Distribuce míst integrace exprimovaných provirů

Pro zajištění účinné exprese svých genů integrují retroviry provirové kopie svých genomů do genomů infikovaných buněk. Epigenetické procesy však mohou narušit a umlčet expresi integrovaných provirů. Takovéto umlčování sice zpomaluje šíření virové infekce, ale vytváří také reservoir latentních provirů, který v důsledku brání účinné léčbě retrovirových (např. HIV-1) infekcí. Umlčování integrujících se retrovirových vektorů navíc omezuje jejich účinou aplikaci v transgenezi či genové terapii. Cílem této práce je popsat interakce mezi expresí retrovirů a hostitelským (epi)genomikým prostředím v místech integrace provirů. Pro splnění stanoveného cíle jsme se rozhodli definovat (epi)genomické prostředí provirů, jejichž exprese není zasažena epigenetickým umlčováním. Jako modelové systémy jsme využili odlišné retrovirové vektory odvozené od ptačího sarkomového a leukózového viru (ASLV), myšího leukemického viru (MLV) nebo lidského viru získané imunodeficience typu 1 (HIV-1), jejichž expresní aktivita v lidských buňkách byla sledována. Za účelem popisu charakteristik míst integrace provirů rezistentních vůči umlčování jsme z infikované populace oddělili buňky nesoucí aktivní proviry, identifikovali místa integrace těchto provirů a porovnali charakteristiky takovýh míst s těmi získanými ze směsné, neselektované...To establish efficient expression of their genes, retroviruses integrate proviral copies into the genomes of the cells they have infected. Epigenetic events, however, silence expression of the integrated proviruses. This silencing protects host cells from harmful viral spread, but also creates a reservoir of latent proviruses that subsequently hinders the cure of retroviral (e.g., HIV-1) infections. Furthermore, the silencing of retrovirus-derived integrative vectors complicates their application in transgenesis and gene therapy. The goal of this thesis is to describe the interaction between retroviral expression and host (epi)genomic environment at the site of proviral integration. To pursue the goal, we sought to define the (epi)genomic environment of the proviruses, which expression is not affected by the epigenetic silencing. Diverse retroviral vectors derived from avian sarcoma and leukosis virus (ASLV), murine leukemia virus (MLV), and human immunodeficiency virus type 1 (HIV-1) were used as model retroviral systems, and expression stability of the vectors in human cell lines was examined. In order to identify the features unique to integration sites of the active proviruses, we sorted the cells positive for the proviral expression, identified their proviral integration sites, and compared them to...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

Expression and Characterization of the Rous Sarcoma Virus Gag Gene Product in Mammalian Cells Using an Sv40 Late Region Replacement Vector

Classical retrovirology had its birth in 1908 with Peyton Rous studying spontaneous sarcomas (tumors of the connective tissue) in chickens. He found that the sarcomas were transmissible and that the tumors showed greater virulence with each passage. He tested cell free filtrates for the presence of the etiological agent, and for the first time demonstrated that the entity involved in the transmissible sarcomas was a virus (Rous, 1911). This virus now bears his name, Rous sarcoma virus. Since Rous's famous discovery of a filterable entity that could induce tumor formation in infected chickens, countless other viruses have also been discovered that induced a variety of neoplastic growths in the infected host. With the new sophisticated techniques of today we have been able to probe the molecular events that occur during a retroviral infection in hopes of elucidating how these viruses not only induce their neoplastic growth but also the molecular events that must occur to allow viral replication. At the time of this thesis, a major obstacle inhibiting detailed molecular studies of Rous sarcoma virus replication is the lack of adequate immortalized avian cell lines and expression vector systems. This thesis therefore is written in two parts. The first part addresses the block of RSV replication in mammalian cells. The idea was to devise a system to study retroviral assembly in immortalized cell lines. If we can elucidate the differences between the avian and murine retroviruses that allow iii the murine viruses but not the avian viruses to replicate in mammalian cells, we might be able to better understand the molecular requirements for viral replication and assembly. In order to study this block to replication of avian retroviruses in mammalian cells, I chose to use the simian virus 40 (SV40) as a vector to express the RSV gag gene (or the viral polyprotein Pr769a9 ) in mammalian cells. The second part of this thesis arose from a problem encountered while using the SV40 virus as an eukaryotic expression vector. The strategy for using this virus as an expression vector is quite simple. The sequence of choice to be expressed, gag, is cloned into the late region of the SV40 virus genome in place of the late genes. The expression of the foreign gene is then driven from the late SV40 promoter. This recombinant vector can then be propagated with the help of a second SV40 virus that supplies the late gene products in trans. The problem was encountered while trying to propagate the recombinant vector. We were able to successfully express Pr76 but unable to passage any newly assembled recombinant virions to fresh monolayers. Instead of then abandoning the project, I chose to pursue the problem in hopes of finding the molecular block in SV40 replication. This is important since finding a sequence or protein that inhibits SV40 replication might prove to be a valuable tool for elucidating the mechanisms of viral replication.Microbiolog

