The mechanism of membrane assembly for Semliki Forest Virus, a Group A Togavirus, was investigated through a series of radioactive pulse chase experiments. Initially a time course for the appearance of virus specified proteins in the microsomal fraction of infected BHK (baby hamster kidney) cells and mature virions was performed. Infected cells were harvested and fractionated at 0, 1, 2, 4, 6, 8 and 11 hours post-infection. Plaque assays were performed on the virus released into the growth medium at these times. It was found that virus production was maximal between 4 and 6 hours. Nucleocapsid was clearly evident at 6, 8 and 11 hours when the microsomal proteins were separated by SDS polyacrylarnide gel electrophoresis. In the next set of experiments infected cells were pulsed 3 for 20 minutes at 5 hours post-infection with H-Leu. Microsomes were prepared from the cells at 0 min., 20 min., 40 min. and 60 min. after removal of H-Leu and subjected to SDS polyacrylarnide gel electrophoresis. Virus was also isolated from the cell medium by sucrose gradient centrifugat-ion. Nucleocapsid protein radioactivity was at levels much greater than the combined peaks of radioactivity due to the membrane proteins E₁ and E₂. Little if any radioactive virus was released into the media during this time of chase. A similar experiment to the one just outlined was performed except that the radioactive chase was extended over the range of 0 hrs., 0.75 hrs., 1.50 hrs., 2.25 hrs. and 3.00 hrs. Levels of ³H- labelled nucleocapsid were again initially higher' than those of the combined E₁ and E₂ radioactive peak. The radioactivity of E₁ E₂ plateaus between 0.75 hrs. to 3.00 hrs. while that in the nucleocapsid continued to increase. This data appears to support the contention that nucleocapsid is synthesized prior to the viral membrane proteins. In hope of chasing the ³H-Leu label into and then out of the microsomes, infected BHK cells were pulsed at 3 hours and chased for 0, 1, 2, 3, 4, 5, and 6 hours after removal of labelled medium. Levels of ³H-Leu increased in both the nucleocapsid and E₁ E₂ protein bands of the SDS acrylamide gels until about 2 hours and then declined over the following time range. Loss of ³H-Leu in the microsomes appeared to correlate with the increase of label incorporated into the virus. Finally, after devising a method for separating plasma membrane (PM) ghosts and endoplasmic reticulum (ER) fragments, another pulse chase experiment was performed. Infected BHK cells were again radioactively pulsed at 3 hours infection and the level chased for 0, 1, 2, 4, 6, 8 and 11 hours following removal of the ³H-Leu. At the various time points labelled cells were harvested and fractionated into PM and ER. The samples of ER and PM were applied to SDS acrylamide gels and the radioactivity incorporated into the virus protein band was quantitated. Virus released into the medium was purified by sucrose gradient sedimentation, assayed for ³H-Leu, and also fractionated by SDS electrophoresis. Label was initially very high in the ER in the form of precursor proteins (NVPI65, NVP97, PE₂), envelope proteins (E₁, E₂), and nucleocapsid protein. This radioactivity was chased from the ER to the PM and then into mature virus. These results appear to indicate that Semliki Forest Virus nucleocapsid does indeed "bud" from the host cell membrane, thus obtaining its envelope. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The mechanism of viral penetration for Semliki Forest Virus into BHK-21 cells was investigated through a series of radioactive pulse-chase experiments. Entry of an enveloped virus such as SF Virus can be visualized to enter host cells both by pinocytosis (viropexis) or by fusion of the viral envelope and plasma membrane. Preliminary experiments were performed to obtain optimum conditions of viral adsorption to host cells. The conditions considered included temperature, time of infection, multiplicity of infection and ionic strength of the inoculum. In subsequent experiments BHK-21 cells were infected one half 35 hour with ³⁵S-Methionine-labeled Semliki Forest Virus. At various time points after removal of unadsorbed ³⁵Met-SF Virus, cells were harvested and fractionated into plasma membrane and endoplasmic reticulum fractions. The fractions were subjected to SDS polyacrylamide gel electrophoresis and analyzed for component proteins of SF Virus. Maximum levels of radioactivity corresponding to the envelope proteins (E₁, E₂ and nucleocapsid protein (NC) were found in the PM fraction at zero minutes of chase. Both E₁, E₂ and NC were found to decline during the chase period (approximately 90% within 60 minutes of chase). On the other hand, high levels of only nucleocapsid protein were observed associated with the endoplasmic reticulum fraction although no general pattern of incorporation was indicated furing the experiment. (There were high levles of NC present in the ER fraction throughout the chase period). The results of these studies are generally inconclusive as they can be rationalized both by the viropexis and fusion mechanisms. The loss of both E₁, E₂ and NC from the PM suggest that viropexis is the mechanism of entry, however, fusion is not eliminated as an alternative since the envelope proteins in the PM may be degraded after internalization of the virus core by host proteases. Although no conclusions have been drawn, this study has demonstrated that radioactive pulse-chase experiments can be performed to augment electron microscopy data concerning the mechanism of viral penetration into animal cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The data from chemical studies and electron microscopy-suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4.5-³H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membrane and extracellular virus was determined. Initial experiments had been conducted previously with microsomes and a precursor-product relationship demonstrated between viral proteins in the microsomes and extracellular virus (CD. Richardson and D.E. Vance, J. Biol. Chem. 251, 5544-5550). Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximum incorporation of [³H]leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this radioactivity had disappeared within 2 hr. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 3 hr into the chase period, and these labeled proteins disappeared from this membrane 8.5 hr after the pulse. At 8.5 hr chase, maximum incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between viral proteins in the endoplasmic reticulum, plasma membrane, and extracellular media. Viral proteins migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance. The possibility that an intact cytoskeletal system might be required for the assembly of Semliki Forest virus was investigated. The microtubules and microfilaments of baby hamster kidney cells (BHK-21) were disassembled with specific drugs and the effect on production of extracellular virus was determined. Colchicine, Nocodazole, dibucaine, and cytochalasin B reduced the production of extracellular virus by 75-90%. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [³H]leucine of [³⁵S] methionine. At various times after addition of one of these drugs, the incorporation of the labeled precursors into viral proteins associated with endoplasmic reticulum or plasma membrane of the cell was evaluated. The results clearly show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the cell where they accumulated. These studies strongly suggested that the cytoskeletal system was involved in the final stages of membrane assembly of Semliki Forest virus at the plasma membrane. Studies were also performed with the cross-linking agents -dimethylsuberimidate (DMS), dithiobis(succinimidyl propionate) (DSP), and dimethylthiobi's(propionimidate) (DTBP) . The proteins of purified virus and infected cells reacted with these agents and the cross-linked proteins were evaluated by one- and two-dimensional SDS electrophoresis. Nucleocapsid protein cross-linked to form up to pentameric complexes, and envelope proteins reacted to yield dimeric species. Nucleocapsid protein did not crosslink with envelope proteins. Cross-linking agents were also utilized to determine the effects of colchicine and dibucaine on the proximity of viral proteins to each other in the plasma membrane of the host cell. Colchicine (which disrupts microtubules) appeared to have no effect on the degree to which [³⁵S]-labeled virus proteins reacted with the agents, while dibucaine (which supposedly disrupts both microtubules and microfilaments) abolished envelope protein dimers dramatically. This result was interpreted to mean that microtubules may not be required for the formation of patches of virus proteins in the plasma membrane prior to budding, while microfilaments may play a more dominant role in this process. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Universiẗat, Diss., 2006--Frankfurt (Main). / Erscheinungsjahr an der Haupttitelstelle: 2005.

