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Expression of anti-HBV primary micro-RNA shuttles using an inducible promoter system.Mlambo, Tafadzwa 28 March 2014 (has links)
Hepatitis B virus (HBV) infection is an important global health concern and chronic carriers of the virus are at high risk of developing hepatocellular carcinoma (HCC) and cirrhosis. Current therapies are only partially effective, which emphasises the need for improved treatment strategies. Harnessing the RNA interference (RNAi) pathway as a treatment strategy against HBV has shown great promise. However, there are obstacles that need to be overcome before RNAi-based treatment of HBV infection is realised. These include problems of liver tissue targeting and dose regulation. This study investigated the use of a liver specific and mifepristone-inducible RNA polymerase (Pol) II promoter system for the specific and precise regulation of anti-HBV sequence expression. The inducible system used consists of two expression cassettes; one containing the
regulator/transactivator protein and another containing the transgene. Natural primary microRNA (pri-miR) mimics, pri-miR-31/5 and pri-miR-31/5/8/9, were used as anti-HBV sequences. Firefly luciferase gene expression was used to test modulation by the inducible system and to determine optimal induction conditions. The pri-miR-31/5, pri-miR-31/5/8/9 and luciferase encoding fragments were incorporated into the plasmid vector pRS17 that bears the inducible promoter, creating pRS-31/5, pRS-31/5/8/9 and pRS-Luc respectively. Firefly luciferase expression with this system was shown to be inducible and mifepristone dose-dependent. Effective knockdown of HBV gene expression was achieved with both pRS-31/5 and pRS-31/5/8/9 in vitro and in vivo. However, with high vector amounts, similar efficiency in silencing of HBV gene expression was observed in the presence and absence of the inducer mifepristone suggesting leaky expression of the pri-miRs. To confirm this, knockdown studies were carried out with the pri-miR-31/5/8/9-expressing cassette separated from the transactivator cassette. HBV gene expression knockdown was observed with the pri-miR-31/5/8/9 cassette alone confirming leaky expression from the inducible system. Leakiness appears to be as a result of the E1B promoter driving the expression of the pri-miRs in the absence of mifepristone. However, reducing the vector amounts decreased basal expression and improved the inducibility of the system in cell culture studies. Successful propagation of an inducible and liver-specific RNAi-activating expression system will address the difficulty of achieving dose control of RNAi effectors and contribute to advancing the use of RNAi for HBV treatment.
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DNA unwinding mechanism of the helicase from hepatitis C virus /Levin, Mikhail Konstantinovich. January 2002 (has links)
No description available.
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Mechanism of Pathogenesis and Replication of an Avian Strain of the Hepatitis E Virus in a Chicken ModelBillam, Padma 02 May 2007 (has links)
Hepatitis E is an acute, enterically transmitted disease of public health importance. The mechanism of pathogenesis of HEV is poorly understood due to the lack of an in vitro cell culture system and an ideal animal model system. With the discovery of avian HEV and its association with a hepatic disease (Hepatitis-Splenomegaly syndrome), chickens provide an excellent small homologous animal model system to study this important virus. The objectives of this dissertation were to utilize chickens as a model system to study the pathogenesis and replication of avian HEV under the natural route of infection, to identify potential extrahepatic replication sites, to determine and analyze the complete genomic sequence of the avirulent strain of avian HEV, and to study the compartive pathogenesis of the two isolates of avian HEV, the prototype pathogenic and avirulent strains of avian HEV.
We attempted to experimentally infect specific-pathogen-free (SPF) adult chickens by the natural fecal-oral route in order to systematically study HEV pathogenesis and replication and to characterize the clinical course and pathological lesions associated with avian HEV infection. Sixty-week-old, specific-pathogen-free (SPF) chickens were inoculated with 5 x104.5 50% chicken infectious dose of avian HEV by oronasal route and IV route. All oronasally- and IV- inoculated chickens had seroconverted to avian HEV antibodies and fecal virus shedding was detected variably from 1 to 20 DPI in the IV group, and from 10 to 56 DPI in the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples earlier during the course of infection in IV-inoculated chickens than in oronasally-inoculated ones. Gross liver lesions including subcapsular hemorrhages and enlargement of right intermediate lobe and microscopic hepatic lesions in the liver characterized by lymphocytic periphlebitis and phlebitis were observed in inoculated chickens. This is the first report of experimental HEV infection via its natural route in a homologous animal model system.