The Role of Human Cytomegalovirus in Transformation and in the Development of Cervical Intraepithelial Neoplasia

The aim of this study was to investigate the role of human cytomegalovirus (HCMV) in transformation and its possible involvement in the development of cervical intraepithelial neoplasia (CIN). Although cell transformation by HCMV is well documented in the literature, the mode of transformation has not yet been elucidated. Nelson et al. (1982) identified a DNA sequence within the HindIII E fragment of the HCMV AD169 genome which when transfected into NIH 3T3 cells, can initiate colony formation in methylcellulose and tumourigenicity in nude mice. It was intended to extend these experiments to investigate the molecular basis for HCMV cell transformation. Repeated attempts to transform 3T3, human embryo lung (Helu) and rat embryo (RE) cells with the HCMV AD169 HindIII E fragment were unsuccessful. However, transformation of RE cells was repeatedly observed after infection of the cells with UV-irradiated HCMV AD169 virus. Southern blot analysis could not detect HCMV DNA in HCMV transformed cell and derived tumour cell lines. This suggested that the retention of viral DNA in the transformed cells may be transient and only necessary to initiate the transformation event. One mechanism by which HCMV DNA could initiate transformation is by activating a cellular proto-oncogene. Experiments were carried out to see if the DNA from a rat tumour induced after inoculation with HCMV transformed cells contained a transforming gene. The rat tumour DNA was found to contain a transforming gene that could be transfected into 3T3 cells and could initiate tumourigenesis when the transfected cells were injected into nude mice. As viral DNA could not be detected in the original HCMV transformed cells, the transforming gene must have been of cellular origin. Preliminary experiments suggested that HCMV may have activated a ras oncogene in the transformed cells. Past seroepidemiological studies have implicated HCMV as an oncogenic agent in the development of CIN and cervical carcinoma. The second aim of this study was to provide molecular evidence for an association between HCMV and CIN. The DNA from biopsies of 43 CIN patients in the West of Scotland was examined for the presence of HCMV DNA sequences by Southern blot analysis. Two biopsies, C2 and C17 were found to contain DNA sequences that hybridized to the HCMV AD169 HindIII E fragment. In C17 the hybridizable DNA sequences were only present at about 0.1 copy/cell and therefore in an amount too low to permit detailed sequence analysis. The hybridizable DNA sequences in C2 were present at about 20 copies/cell and were found to contain BamHI restriction fragments that comigrated with the BamHI P, W, c and e fragments of HCMV AD169. The hybridizable CIN DNA sequences may represent HCMV sequences that were retained in the tissue after HCMV infection and may have instigated the development of CIN. Two other BamHI fragments of C2 DNA were detected using the HCMV AD169 HindIII E fragment as a probe and these may represent rearranged sequences in the retained HCMV DNA. Rearrangements could have occurred when the viral DNA became integrated into the cellular DNA of this patient. As only a small amount of DNA was available from the C2 biopsy, further analysis of the viral sequences retained and the nature of the rearrangements was only possible if the relevant sequences could be cloned. A l library of the C2 DNA was constructed using the vector EMBL3 and three clones were isolated that contained C2 sequences that hybridized to the cloned HCMV AD169 HindIII E fragment. One of the clones was found to contain C2 sequences that hybridized to pAT vector sequences present in the probe. The other two clones contained C2 sequences that hybridized specifically to the HCMV AD169 HindIII E fragment but were unfortunately lost during the purification procedure after only 2 or 3 rounds of replication. Since HSV-2 and HPV have also been implicated in the development of CIN and cervical carcinoma, the same 43 CIN biopsies were analysed for the presence of HSV-2 and HPVII DNA sequences. Authentic HPVII DNA was detected in two CIN biopsies (C13 and C19) but no conclusive evidence of HSV-2 specific DNA could be found, although sequences that hybridized to vector pBR322 were detected. (Abstract shortened by ProQuest.)

