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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
111

Characterization Of Structural And Non-structural Proteins Of Positive Sense, Single-stranded RNA Plant Viruses

Mathur, Chhavi 06 1900 (has links) (PDF)
In the present thesis, two positive sense single-stranded RNA viruses have been used as models to understand the structure and function of viral-encoded proteins. One of them, Pepper Vein Banding Virus (PVBV; genus Potyvirus; family Potyviridae) is a flexuous, rod-shaped virus that encodes for a polyprotein of size ~340 kDa. The polyprotein undergoes proteolytic processing by viral-encoded proteases, of which Nuclear Inclusion-a Protease (NIa-Pro) is the major protease. It is a serine-like cysteine protease which cleaves between a Q/A or Q/S, present in the context of the heptapeptide recognition sequence. The temporal regulation of intermediates and mature proteins released by NIa-Pro cleavage is crucial for a successful infection. In the present study, histidine-tagged NIa-Pro, Viral Protein genome-linked (VPg), and the cleavage site mutant (E191A) VPg-Pro were over-expressed in E. coli and purified. The protease activity of NIa-Pro was monitored using an HPLC-based protease assay developed using a peptide substrate. NIa-Pro protease activity was found to get modulated upon interaction with VPg and upon undergoing phosphorylation. Both these events have been found to involve the face of NIa-Pro which contains the solvent-exposed Trp143. Mutational studies and molecular dynamics analyses provide evidence that this residue is buried upon interaction of NIa-Pro with VPg, and any perturbation of its orientation influences the active site Cys151 via an extensive interaction network. This interaction was found to enhance the velocity of NIa-Pro protease activity, especially if the two domains were present in trans (VPg+Pro). In addition, the main-chain –NH2 group of Trp143 was found to be hydrogen-bonded to the side chain –OH group of Ser129, the residue which was identified to undergo phosphorylation by host plant kinases. Interestingly, when the two domains were present in cis (E191A VPg-Pro), no phosphorylation was observed. Mutations of Ser129 (to phosphorylation-mimic Asp or phosphorylation-deficient Ala residues) which affected this H-bond were found to disturb Trp143 and Cys151 orientation, which drastically reduced the protease activity of NIa-Pro. Within the polyprotein, VPg is present at the N-terminus of NIa-Pro and the cleavage site between them is suboptimal (E/A). In the present study, VPg-Pro was shown to be covalently linked to the genomic RNA present in the virions. Interestingly, during purification, VPg could only be purified from the soluble when it was expressed at the N-terminus of NIa-Pro. A series of bioinformatics and biophysical analysis of VPg showed that PVBV VPg, like other potyviral VPgs, exists as a molten-globule. Moreover, while VPg was shown to harbour the Walker motifs, it was found to exhibit an ATPase activity only when it was present with the NIa-Pro (especially in cis). Lys47 and Asp88:Glu89 were found crucial for optimal activity. Over all the results demonstrated that there is a reciprocal modulation of structure and function of the VPg and NIa-Pro domains. These results can explain the possible significance of an impeded cleavage rate between the two domains of VPg-Pro during PVBV infection. The precursor, VPg-Pro, could offer the advantage of evading the inhibitory phosphorylation of NIa-Pro by the host, as well as drive certain viral processes by virtue of its ATPase activity. And subsequent cleavage of the domains and their trans interaction could offer a higher turnover rate which might assist sufficient CP production required for viral morphogenesis. Another virus, Tobacco Streak Virus (TSV) that belongs to the Ilarvirus genus of the Bromoviridae family is a spherical virus which forms pleiomorphic icosahedral virus particles. It has a tripartite genome and each RNA is encapsidated individually. In the present thesis, TSV was used as a model to understand the properties of its structural protein-the coat protein (CP), with the aim of deciphering TSV assembly process. Thus, the CP gene from TSV RNA 3 was cloned and over-expressed in E. coli. The coat protein thus expressed formed virus-like particles (VLPs), which could be disassembled into dimers using high CaCl2 concentrations. Reassembly of VLPs was possible from dimers even in the absence of any nucleic acid. Mutational analysis of the N-terminal disordered domain showed that 26 amino acid residues from the amino-terminus could be crucial for capsid heterogeneity while, zinc-binding domain was essential for assembly. Overall, the present study shows that the flexible W-C loop of PVBV NIa-Pro, the disordered N-terminal region of PVBV VPg and the disordered N-terminal region of TSV CP harbour residues crucial for regulation of protein function. Such regulatory elements would ultimately allow viruses to maintain a smaller protein number, and thus a smaller genome size.
112

