNAJ B14. Polyomaviruses probably enter the nucleus through nuclear pores with the assistence of importins. These processes were mainly studied with SV40. In this work, we show that MPyV infection induces a change in distribution of the DNAJ B14 protein, which became clustered into foci, where it co-localizes with the viral capsid protein, VP1. The occurrence of foci varies during infection. With use of proximity ligation assay, we have shown that during an early fase of MPyV infection, DNAJ B14 and BiP get in the close proximity with VP1. It is suggested that negatively charged amino acids at the N-terminus of the minor capsid protein, VP2, are required for targeting virions to translocon and proteins associated with ERAD. We created MPyV with VP2 mutated in these amino acids. The negatively charged amino acid at position 17 is not necessary for successful...
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Contribution of gag region to overall HIV replicative fitness in patients with different disease progression
Suchý, Tomáš ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Human immunodeficiency virus (HIV) is globally spread virus without available cure. Since its life-long presence, virus is carefully monitored as well as patient's immunological status. Replicative fitness of the virus is one of important aspects which can be taken into account, when monitoring HIV. Here, we are measuring HIV replicative fitness of gag recombinant viruses and comparing the results with replicative fitness of primary isolates. Further, we are comparing our findings of replicative fitness change over time with disease progression in the patient. We found that gag can be major contributor to overall fitness, although not in all cases. Additionally, we observed a correlation of replicative fitness development and slope of patient's CD4+ T cells. Moreover, this relation was even more noticeable in patients with slow disease progression or in carriers of protective alleles. In summary, our results extend the understanding of replicative fitness and its role in disease progression; and pave the way to use the recombinant HIV for replicative fitness measurement in clinical practice. Keywords: HIV, replicative fitness, recombinant virus, HIV disease progression, gag
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The role of capsid protein in hepatitis B virus life cycle
Eliáš, Vratislav ; Weber, Jan (advisor) ; Drda Morávková, Alena (referee)
Hepatitis B virus (HBV) can cause either an acute or a chronic infection of hepatocytes, often leading to a hepatocellular carcinoma or a liver cirrhosis. HBV encodes seven proteins in its 3.2kb genome. Two of these proteins are transcribed from the same ORF (HBcAg and HBeAg). HBeAg, as a soluble variant of the core protein, plays a crucial role in virus immunogenicity. HBcAg, apart from building viral capsid, represents a pleiotropic protein engaging in multiple viral stages. Its primary structure is divided into two domains: an assembly domain and a C-terminal domain (CTD). The latter, containing four arginine clusters, is an important life cycle regulation element. By interaction with multiple viral and cellular factors, it directs the virus through a successful replication. In this thesis, we have collected the available data about the various roles of HBcAg in viral life cycle. We believe that further investigation of this field could lead to therapeutic advances resulting in the decrease of HBV infections worldwide, subsequently to a lower lethality connected with the diseases caused by HBV. Powered by TCPDF (www.tcpdf.org)
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