Very little is known about HEV pathogenesis and it has been hypothesized that HEV replicates in tissues other than liver. The replicating negative-strand viral RNA was detected by negative-strand-specific RT-PCR in liver, serum, colon, cecum, jejunum, ileum, duodenum and cecal tonsils,but not in other non-GIT tissues. Immunohistochemistry using an avian HEV capsid protein-specific anti-peptide antibody revealed positive signal in liver and GIT tissues including colon, jejunum, ileum, cecum, cecal tonsils and pancreas. The detection of avian HEV capsid antigen and replicative negative-strand viral RNA in the GIT tissues indicates that HEV replicates in the GI tract following infection by fecal-oral route.
The complete genomic sequence of an avirulent strain of avian HEV was determined using primer walking strategy. The full-length genome of the avirulent strain is 6649 nts in length and has a nucleotide sequence identity of 90.1% with the prototype pathogenic strain. Numerous non-silent mutations were observed in ORF1, the region coding for the nonstructural proteins. Six unique non-silent mutations were identified in the capsid-encoding ORF2 region and the ORF3 had four non-silent mutations. Phylogenetic analysis based on full-length genomic sequence revealed that the avirulent strain is clustered together with the pathogenic avian HEV and represents a branch distinct from mammalian HEVs.
In order to study the comparative pathogenesis between the pathogenic and avirulent strains of avian HEV, an infectious stock of the avirulent avian HEV was generated and infectivity titer was determined to be 5 x 102.5 CID50 per ml by experimentally infecting young SPF chickens. Six-week-old SPF chickens were inoculated with one of two strains of avian hepatitis E viruses, pathogenic avian HEV recovered from a chicken with HS syndrome and avirulent avian HEV isolated from a healthy chicken to study comparative pathogenesis. Most of the chickens seroconverted by 3 wpi in both pathogenic avian HEV and avirulent avian HEV groups. Avian HEV RNA was detected in feces and serum of the chickens from both the inoculated group from 1 wpi. Microscopic liver lesions included lymphocytic periphlebitis and phlebitis the overall hepatic lesion mean score was higher for the pathogenic avian HEV group compared to the avirulent avian HEV and control groups, suggestive of attenuation
In summary, SPF chickens were experimentally infected with avian HEV by natural route to study the systematic pathogenesis and replication. Non-liver replication sites of avian HEV were also identified in a chicken model. The complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus was determined and the comparative pathogenesis of avian hepatitis E virus isolates from a chicken with HS syndrome and from a healthy chicken was also studied by experimental infections in young SPF chickens. The results from this dissertation research have important implications for the understanding of HEV pathogenesis. / Ph. D.
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Hepatitis-B-associated glomerular disease : a clinicopathological study of Hepatitis B virus associated Membranous Glomerulonephritis in Namibian and South African children 1974 - 2005 and a comparison with Hepatitis B associated Membranous Glomerulonephritis as well as Idiopathic Membranous Glomerulonephritis in adultsBates, William D. 12 1900 (has links)
Thesis (DMed)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe
proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease
(MCD). This is also the pattern observed in white and Indian children in South Africa
(SA). By contrast, black and mixed race/coloured children of Southern Africa in the
1960s to 1990s were shown to have a different pattern of NS. One of the main
differences was the frequency of hepatitis B virus (HBV) associated
glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of
this project was a clinicopathological study of this subgroup of nephrotic children to
document the disease further and in particular to seek correlations between
pathological and clinical features including prognosis. A central focus was to
document the detailed ultrastructural examination of the renal biopsies of these
children and to correlate the spectrum of pathological features with demographic,
clinical, laboratory and prognostic features.
The hypothesis was that the clinicopathological features of HBV MGN in
children differed substantially from idiopathic MGN in general (children and
adults) and also from HBV MGN in adults and that HBV MGN in children should
be viewed as a distinct disease.
Patients and methods: The childhood (12 years and younger) patient cohort was
309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg
Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from
Namibia. The study group was 71 children with HBV MGN who were followed up to
2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN
in childhood as this centre only had 2 such patients during the study period.)
Demographic, clinical, laboratory and renal pathology data were collected, compared
and correlated.
Results: HBV associated MGN was the most frequent cause of NS in the Namibian
subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood
cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood
nephrotic cohort, by far the dominant subgroup.
The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years)
at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen
(HBeAg) was identified in the serum of 87% of children tested. Laboratory features
different from idiopathic MGN included more prominent haematuria, mildly raised
serum transaminases and more frequently lowered serum C3 and C4 levels. Light
microscopic examination of renal biopsies showed mesangial proliferation in all
patients but with minimal glomerular sclerosis and interstitial disease. On
ultrastructural examination mesangial and subendothelial deposits were common and
prominent as was mesangial interposition. The MGN of HBV in children therefore
frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition
to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies
displayed severe mesangial interposition in addition to the subepithelial deposits of
MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like
bodies and tubuloreticular inclusion bodies were both found in more than 80% of
biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits
in the glomeruli. This was the first time this feature was demonstrated in Africa. The 46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort
developed chronic renal failure (CRF). Age of 6 years and above at presentation and
severe mesangial deposits on biopsy correlated with fewer remissions and poorer
outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset
of CRF was more than 19 years with the longest being 23 years. The 358 cases of
childhood HBV MGN from Southern Africa constitute 37% of the reported childhood
patients.
