Structure biology concerns the study of the molecular structures of biological macromolecules, such as proteins, and how these relate to the function. Protein structures are also of importance in structure-based drug design. In this thesis, the work has been carried out in two different projects. The first project concerns structural studies of proteins from the bacterium Escherichia coli and the second of proteins from five different herpesviruses.  The E. coli project resulted in the structural characterization of three proteins: CaiB, RibD, and YhaK. CaiB is a type-III CoA transferase involved in the metabolism of carnitine. Its molecular structure revealed a spectacular fold where two monomers were interlaced forming an interlocked dimer. RibD, a bi-functional enzyme, catalyzes two consecutive reactions during riboflavin biosynthesis. In an attempt to characterize the mechanism of action of the N-terminal reductase domain, the structure of RibD was also determined in two binary complexes with the oxidized cofactor, NADP+, and with the substrate analogue ribose-5-phosphate. YhaK is a protein of unknown function normally found in low abundance in the cytosol of E. coli and was previously annotated to be a member of the Pirin family. However, some structural features seem to distinguish YhaK from these other Pirin proteins and we showed that YhaK might be regulated by reactive oxygen species.  The Herpesvirus project resulted in the structural determination of two proteins, the SOX protein and ORF60 from Kaposi’s sarcoma associated herpesvirus (KSHV). SOX, a bi-functional shutoff and exonuclease protein, is involved in the maturation and packaging of the viral genome into the viral capsid and in the host shutoff of cellular proteins at the mRNA level. The SOX structure was also used for modeling DNA binding. The crystallization and preliminary structural studies of ORF60, the small R2 subunit of the ribonucleotide reductase (RNR) from KSHV is also discussed. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript.

El objetivo del presente estudio fue determinar la permisibilidad de los cultivos celulares secundarios de alpaca y llama a la infección por distintos agentes virales de conocida seroprevalencia en este tipo de ganado. Se establecieron dos líneas celulares de cornete nasal y piel de alpaca y llama infectándose con Virus de la diarrea viral bovina (VDVB), Virus Herpes Bovino tipo 1 (VHB-1), Virus respiratorio Sincitial Bovino (VRSB) y Virus Parainfluenza bovino tipo 3(VPI-3). Se determinó y caracterizó la presentación de efectos citopatogénicos (ECP) por medio de microscopia óptica de las monocapas teñidas con Hematoxilina-Eosina (HE). Se confirmó la presencia de antígenos virales por medio de la prueba de Inmunofluorescencia Directa (IF). Los cultivos celulares secundarios de piel y cornete nasal de llama y alpaca fueron permisibles a la infección de los distintos virus, presentando los ECP característicos de cada uno de ellos. Esto demuestra que las células de alpaca y llama cultivadas in vitro presentan receptores homólogos a los presentes en células bovinas y determina que este tipo de cultivos es un modelo apropiado para ensayos de infección viral. Palabras Claves: alpacas, llamas, líneas celulares, permisibilidad, Virus de la diarrea viral bovina (VDVB), Virus Herpes Bovino tipo 1 (VHB-1), Virus respiratorio Sincitial Bovino (VRSB) y Virus Parainfluenza bovino tipo 3(VPI-3), Efectos citopatogénicos (ECP). / In order to determine the permisibility of alpaca and llama cell cultures to infection by various viral agents of known seroprevalence, nasal turbinate and skin cell lines of alpaca and llama were established and infected with Bovine Viral Diarrhoea Virus (BVDV), Bovine Herpes Virus type 1 (BHV-1), Bovine respiratory Sincitial Virus (BRSV) and Bovine Parainfluenza Virus type 3 (BPIV-3). Presentation of citopathogenic effect (CPE) was determined and characterized by optical microscopy of Hematoxilin-Eosine stained monolayers. The presence of viral antigen was confirmed by Direct Immunofluorescence. Every cell line was permisible to infection with the four viral strains, showing the characteristic CPE. These results prove that alpaca and llama cells cultured in vitro show homologue receptors to those found in bovine cells and determine that these type of cultured cells repesent an appropriate model for viral infection assays. Key Words: alpaca, llama, cell lines, permisibility, Bovine Viral Diarrhoea Virus (BVDV), Bovine Herpes Virus type 1 (BHV-1), Bovine respiratory Sincitial Virus (BRSV) and Bovine Parainfluenza Virus type 3 (BPIV-3), Citopathogenic effect (CPE).