Reconstitution and Characterization of Human Endogenous Retrovirus-K

Retroviruses are a family of clinically significant and scientifically fascinating viruses that infect a wide array of organisms from all vertebrate classes. The two hallmark events in the life cycle of retroviruses are the reverse transcription of the single stranded RNA (ssRNA) genome generating a double stranded DNA (dsDNA) and the integration of this dsDNA into the host genome. Because integration is irreversible and the infected cells are usually difficult to target for elimination in the host, the infection is generally permanent. HIV-1, the most important and well-studied member of all retroviruses, is the causative agent of acquired immune deficiency syndrome (AIDS) for which no vaccine or cure is known. Since recognition of the AIDS epidemic, around 25 million people have died from HIV-1 related causes, including 2 million in 2007. Currently, 33 million people are believed to be living with the virus, with most of these people living in sub-Saharan Africa, where 67% of all infected people reside and 75% of AIDS deaths occurred in 2007. When retroviruses infect germ cells or germ cell progenitors, the virus can become endogenized. These viruses, called endogenous retroviruses (ERV), make up more than 8% of the human genome. The integrated virus will be present in the genome of all cells of the individual derived from the infected germ cell, and be passed on to progeny in a Mendelian manner to following generations. Both chance and the insertion’s effect on the fitness of the host can determine the allelic frequency in the population. Hence, elements which produce large quantities of viral proteins and progeny or elements that insert into a necessary gene will likely reduce the fitness of the host and as an allele will be negatively selected in the host population. Currently, there is no known replication competent HERV, as most proviruses are filled with deletions and premature stop codons. However, one family of Class II HERV, HERV-K(HML-2), seems to have been replicating until recently. The HERV-K(HML-2) family includes human specific members and elements that are polymorphic in the human population, suggesting replication since the divergence of humans from chimpanzees 6 million years ago and potentially more recently as well. In this body of work, the problem of the lack of a replication competent virus sequence is circumvented by deducing a consensus sequence from the youngest set of HERV proviruses. Named HERV-KCON, we find that many of its components are functional individually and together enable infection of target cells in a single-cycle infection system. Using this system, we have characterized the previously unknown aspects of HERV-K(HML-2) life cycle, such as location of assembly and budding, dependency on cell replication, and more extensively, its ntegration site preference. HERV-KCON’s interaction with current anti-retroviral host proteins is accessed, and evidence of the same interaction occurring in vivo is presented in the context of APOBEC3G