Comparative data
A comparison was made between the 71 children with HBV MGN, 12 adults with
HBV MGN and 33 adults with idiopathic MGN. The main differences were that both
HBV MGN groups included only coloured and black patients and were more
predominantly male while the idiopathic MGN group included all races. In the HBV
patients, haematuria was more frequent and severe, liver enzymes were frequently
raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of
adult MGN patients had normal C4 levels while the childhood HBV MGN group had
reduced C4 levels.
The immune complex pattern in both of the HBV MGN adult and childhood groups on
biopsy was similar with more mesangial and subendothelial deposits as well as
mesangial interposition than the idiopathic group. Despite this similarity between the
two HBV groups, both adult groups showed more glomerular sclerosis and interstitial
disease than the childhood group. The clinical outcome of the children’s cohort was
better than the other 2 groups with remission (52%) more frequent at 4 years (p<0.01) and better renal and patient survival.
Including the 83 cases from this series, at least 1243 renal biopsy proven cases of
HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar
(children 79%; adults 84%) and significantly greater than for idiopathic MGN.
Conclusions: The findings confirm that HBV MGN in children is a distinct form of
GN which broadens the classical morphologic description of MGN by often including
a number of mesangiocapillary GN features. The subgroup of renal biopsies with the
most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary
GN group. The MGN spectrum as a whole comprised 86% of the
HBV positive childhood group. HBV MGN was the most frequent association with
NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group
(19%) in the SA children. It showed a relatively high spontaneous remission rate but
at least 10% of the children developed renal failure. Age of 6 years and above at
presentation and severe mesangial deposits on biopsy correlated with fewer
remissions and poorer outcome. Extended follow up (more than 15 years) was
required to demonstrate renal failure in some patients in the poor outcome group.
Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated
routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this
disease in SA. HBV MGN has been a valuable and possibly unique model of human
GN and MGN in particular in that the HBeAg has been identified in both the serum
and glomeruli enabling confirmation of the aetiological role of HBeAg. het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar
getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking
(KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer.
Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV
ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare
HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder
pasiënte.
Vergelykende data
’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met
HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide
HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik
was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was
hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer
dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN
pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN
verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die
HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en
subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese
groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee
volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep
vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee
groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met
beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde
gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse
literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV
MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir
idiopatiese MGN.
Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike
vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene
insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier
biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre
GN groep geklassifiseer. Die MGN spektrum in geheel het 86%
van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene
assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde
grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane
remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking
ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer.
Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van
die swak uitkomsgroep aan te toon.
Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met
algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp
daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n
waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die
HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol
van HBeAg te bevestig. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese
sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon
kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is
aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die
jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van
die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV)
geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN).
Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep
van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies
te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die
gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die
korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese,
laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea.
Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in
kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en
volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders
as ’n afsonderlike siekte beskou behoort te word.
Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met
erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel
was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië.
Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12
HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN
in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte
tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en
nierpatologie inligting is versamel, vergelyk en gekorreleer.
Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die
Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die
kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder
nefrotiese kohort en verreweg die oorheersende subgroep.
Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was
6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B
omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87%
van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese
MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum
transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese
ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon,
maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele
ondersoek was mesangiale en subendoteliële neerslae asook mesangiale
interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van
mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae
van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale
interposisie aangetoon het asook die subepiteliale neerslae van MGN is die
gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies
en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies
bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste
keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders
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Mucosal DNA vaccines for regionally unique pathogens: hepatitis B virus and penicillium marneffeiWong, Lei-po., 黃利寶. January 2002 (has links)
published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy
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Association of cytokine gene polymorphisms with susceptibility and disease progression in chronic hepatitis B virus (HBV) infectionLee, Wing-yan, 李穎欣 January 2007 (has links)
published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy
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Relationship of serological markers, basic core promoter and precore mutations to genotypes of Hepatitis B virusLo, Kin-hang, Ken., 盧建恆. January 2009 (has links)
published_or_final_version / Medicine / Master / Master of Medical Sciences
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Investigating the modulation of viral translation by the Hepatitis C virus nonstructural protein 5A2015 April 1900 (has links)
Hepatitis C virus NS5A is a multi-functional viral protein essential for viral replication and assembly, although the exact role the protein plays in the viral lifecycle remains unclear. A vast array of functions have been attributed to NS5A in recent years, despite the lack of enzymatic activity. NS5A has been found to interact with over 130 host proteins including many which are central to cellular signaling pathways. NS5A is composed of three domains separated by regions of low complexity. All three domains perform important functions in the viral lifecycle. Domains I and II are essential for viral replication whereas domain III is required for viral assembly. However, the role that NS5A and its individual domains may play in modulating viral translation remains controversial. Previous studies have utilized translation reporter systems that do not accurately reflect the role of the viral 3´-UTR in modulating viral translation. We and others have shown that NS5A binds to the poly-U/UC region of the 3´-UTR. In addition to serving as the initiation site for negative strand synthesis the 3´-UTR functions to significantly enhance viral translation. The mechanism of translation enhancement remains unclear but may involve long range RNA-RNA interaction with the IRES, the binding of cellular proteins which stimulate translation and/or the recycling of ribosomes. Therefore, the protein-RNA interaction between NS5A and the poly-U/UC region has the potential to modulate viral translation. Therefore we set out to determine the role of NS5A and its individual domains in modulating viral translation and the role of the NS5A-poly-U/UC region interaction in this modulation.
Utilizing monocistronic RNA reporters which contain the viral 5´- and 3´-UTRs and an internal Renilla luciferase reporter gene, we determined that NS5A specifically down-regulates viral translation in a dose-dependent manner through a mechanism dependent upon the presence of the poly-U/UC region in the viral 3´-UTR. Furthermore, we have re-tested the effect using full-length HCV genomic RNA reporters. These results suggest that NS5A is able to interfere with the stimulation of viral translation exerted by the 3´-UTR. This down-regulatory function of NS5A may function in mediating a switch from translation to replication, a step required in the lifecycle of a positive sensed RNA virus. Having established a role for NS5A in modulating viral translation, we then aimed to determine which region of NS5A was responsible for this effect. We found that each of NS5A domains was capable of this modulatory effect on viral translation independently. Although surprising, this finding is not entirely unexpected as each domain has been shown to retain the ability to bind to the poly-U/UC region.
Within NS5A domain I we identified a 61 aa. region sufficient for translation down-regulation. Furthermore, we have identified a number of positively charged residues within this region involved in the modulation of viral translation, in particular arginine 112 (R112). This residue has previously been found to be at the domain I dimer contact interface and to form an intermolecular hydrogen bond with glutamic acid 148 (E148). We found that mutations R112A and E148A individually negate the ability of domain I to modulate viral translation and these mutations impede the formation of domain I dimers. Additionally, the R112A mutation appears to affect the ability of domain I to interact with the poly-U/UC region of the viral 3´-UTR alluding to the possible mechanism of translation modulation. Finally this mutation was lethal in an HCV subgenomic replication, confirming the link between NS5A dimerization, RNA binding and viral replication. These results collectively point to a crucial role for the NS5A arginine 112 residue in the modulation of HCV lifecycle by NS5A.
Within NS5A domain II, we identified a 47 aa. region sufficient for translation modulation. Through the mutation of positively charged amino acids within this region, we found that lysine 312 (K312) was essential for this effect. The ability of this domain to modulate viral translation was completely lost when K312 was mutated within a full domain II protein fragment. The mechanism behind this modulation remains unclear but the 47 aa. region identified has been previously found to contain a region proposed to make contact with poly-U RNA and the K312 residue was suspected to interact directly with such RNA. Furthermore, this region interacts with the host protein cyclophilin A, an interaction that enhances the RNA binding ability of domain II. These findings strongly suggest that domain II modulates viral translation by binding within the poly-U/UC region.
While investigating the modulation of viral translation by NS5A domain III we determined that the C-terminal 31 aa. are sufficient for the effect of this domain on viral translation. Through alanine scanning mutagenesis we identified glutamic acid 446 (E446) as playing a key role in the modulatory function of this region. Within a domain III protein fragment mutation of this E446 residue abolishes the modulatory function of this domain towards HCV translation. The mechanism behind this modulation and the role of E446 in this effect remains to be determined.
These findings suggest that in addition to being essential for viral replication and assembly, NS5A has an important role in modulating viral translation through a mechanism requiring the poly-U/UC region of the viral 3´-UTR. Furthermore, each domain of NS5A appears to contribute to this effect. These results support the description of NS5A as a multi-functional protein and the further characterization of its functions may aid in the development of novel antivirals targeting the numerous functions of this complex, and at times puzzling, viral protein.