El Virus Herpes Canino tipo 1 (VHC-1) es responsable de la enfermedad hemorrágica canina en cachorros menores de cuatro semanas de vida y de algunos problemas reproductivos en perras adultas. En el Perú la enfermedad no ha sido reportada, aunque existen hallazgos que sugieren su presencia. El objetivo del presente estudio fue demostrar la presencia de anticuerpos contra el VHC-1, en la población canina de la provincia de Lima con antecedentes asociados a problemas reproductivos. Para ello se recolectaron muestras de suero de 28 animales procedentes de 7 distritos de la provincia de Lima que tuvieron algún antecedente relacionado a problemas reproductivos y/o mortalidad neonatal para someterlos a la prueba de Inmunofluorescencia indirecta (IFI). De los 28 animales muestreados, 9 (32 ± 17 %) resultaron positivos a la prueba, de estos, 5/9 (56%) fueron machos y 4/9 (44%) hembras. De acuerdo al distrito, 5/7 (71%) de los distritos muestreados tenían algún animal positivo y las edades de los mismos fluctuaban entre 8 meses y 10 años. / The canine Herpesvirus type 1 (CHV-1) is responsible for the canine hemorrhagic disease in puppies aged less than four weeks and for any reproductive problems in adult bitches. In Peru, this disease has been not reported, althoug there is findings suggesting its presence. Thus, the aim of this study was to show the presence of antibodies against CHV-1 among the canine population associated with antecedents to reproductive problems in the province of Lima. Blood serum samples coming from 28 animals belonging to 7 districts of the province of Lima were collected. These samples that have been related to any reproductive problems antecedent and/or neonatal mortality were submited to the indirect inmunofluorescens test (IFAT). From the total samples, 9 of them (32 ± 17%) turned out to be positive to the test, besides 5/9 (56%) were male and 4/9 (44%) were female. According to the district, 5/7 (71%) of them had some positive animal which were aged 8 months to 10 years.

Epstein-Barr Virus (EBV) has two states of infection, latent and lytic. During the latent state the viral genome remains stable in cells as episomes and replicates with cellular DNA. During the lytic cycle the viral DNA becomes amplified and packaged in newly formed virions. An unsolved problem is whether newly replicated EBV DNA produced upon lytic cycle activation is associated with histones, and if so, whether these histones are acetylated. This question has biological significance as knowing the chromatin structure of genes is important in determining their function and expression profile. Our hypothesis is that newly synthesized EBV lytic DNA is associated with histones and the histone tails are selectively acetylated. To investigate our hypothesis we performed chromatin immunoprecipitation (ChIP) in HH514-16 cells, a Burkitts Lymphoma cell line, during latent and lytic replication. We used quantitative PCR (qPCR) to detect the relative concentration of DNA among the different samples. We tested three different variables: type of inducing agent, duration of treatment, and different regulatory regions in the genome of Epstein-Barr Virus. We found that in cells induced into the lytic cycle with Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), association of newly replicated EBV DNA with acetylated histone 3 (H3) increased ~ 6-10 fold. This increase in association was greatest 72 hrs after treatment. Furthermore, activation of lytic viral replication in HH514-16 cells using a different inducing agent, Azacytidine (AZC), which is known to function as a DNA methyltransferase inhibitor, increased binding of H3 with viral DNA ~8 fold. However, unlike TSA, AZC increased the acetylation state of histones bound to newly synthesized viral DNA only ~ 2 fold. Changing the regulatory region of the EBV genome analyzed in qPCR did not affect our results. Our results suggest that newly replicated viral DNA is associated with histones, a fraction of which are acetylated. The degree of acetylation likely depends on the agent used to induce the lytic cycle. H3 is highly acetylated when an HDACi is used and less acetylated when AZC is used. Our study provides new insight on the epigenetic profile of newly replicated viral DNA during the lytic cycle. It remains to be determined whether histones are packaged together with viral genomes into virions and whether the chromatin state of virion DNA affects gene expression after the virus enters uninfected cells.