Univ., Diss., 2006--Frankfurt (Main)

Arboviruses are transmitted between vertebrate hosts by arthropod vectors, such as mosquitoes or ticks. In vertebrates arboviruses cause cytopathic effects and disease, however, arbovirus infection of arthropods usually results in persistence. Control of arboviral infection is mediated by the arthropod’s immune system. Pathways such as RNAi, JAK/STAT, Toll and IMD have previously been implicated in controlling arbovirus infections. In contrast, the antiviral role of other pathways in mosquitoes, such as melanisation, is unknown. Using high through output 454 sequencing the transcriptome of U4.4 cells infected with the model arbovirus Semliki Forest virus (SFV)(Togaviridae, Alphavirus) was generated. This experiment revealed intriguing patterns of differential transcript abundance that suggest a broad impact of SFV infection in U4.4 cells, such as in metabolism, cell structure and nucleic acid processing. SFV infection induces differential expression of genes in pathways such as apoptosis, stress response and cell cycle. Most interestingly, this study indicated that melanisation might have an antiviral role in mosquitoes. In arthropods, melanisation is a process involved in wound healing and antimicrobial defences. Phenoloxidase (PO), a key enzyme involved in melanisation, is cytotoxic and therefore kept in its inactive form, prophenoloxidase (PPO), until activation is triggered. The PPO activation process is tightly regulated by serine protease inhibitors (serpins) which inhibit the proteolytic activation reaction. In this thesis I demonstrate that the supernatant of cultured Aedes albopictus-derived U4.4 cells contains a functional proPO-activating system, which is activated by infection with bacteria and virions of SFV. Activation of this pathway reduces the spread and infectivity of SFV in vitro and in vivo. In order to further characterise the PO cascade and its antiviral role the serpins in Ae. albopictus were also investigated. Using the transcriptome sequencing and bioinformatics we identified and classified 11 serpins. We silenced each of the serpins and monitored PPO levels and antiviral activity showing that homologues to drosophila’s serpin- 27a plays a role in melanisation against SFV in vitro. Collectively, these results characterise the mosquito PO cascade as a novel immune defence against arbovirus infection in mosquitoes.