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Development of an intra- and intergenotypic HCV cell culture method to phenotype and assess antiviral susceptibilities and resistance development of HCV NS3 protease genes from HCV genotypes 1-6Imhof, Ingrid January 2010 (has links)
The development of specific antiviral drugs directly targeting the hepatitis C virus (HCV) is clinically important, as the current standard interferon/ribavirin combination treatment is only partially effective, expensive and often associated with severe side effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternative or additional therapy. To date, the development and in vitro evaluation of PIs is restricted to the genotype 1/2 based replicon and the genotype 2a full length viral cell culture system. However, proteases of the different HCV genotypes vary substantially in their amino acid sequence and secondary structure and require separate evaluation of their efficacy before they go into clinical trials. To address this issue, a panel of intra- and intergenotypic recombinants based on the recombinant infectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. The viability of these recombinants was assessed in the Huh7.5 cell culture system, where replicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinants containing genotype 1a, 1b, 3a, 4a and 6a derived proteases were replication defective, whereas the recombinant with genotype 5a derived protease replicated efficiently after acquiring cell culture adaptive mutations. The replacement of not only the NS3 protease gene region, but also its cofactor NS4A, allowed the generation of replication competent intra- and intergenotypic recombinants for all 6 major genotypes. Replacing the NS3 protease of the recombinants with that of patientderived proteases also generated replicating recombinants, greatly expanding the panel of intergenotypic recombinants available for phenotyping and PI evaluation. However, intra- and intergenotypic recombinants showed substantial differences in their replication kinetics, which may be influenced by naturally occurring polymorphism between genotypes and the differential requirement of adaptive/attenuating cell culture mutations. Genotype 1a recombinants replicated very poorly, which may be due to incompatibilities between the type 1a NS3/4A protease and the type 2a backbone. 50% inhibitory concentrations (IC50) of different PIs were measured using Foci Forming Units/ml (FFU/ml) reductions and replication inhibition assays. The different recombinants showed consistent, genotype-associated differences in their susceptibility to the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showing approximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants. These observations are consistent with major differences in response rates found in recent treatment trials of genotype 1, 2 and 3 infected patients. Differences in susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derived recombinants being twice as susceptible than genotype 3a, 4a and 5a derived recombinants. Passaging the intra- and intergenotypic recombinants under increasing concentrations of PI allowed the identification of PI resistance mutations. Resistance mutations to BILN 2061 mapped to the previously identified positions 156 and 168 within the NS3 protease, with a great diversity of amino acid substitutions observed within each genotype. Reintroduction of the identified resistance mutations into the original recombinant viruses conferred increased resistance towards BILN 2061 and some mutations also affected replication kinetics of the recombinants. The developed system will be of major value for the phenotypic characterisation of naturally occurring and treatment induced resistance mutations within all 6 major HCV genotypes towards different PIs. This will allow treatment response predictions for newly developed PIs before they enter clinical trials and the development of individually tailored antiviral treatment regimes.
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Recombinant expression and bioinformatic analysis of the Hepatitis B virus X proteinThompson, Liam Jed 18 September 2012 (has links)
There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which
approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer.
The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer
and has been reported to manipulate several critical cellular pathways. These include the cell cycle,
the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role
of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of
animal models and infectable cell lines together with solubility and stability issues related to the
HBx protein have made progress difficult. The reliance on approximate cellular and animal models
has yielded many discordant studies that have confounded our interpretations of the role of HBx.
There have been no novel approaches attempting to express HBx at a quantity and quality sufficient
for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no
bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be
responsible for these challenges have not been reported.
This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble
and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus
(SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein
protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors
showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and
human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed
HBx. This result was despite the positive visualisation of HBx using the same antibodies
and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity
of HBx. Furthermore these findings supported the decision to develop eukaryotic-based
HBx expression vectors as results suggested structural differences between eukaryote and prokaryote
expressed protein. HBx was subsequently detected and purified in a soluble and active form
using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point
from which further work at optimising HBx expression and structural elucidation can occur.
Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive
sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on
HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly
regulated and thus experimental systems employing overexpression methodologies may encourage
cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary
analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding
synonymous and non-synonymous substitutions at the overlapping and non-overlapping
domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution
occurred in the overlapping partner gene. The presence of sites under positive, neutral and
negative selection were identified across the length of HBx. The different genotypes showed positive
selection indicating selective pressures unique to each, thus offering a contributing explanation for
the variable disease severity observed between the subtypes.
Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and
mammalian cells. These methods, together with an increased understanding of the nature of HBx
sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological
assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the
progression to liver cancer.
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