Studies On Phosphorylation And Oligomerization Of Rotavirus Nonstructural Protein 5 (NSP5) And Cellular Pathways That Regulate Virus Replication

Namsa, Nima Dondu 07 1900 (has links) (PDF)
Rotavirus is one of the leading etiological agents of gastroenteritis in young of many species including humans worldwide and is responsible for about 600,000 infant deaths per annum. Rotavirus belongs to the Reoviridae family, and its genome is composed of 11 double-stranded RNA segments that encode six structural proteins and six nonstructural proteins. Rotavirus replication is fully cytoplasmic and occurs within highly specialized regions called viroplasms. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm¬like structures. A recent study suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins, containing four structural (VP1, VP2, VP3 and VP6) and two nonstructural (NSP2 and NSP5) proteins. NSP5, the translation product of gene segment 11 has a predicted molecular eight of 21 kDa. NSP5 has been reported to exist in multiple isoforms ranging in size from 28-and 32-35 kDa from a 26-kDa precursor has been attributed to O-glycosylation and hyperphosphorylation. To study different properties of the protein, recombinant NSP5 containing an N-terminal hisidine tag was expressed in bacteria and purified by affinity chromatography. A significant observation was the similarity in phosphorylation property of the bacterially expressed and that expressed in mammalian cells. While the untagged recombinant protein failed to undergo phosphorylation in vitro, addition of His tag or deletions at the N-terminus promoted phosphorylation of the protein in vitro, which is very similar to the reported properties exhibited by the corresponding proteins expressed in mammalian cells. Phosphorylation of NSP5 in vitro is independent of the cell type from which the extract is derived suggesting that the kinases that phosphorylate NSP5 are distributed in all cell types. Among the C-terminal deletion mutants studied, NH-∆C5 and NH-∆C10 were phosphorylated better than full-length NSP5, but NH-∆C25 and NH¬∆C35 showed substantial reduction in the level of phosphorylation compared to full-length NSP5. These results indicate that the C-terminal 30 residues spanning the predicted α-helical domain of NSP5 are critical for its phosphorylation in vitro which is in correspondence with previous findings that C-terminal 21 amino acids of NSP5 direct its insolubility, hyperphosphorylation, and VLS formation. The results revealed that though the tagged full-length and some of the mutants could be phosphorylated in vitro, they are not suitable substrates for hyperphosphorylation unlike the similar proteins expressed in mammalian cells or infected cells. Analysis by western blot and mass spectrometry revealed that the bacterially expressed NH-NSP5 is indeed phosphorylated. It appears that prior phosphorylation in bacteria renders the protein conformationally not amendable for hyperphosphorylation by cellular kinases in vitro. Mutation of the highly conserved proline marginally enhanced its phosphorylation in vitro but the stability of protein is affected. Notably, mutation of S67A, identified as a critical residue for the putative caesin kinase-I and-II pathways of NSP5 phosphorylation, affected neither the phosphorylation nor the ATPase activity of NSP5. These results suggest that bacterially expressed NSP5 by itself has undectable auto-kinase activity and it is hypophosphorylated. Purified recombinant NSP5 has been reported to possess an Mg¬ 2+-dependent ATP-specific triphosphatase activity. The results indicated that deletion of either C-terminal 48 amino acids or N-terminal 33 residues severely affected the ATPase activity of recombinant NSP5, underlying the importance of both N-and C-terminal domains for NSP5 ATP hydrolysis function. NSP5 expressed in rotavirus infected cells exists as inter-molecular disulfide-linked dimeric forms and it appears that the 46 kDa isoforms, that are phosphorylated, corresponds to dimer as revealed by western blotting. Analytical gel filtration analysis of NH-NSP5, NH-ΔN43 and NH-ΔN33-ΔC25 showed most of the proteins in void volume, but an additional peak corresponding to the mass of dimeric