Molecular Genetic Changes During Tumour Progression in Mouse Skin

This thesis describes the development of a model to analyse the genetic changes associated with tumour progression in mouse skin. Tumours were induced in F1 hybrid mice, thereby permitting the use of heterozygous DNA markers (restriction fragment length polymorphisms) to determine the role of allele loss in papilloma and carcinoma development. Frequently, initiation of mouse skin carcinogenesis involves H-ras activation. This gene is located on mouse chromosome 7. The F1 hybrid tumour model was used to demonstrate that tumours with this mutation also show loss of heterozygosity (LOH) or imbalance of alleles on chromosome 7 at a very high frequency. Thus LOH may indicate the presence of an oncogene, although it is often equated with tumour suppressor gene loss. Most frequently the alterations involved non-disjunction, but in some cases mitotic recombination or deletion was detected. These gross chromosome changes were not observed in mouse skin tumours lacking activated H-ras. Thus, it is clear that the initiation event can influence the type of alterations which occur at later stages of tumour progression. In the majority of cases, gross chromosome 7 changes result in an increased copy number of mutant H-ras and under-representation or loss of the normal allele, indicating that mutant H-ras is involved in both the initiation and progression of mouse skin tumours. It may be that elevation of the mutant signal is required to overcome a suppressive effect of the normal allele. In addition, because elevation of mutant H-ras gene copy number occurs by gross chromosomal mechanisms, it is possible that another chromosome 7 gene is also involved in tumour progression. In support of this, mitotic recombination or deletion was detected distal to H-ras in 4/26 of the chemically induced tumours with activated H-ras. In addition, a chromosome 7 alteration was detected in a v-H-ras initiated tumour, further evidence that a gene other than H-ras on this chromosome is involved in tumour progression. Human tumours frequently demonstrate LOH at the chromosomal region 11p 15.5, which is syntenic with the part of mouse chromosome 7 that encompasses the H-ras locus. Thus, the homologue of a tumour suppressor gene in this region of human chromosome 11 may be involved in mouse skin tumour development. The Wilms' tumour locus, also on human 11p, is on mouse chromosome 2. RFLP analyses provided no evidence that this gene has a role in mouse skin tumorigenesis. The non-random nature of chromosome 7 changes was supported by the low frequency of alterations on chromosomes 2 and 11. Two carcinomas did show LOH of a marker on the latter. Interestingly, this chromosome contains a region homologous to human chromosome 17p, which is involved in colorectal cancer. Minisatellite analysis also supported the non-random nature of chromosome 7 changes. Loss or rearrangement of minisatellite bands tended to involve hypervariable loci, suggesting that these were random rearrangements at unstable loci. In some human cancers genomic imprinting influences the direction of allele loss on 11p. However, this did not appear to be the case with LOH on chromosome 7 in mouse skin carcinomas. The parental strain also did not influence which alleles were under-represented in these tumours. Some important differences were detected between the genetic changes associated with carcinomas induced by initiation/promotion and those seen in carcinomas obtained by repeated carcinogen treatment. A similar proportion of MNNG/TPA and MNNG/MNNG carcinomas were positive for mutant H-ras. However, whereas non-disjunction of chromosome 7 had also occurred in the former, no chromosome 7 changes were detected in carcinomas induced by repeated MNNG treatment. This carcinogen may remove the need for additional chromosome 7 changes by mutating the gene(s) affected by these events in TPA-promoted tumours, or by altering entirely separate loci. In contrast, tumours induced with repeated DMBA treatment which were positive for activated H-ras also had chromosome 7 changes. However, the frequency of events such as mitotic recombination or deletion was much higher in these tumours than in carcinomas induced by an initiation/promotion regime. The major difference between DMBA/DMBA carcinomas and DMBA/TPA carcinomas was that the latter contained a much higher proportion of tumours which lacked activated H-ras. Thus it appears that repeated DMBA treatment stimulates the growth of initiated cells which are insensitive to TPA. Analysis of papillomas showed that gross chromosome 7 changes occur at a premalignant stage of tumorigenesis. This may suggest a tumour promoter-related genetic effect

Studies of the pathogenesis of Jembrana disease virus infection in Bos javanicus

Jembrana disease was reported initially in Bali cattle (Bos javanicus) on Bali island in 1964 and the causative agent was subsequently identified as a bovine lentivirus and designated Jembrana disease virus (JDV). This atypical lentivirus causes an acutely pathogenic disease that is associated with clinical signs and pathological lesions attributable to a disease primarily affecting the lymphoid system. Based on the intense proliferation of cells in the parafollicular (T-cell) areas of lymphoid tissue it has been assumed that the cellular tropism of the virus was for T-cells. An initial investigation of the pathological changes following JDV infection provided morphological evidence that JDV infection occurred not in T-cells but probably in centroblast-like cells containing IgG and presumably of B-cell lineage. The identity of the infected cells was confirmed by double immunofluorescence labelling techniques as being IgG-containing CD79α+ cells, indicating that the virus replicated in mature B-cells. Unlike other lentiviruses, no evidence of infection in T-cells or macrophages was obtained. These observations provide an explanation for suppression of the JDV-specific antibody response associated with JDV infection and the unique nature of the pathological response of Bali cattle to JDV infection. Flow cytometric analysis of peripheral blood leucocyte populations was used to further the understanding of the pathogenesis of JDV infection. Changes in lymphocyte subsets during the course of Jembrana disease were investigated and analysis of the results showed that lymphopenia, a characteristic of the acute febrile phase of Jembrana disease, was at least partly due to a significant decrease in CD4+ and CD8+ T-cells and CD21+ B-cells. In the immediate post-febrile recovery phase, virus-infected cells were not detected in lymphoid tissue but both CD8+ T-cells and CD21+ B-cells increased significantly and CD4+ T-cells remained below normal levels resulting in a significantly reduced CD4+:CD8+ ratio. Changes in expression of CD8+ T-cell regulated cytokine genes was examined during the course of the acute disease process by quantifying cytokine mRNA expression using real-time reverse-transcription polymerase chain reaction (RT-PCR). The results showed that both IL-2 and IFN-γ cytokine mRNA were strongly expressed during the febrile and early post-febrile recovery phases, which coincided with the significant increase of CD8+ T-cells and reduction of viraemia during this phase. The results suggested the CD8+ T-cell–associated cytokines IL-2 and IFN-γ probably play a significant role in the recovery process