Reproduction de : Thèse d'exercice : Médecine générale : Nancy 1 : 2002. / Thèse : 2002NAN11055. Titre provenant de l'écran-titre.

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on Mar. 4, 2010). Includes bibliographical references (p. 65-72).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 77-89).

Human cytomegalovirus (HCMV), a beta herpesvirus, persists indefinitely in the human host through a life-long, latent infection. HCMV is associated with life threatening pathologies in the immune naïve or compromised and, therefore, understanding of the mechanisms of viral persistence is imperative to human health. The ULb' region of the HCMV genome is selectively lost in high-passage strains of the virus, yet retained in low-passage strains. As such, the ULb' is hypothesized to play a role in immune evasion, pathogenesis, latency, and dissemination. ULb' encoded viral products are poorly characterized, hindering a mechanistic understanding of HCMV persistence. We previously defined a 3.6-kb locus spanning UL133-UL138 within the ULb' region important to viral latency. UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and translation mechanisms. We mapped the origins of each isoform through advanced bacterial artificial chromosome recombineering, where each ATG was disrupted and the resulting UL136 recombinant virus was screened for altered expression of the pUL136 isoforms. Remarkably, 8 of the 11 potential translation initiation sites encoded within the ORF are utilized to create the pUL136 isoforms. The pUL136 isoforms have distinct localization and trafficking patterns within the cell, including varying degrees of Golgi association, suggesting each isoform may interface with different cellular components and pathways. Further characterization of UL136 recombinant viruses revealed a complex, antagonistic relationship between the pUL136 isoforms. In endothelial cells, which are important to viral persistence and dissemination due to their ability to maintain a slow, "smoldering" infection, the 33- and 26-kDa isoforms promote replication, while the 25-kDa isoform enhances their combined activity, and the 23-/19-kDa isoforms repress the activity of the 25-kDa isoform. The pUL136 isoforms are also required for virus maturation in endothelial cells, where the 33-kDa is required both for virion envelopment and efficient formation of the perinuclear viral assembly compartment. In both an in vitro CD34⁺ cell culture model of latency and an in vivo NOD-scid IL2Rɣc^(null) humanized mouse model, a virus lacking the 23-/19-kDa isoforms fails to establish latency, instead replicating and disseminating with increased efficiency while viruses lacking the 33- and 26-kDa isoforms fail to efficiently reactivate or disseminate. Our data suggest that the interplay between the pUL136 isoforms maintains an intricate balance of infection that governs replication, latency, and virus dissemination, which ultimately contributes to the role of the UL133/8 locus in mediating outcomes of HCMV infection.

In response to viral infection, host cells activate various antiviral defence mechanisms to inhibit virus replication. Therefore in order for a virus to replicate efficiently, it must counteract cellular antiviral defence. Promyelocytic leukemia protein (PML) is a cellular protein involved in intrinsic immunity. It inherently forms nuclear bodies (PML-NBs) that assemble at the site of viral genomes. Proteins related to epigenetic regulation are recruited to PML-NBs, and silence viral gene transcription. This study focuses on the role of two herpes simplex virus type 2 (HSV-2) proteins, ICP0 and Us3, in disrupting PML-NBs and counteracting cellular antiviral defence. En passant mutagenesis was used to create recombinant HSV-2 viruses lacking ICP0, Us3, or both ICP0 and Us3. Growth analysis of these recombinants indicates no growth defects for the ICP0Δ virus, while the Us3Δ virus grows to one log lower titres than wild type virus (WT). By contrast, the ICP0Δ virus displays a delay in PML-NB disruption, but the Us3Δ virus is as efficient as WT. However, Us3 is still important for PML-NB disruption, since the ICP0Δ/Us3Δ double mutant exhibits a greater delay than the ICP0Δ single mutant. Although PML is a mediator of the interferon (IFN) response and it was predicted that ICP0 and Us3 interfere with the IFN response through disruption of PML-NBs, my results show that only some HSV-2 Us3Δ clones are hypersensitive to the effects IFN, and others are resistant. Us3 affects more than one cellular pathway, and those cellular pathways are affected by more than one viral protein. I conclude that the activities of multiple viral proteins create a fine balance between activating cellular pathways to promote virus replication, and inhibiting cellular antiviral defence. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-01-23 10:55:16.715

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.