species further supports that NSP5 is basically a dimer that undergoes oligomerization. Analysis by sucrose-gradient fractionation revealed that NH-NSP5 and NH-ΔN43 proteins were mainly distributed in the lower fraction of the gradient suggesting the existence of high molecular weight complexes or higher oligomers. The multimeric nature of NSP5 and its mutants was further confirmed by dynamic light scattering which suggests that high molecular weight complexes are of homogenous species. The correlation curves showed a low polydispersity distribution and a globular nature of recombinant NH-NSP5 proteins. The present results clearly demonstrate that dimer is the basic structural unit of NSP5 which undergoes oligomerization to form a complex consisting of about 20-21 dimers. The nonstructural protein 5 is hyperphosphorylated in infected cells and cellular kinases have been implicated to be involved in its phosphorylation. NSP5 contains multiple consensus sites for phosphorylation by several kinases, but the cellular kinases that specifically phosphorylate NSP5 in infected cells are yet to be identified. Previous studies from our laboratory using signaling pathway inhibitors revealed that recombinant NH¬NSP5 and its deletion mutants can be phosphorylated in vitro by purified cellular kinases and by mammalian cell extracts. These studies also showed the involvement of PI3K-Akt and MAPK signaling pathways in NSP5 phosphorylation and a negative role for GSK3β in the phosphorylation of bacterially expressed recombinant NSP5 in vitro. In the present work, using phospho-specific anti-Ser9 GSK3β antibody, we observed that GSK3β is inactivated in a rotavirus infected MA104 cells in a strain-independent manner. GSK3β¬specific small interfering RNA (siRNA-GSK3β) reduced GSK3β levels leading to increased level of synthesis of the structural rotavirus protein VP6 and NSP5 hyperphosphorylation compared to control siRNA. The pharmacological kinase inhibitors (LY294002, Genistein, PD98059, and Rapamycin) studies at the concentrations tested did not significantly affect rotavirus infection as seen from the number foci, while U0126 severely affected rotavirus replication. The results clearly demonstrated the importance of the MEK1/2 signaling pathway in the successful replication of rotavirus and NSP5 hyperphosphorylation in rotavirus-infected cells. In contrast inhibition of GSK3β activity by LiCl, increased in general, the number of foci by greater than 2-fold for all viral strains studied. These results suggest that MEK1/2 pathway majorly contributes to GSK3β inactivation in rotavirus infected cells. Thus, our results reveal that rotavirus activates both the PI3K/Akt and FAK/ERK1/2 MAPK pathways and appears to utilize them as a strategy to activate mTOR, and inhibit GSK3β through phosphorylation on serine 9, the negative regulator of rotavirus NSP5 phosphorylation, and thus facilitate translational competence of rotaviral mRNAs during virus replication cycle. It was shown previously in the laboratory by co-immunoprecipitation assay that Hsp70 interacts with rotaviral proteins VP1 and VP4 in rotavirus-infected mammalian cells. In this study, the interactions between Hsp70 with VP1 and VP4 were further evaluated in vitro by GST-pull down assay. It was observed that the N-terminal ATPase and C-terminal peptide-binding domain of Hsp70 is necessary for its direct interaction with VP1 and VP4. The presence of Hsp70 in purified double-and triple-layered virus particles further supported the observed interactions of rotaviral proteins VP1 and VP4 with Hsp70. However, the specific interaction observed between Hsp70 and rotaviral capsid proteins, VP1 and VP4 in viral particles suggests that Hsp70 has an important role during rotavirus assembly which requires further investigation.
113

Control of Bovine Papillomavirus E2 Function By Acetylation and the Novel E2 Interacting Protein RINT1: A Dissertation

Quinlan, Edward J. 27 January 2012 (has links)
Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.
114

Participação de proteínas da via secretória no tráfego e montagem do vírus sincicial respiratório / Participation of proteins in secretory route traffic and assembling of respiratory syncytial virus