Allosteric Integrase Inhibitors Reveal a Role for Integrase During HIV-1 Maturation

Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation.Medical Science

Investigation of apoptosis by conditional gene expression

Apoptosis is a form of cell death that occurs in individual cells in normal and diseased tissues. It occurs as a consequence of activation of a program of molecular events that results in the dismantlement and clearance of cells, often without induction of an inflammatory response. Many of the molecular components that regulate and execute apoptosis have been identified. Some of these regulators are, or are related to, genes that are altered in oplasia, such as c- myc,p53 and Bcl -2. The effector molecules belong to a class of cysteine proteases, known the ICE -like proteases (e.g. Nedd2). Whilst the known components can have identifiable activity in apoptosis id (or) cell cycle control, it has been suggested that they interact to control apoptosis. Further, it is not known nv the regulatory molecules interact upon downstream effectors to control apoptosis induction. Until recently, has not been possible to examine the role of apoptosis genes in combination other than by combinatorial mouse lockouts - a very time- consuming and expensive exercise. It was therefore decided to direct the expression of , veral apoptosis -related genes (namely c -myc, p53, p21 WAF /CIP, and Nedd2) in tissue culture in order to .ovide useful tools to answer questions about the role of such genes in the control of apoptosis.Owing to the limitations of existing conventional expression technology, where test genes are constitutively ,overexpressed from a strong viral promoter often in transient expression assays, use was made of conditional inigenes: 1) A temperature- sensitive (ts) murine p53 (p53va1135) was employed to investigate the sensitivity of :tivated c- Ha -ras- transformed rat embryo fibroblasts (Clone 6) to DNA -damage induced by the genotoxic iemotherapeutic drugs etoposide and bleomycin; 2) An oestrogen -regulable c- myc -oestrogen receptor hormone nding domain fusion protein (myc -ER) was used in conjunction with p53va1135 in order to investigate whether ,rced expression of phenotypically wild -type p53 was sufficient to trigger apoptosis by c -myc in Clone 6 and at -1 fibroblasts; and 3) Vectors were constructed that contain apoptosis genes under the control of semi - rnthetic promoters based upon the inducible E. coli lac operator- repressor system or a promoter containing east Gal-4 binding sites inducible by a tamoxifen -sensitive VP16GaElen chimaeric trans- activator protein.Results showed that: 1) Expression of ts p53 at the permissive temperature protected Clone 6 cells from rtotoxic drug- induced apoptosis, probably by enforcing a cell cycle arrest in G1. 2) Co- expression of myc -ER id ts p53 yielded no stable cell lines probably due to biologically significant basal activation of both p53val 135 id myc -ER under the culture conditions used. This is consistent with a co- operative role for c -myc and p53 in apoptosis triggering. 3) Control of expression in Rat -1 cells of the ICE -like protease Nedd2 (the mouse 3mologue of human ICH- I) by the VP I6GalER`m system was tight enough to allow development of stably - ansfected cells, which upon induction with 4- hydroxytamoxifen, rapidly underwent apoptosis. In contrast, ansfections with a LacI- repressible Nedd2 expression vector could not produce repressed stable expression vets that were low enough to be compatible with colony survival following selection in tissue culture.In this work, conditional expression technology was applied to the problem of control of apoptosis and shows tat gene expression experiments that increase the susceptibility of cells to apoptosis can be carried out in a :gulated fashion. Using this approach, a cytoprotective role of wild type p53 -mediated growth arrest was discovered that was abrogated by c -myc. In addition, cell lines were developed that are suitable for the biochemical characterisation of the action of Nedd2 protease