The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

No description available.

Made available in DSpace on 2014-06-12T22:58:02Z (GMT). No. of bitstreams: 2 arquivo4051_1.pdf: 1604491 bytes, checksum: 8aeeece559f4a22d47226afdc43c3681 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A infecção oral pelo vírus herpes simples (HSV) é comum em indivíduos co-infectados pelo vírus da imunodeficiência humana (HIV). Entretanto, esta infecção é eventualmente assintomática. A lectina ligadora de manose (MBL) é uma proteína que apresenta um importante papel na imunidade inata, pois se liga a vários patógenos, mediando a opsonofagocitose diretamente e através da ativação do sistema complemento. Existem três tipos de polimorfismos do gene MBL-2 que resultam em baixos níveis plasmáticos da MBL, que têm sido relacionados com maior índice de infecções recorrentes, principalmente nos grupos de pacientes imunocomprometidos. O objetivo do presente estudo é avaliar a possível relação entre o polimorfismo do exon 1 do gene MBL-2 e as manifestações orais do HSV em portadores do HIV. Materiais e métodos: Foi realizado um estudo observacional do tipo caso-controle com uma amostra constituída de 64 indivíduos infectados pelo HIV e 65 com sorologia desconhecida para o HIV. Foi realizado exame clínico da mucosa e dos tecidos da face, além de questionamento em relação à presença de manifestações orais do HSV e posteriormente foram coletadas células de descamação da mucosa oral. A detecção dos polimorfismos do gene MBL-2 e a presença do HSV-1 DNA foram realizadas através da curva de Melting após amplificação pela metodologia da PCR em tempo real. Resultados: Dentre os 64 pacientes HIV positivos 19 (29,6%) relataram sinais e sintomas compatíveis com a infecção pelo HSV na cavidade oral. Destes, foi possível amplificar o HSV-1 DNA em 21% (4/19), e em 13,3% (6/45) dos assintomáticos. Não houve diferença estatisticamente significativa entre os sintomáticos (p=1) e os assintomáticos (p=0,5292) do grupo de estudo e o grupo controle. Com relação às alterações do gene MBL-2, observou-se que 48,8% dos pacientes HIV positivos assintomáticos para o HSV mostraram um genótipo homozigoto dominante (A/A), 35,5% heterozigoto (A/0), e 15,5% do tipo 0/0. Enquanto que nos sintomáticos a ocorrência foi de 52,6% para o genótipo A/A, 26,3% A/0, e 21% 0/0. Os diferentes genótipos encontrados não contribuíram para as manifestações orais do HSV tanto no grupo de portadores do HIV (p=0,8144) como no grupo controle (p=0,4513), não havendo diferença estatisticamente significante entre eles (p=0,1937).Conclusão: Não foi encontrada relação estatisticamente significativa entre as alterações polimórficas no exon-1 do gene MBL-2 e manifestações orais do HSV em pacientes HIV positivos

Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.

No description available.

No description available.

No description available.

No description available.