Cardoso, Ricardo de Souza 11 March 2016 (has links)
O vírus sincicial respiratório humano (HRSV) é o mais frequente agente patogênico da família Paramyxoviridae. Apesar de sua grande importância e impacto em saúde pública, alguns aspectos demandam elucidação. Entre eles, estão os mecanismos de tráfego intracelular de proteínas virais para o sitio de montagem. Baseado nisso, fizemos um estudo de imunofluorescência tentando contribuir para o entendimento da participação da via secretória no tráfego de proteínas estruturais de HRSV que não são glicosiladas: proteínas de matriz (M) e de nucleocapsídeo (N). Pudemos observar que essas proteínas seguem rota similar àquelas que são glicosiladas no Golgi, como a proteína de fusão (F). Ademais, as proteínas M e N, além de colocalizarem com proteínas celulares da via secretória, tais como trans-Golgi network-46 (TGN46) e sorting nexin-2 (SNX2), também influem no recrutamento de proteínas celulares para os corpos de inclusão virais, como mostrado no caso da proteína Glut1. Os dados indicam que proteínas M e N de HRSV seguem pela via endocítica inicial, acumulam-se em corpos de inclusão que seriam fábricas virais e, no caso de TGN46, podem ser incorporadas aos vírus em brotamento / Human respiratory syncytial virus (HRSV) is the most relevant cause of respiratory infection in children worldwide. Despite its importance in public health, some aspects of the mechanisms of the trafficking of viral structural proteins remain unclear. In the present study, immunofluorescence was used to understand how the virus matrix (M) and nucleocapsid (N) proteins, which are non-glycosylated , are addressed to inclusion bodies in Hep-2 cells (MOI=3). M and N proteins followed similar intracellular trafficking routes as compared to the glycosylated fusion (F) viral protein. Moreover, M and N proteins colocalized with two key elements of the secretory pathway: trans-Golgi network- 46 (TGN46) and sorting nexin-2 (SNX2). Viral proteins M and N appear to be involved in the recruitment of cell proteins at the formation of virus inclusion bodies, as shown for Glucose Transporter Type 1 (Glut1). The data suggest that HRSV M and N proteins follow the secretory pathway, initiating in early endosomes, as indicated by the co-localization with TGN46 and SNX2. In addition, these host cell proteins accumulate in inclusion bodies that are viral factories, and can be part of budding viral progeny. Therefore, HRSV M and N proteins, even though they are not glycosylated, take advantage of the secretory pathway to reach virus inclusion bodies. Confocal images suggest that SNX2, which is known for its membrane-deforming properties, could play a pivotal role in HRSV budding
115

Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication

Boyne, J. R., Colgan, K. J., Whitehouse, A. January 2008 (has links)
A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.
116

The in silico prediction of foot-and-mouth disease virus (FMDV) epitopes on the South African territories (SAT)1, SAT2 and SAT3 serotypes

Mukonyora, Michelle 24 January 2017 (has links)
Foot-and-mouth disease (FMD) is a highly contagious and economically important disease that affects even-toed hoofed mammals. The FMD virus (FMDV) is the causative agent of FMD, of which there are seven clinically indistinguishable serotypes. Three serotypes, namely, South African Territories (SAT)1, SAT2 and SAT3 are endemic to southern Africa and are the most antigenically diverse among the FMDV serotypes. A negative consequence of this antigenic variation is that infection or vaccination with one virus may not provide immune protection from other strains or it may only confer partial protection. The identification of B-cell epitopes is therefore key to rationally designing cross-reactive vaccines that recognize the immunologically distinct serotypes present within the population. Computational epitope prediction methods that exploit the inherent physicochemical properties of epitopes in their algorithms have been proposed as a cost and time-effective alternative to the classical experimental methods. The aim of this project is to employ in silico epitope prediction programmes to predict B-cell epitopes on the capsids of the SAT serotypes. Sequence data for 18 immunologically distinct SAT1, SAT2 and SAT3 strains from across southern Africa were collated. Since, only one SAT1 virus has had its structure elucidated by X-ray crystallography (PDB ID: 2WZR), homology models of the 18 virus capsids were built computationally using Modeller v9.12. They were then subjected to energy minimizations using the AMBER force field. The quality of the models was evaluated and validated stereochemically and energetically using the PROMOTIF and ANOLEA servers respectively. The homology models were subsequently used as input to two different epitope prediction servers, namely Discotope1.0 and Ellipro. Only those epitopes predicted by both programmes were defined as epitopes. Both previously characterised and novel epitopes were predicted on the SAT strains. Some of the novel epitopes are located on the same loops as experimentally derived epitopes, while others are located on a putative novel antigenic site, which is located close to the five-fold axis of symmetry. A consensus set of 11 epitopes that are common on at least 15 out of 18 SAT strains was collated. In future work, the epitopes predicted in this study will be experimentally validated using mutagenesis studies. Those found to be true epitopes may be used in the rational design of broadly reactive SAT vaccines / Life and Consumer Sciences / M. Sc. (Life Sciences)
117

Host-Pathogen Interactions in Hepatitis C Virus Infection : Deciphering the Role of Host Proteins and MicroRNAs

Shwetha, S January 2015 (has links) (PDF)
Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.
118

Participação de proteínas da via secretória no tráfego e montagem do vírus sincicial respiratório / Participation of proteins in secretory route traffic and assembling of respiratory syncytial virus

Ricardo de Souza Cardoso 11 March 2016 (has links)
O vírus sincicial respiratório humano (HRSV) é o mais frequente agente patogênico da família Paramyxoviridae. Apesar de sua grande importância e impacto em saúde pública, alguns aspectos demandam elucidação. Entre eles, estão os mecanismos de tráfego intracelular de proteínas virais para o sitio de montagem. Baseado nisso, fizemos um estudo de imunofluorescência tentando contribuir para o entendimento da participação da via secretória no tráfego de proteínas estruturais de HRSV que não são glicosiladas: proteínas de matriz (M) e de nucleocapsídeo (N). Pudemos observar que essas proteínas seguem rota similar àquelas que são glicosiladas no Golgi, como a proteína de fusão (F). Ademais, as proteínas M e N, além de colocalizarem com proteínas celulares da via secretória, tais como trans-Golgi network-46 (TGN46) e sorting nexin-2 (SNX2), também influem no recrutamento de proteínas celulares para os corpos de inclusão virais, como mostrado no caso da proteína Glut1. Os dados indicam que proteínas M e N de HRSV seguem pela via endocítica inicial, acumulam-se em corpos de inclusão que seriam fábricas virais e, no caso de TGN46, podem ser incorporadas aos vírus em brotamento / Human respiratory syncytial virus (HRSV) is the most relevant cause of respiratory infection in children worldwide. Despite its importance in public health, some aspects of the mechanisms of the trafficking of viral structural proteins remain unclear. In the present study, immunofluorescence was used to understand how the virus matrix (M) and nucleocapsid (N) proteins, which are non-glycosylated , are addressed to inclusion bodies in Hep-2 cells (MOI=3). M and N proteins followed similar intracellular trafficking routes as compared to the glycosylated fusion (F) viral protein. Moreover, M and N proteins colocalized with two key elements of the secretory pathway: trans-Golgi network- 46 (TGN46) and sorting nexin-2 (SNX2). Viral proteins M and N appear to be involved in the recruitment of cell proteins at the formation of virus inclusion bodies, as shown for Glucose Transporter Type 1 (Glut1). The data suggest that HRSV M and N proteins follow the secretory pathway, initiating in early endosomes, as indicated by the co-localization with TGN46 and SNX2. In addition, these host cell proteins accumulate in inclusion bodies that are viral factories, and can be part of budding viral progeny. Therefore, HRSV M and N proteins, even though they are not glycosylated, take advantage of the secretory pathway to reach virus inclusion bodies. Confocal images suggest that SNX2, which is known for its membrane-deforming properties, could play a pivotal role in HRSV budding
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Development of a novel cell-based screening platform to identify inhibitors of viral interferon antagonists from clinically important viruses

Vasou, Andri January 2016 (has links)
All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.
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Mechanism Of Replication Of Sesbania Mosaic Virus (SeMV)

Govind, Kunduri 02 1900 (has links) (PDF)
No description available.

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