A glicoproteína do vírus rábico (RVGP) é o antígeno capaz de induzir a formação de anticorpos neutralizantes e a resposta imune protetora contra a infecção pelo vírus rábico. Estudamos as cinéticas de expressão da RVGP e de seu RNA mensageiro (RVGPmRNA) em dois sistemas distintos de expressão recombinante: células de Drosophila melanogaster (S2) e células BHK-21 infectadas com vírus Semliki Forest Virus (SFV). Para isso, fizemos um trabalho de padronização do tratamento de amostras de cultivos celulares, adequando-as a um teste de ELISA para dosagem da RVGP e estabelecemos um método de RT-PCR quantitativa (qRT-PCR) para a dosagem do RVGPmRNA. Desenvolvemos também um novo método de titulação de partículas SFV por qRT-PCR, aplicável a praticamente qualquer construção de SFV recombinante. Em ensaios preliminares, as preparações de RVGP recombinante utilizadas para a imunização de camundongos foram capazes de induzir a formação de anticorpos neutralizantes e de conferir um bom grau de proteção ao teste de desafio intracerebral com vírus rábico. / The rabies virus glycoprotein is the major antigen able to induce a neutralizing antibody response and survival after challenge against rabies virus infection. We have studied the kinetic expression of RVGP and its messenger RNA (RVGPmRNA) in two different recombinant expression systems: stably transfected Drosophila melanogaster cells (rS2) and BHK-21 cells infected with Semliki Forest Virus carrying RVGP genetic information (SFV-RVGP). We have done a work of standardization of the cell culture samples treatment prior to RVGP quantification by ELISA, and we have developed and standardized a quantitative RT-PCR (qRT-PCR) to quantify the RVGPmRNA. We have also developed a new method of SFV particles titration by qRT-PCR, which is applicable to other constructions of recombinant SFV. We utilized the RVGP expressed by rS2 and SFV-RVGP systems on preliminary in vivo assays. The RVGP samples used to mice immunization were able to induce neutralizing antibodies and to lead to a nice level of protection against the intracerabral rabies virus challenge.

A glicoproteína do vírus rábico (RVGP) é o antígeno capaz de induzir a formação de anticorpos neutralizantes e a resposta imune protetora contra a infecção pelo vírus rábico. Estudamos as cinéticas de expressão da RVGP e de seu RNA mensageiro (RVGPmRNA) em dois sistemas distintos de expressão recombinante: células de Drosophila melanogaster (S2) e células BHK-21 infectadas com vírus Semliki Forest Virus (SFV). Para isso, fizemos um trabalho de padronização do tratamento de amostras de cultivos celulares, adequando-as a um teste de ELISA para dosagem da RVGP e estabelecemos um método de RT-PCR quantitativa (qRT-PCR) para a dosagem do RVGPmRNA. Desenvolvemos também um novo método de titulação de partículas SFV por qRT-PCR, aplicável a praticamente qualquer construção de SFV recombinante. Em ensaios preliminares, as preparações de RVGP recombinante utilizadas para a imunização de camundongos foram capazes de induzir a formação de anticorpos neutralizantes e de conferir um bom grau de proteção ao teste de desafio intracerebral com vírus rábico. / The rabies virus glycoprotein is the major antigen able to induce a neutralizing antibody response and survival after challenge against rabies virus infection. We have studied the kinetic expression of RVGP and its messenger RNA (RVGPmRNA) in two different recombinant expression systems: stably transfected Drosophila melanogaster cells (rS2) and BHK-21 cells infected with Semliki Forest Virus carrying RVGP genetic information (SFV-RVGP). We have done a work of standardization of the cell culture samples treatment prior to RVGP quantification by ELISA, and we have developed and standardized a quantitative RT-PCR (qRT-PCR) to quantify the RVGPmRNA. We have also developed a new method of SFV particles titration by qRT-PCR, which is applicable to other constructions of recombinant SFV. We utilized the RVGP expressed by rS2 and SFV-RVGP systems on preliminary in vivo assays. The RVGP samples used to mice immunization were able to induce neutralizing antibodies and to lead to a nice level of protection against the intracerabral rabies virus challenge.

Arboviruses are transmitted to vertebrates by arthropod vectors such as mosquitoes or ticks. The replication of Semliki Forest virus (SFV) (Togaviridae; Alphavirus) in vertebrate cells is well established and triggers cell death. SFV infection of Aedes albopictus mosquito cells was characterised. Virus growth curves were compared in three cell lines. Infection of U4.4 cells was persistent and did not affect growth of the culture. In contrast, infection of C6/36 and C7-10 cells resulted in a static culture with no cell division and no cell death. The response of U4.4 cells was characterised in greater detail using viruses containing fluorescent or luciferase markers within the replicase or structural open reading frame of the virus genome. Activation of the STAT/IMD pathway prior to SFV infection significantly reduced virus driven luciferase expression and virus production. Activation of the Toll pathway prior to SFV infection had no effect. However, activation of Toll in addition to STAT/IMD had a cumulative effect on luciferase expression and virus production. viRNAs were characterised by Illumina Solexa sequencing. Two percent of the small RNA species found in virus infected cells were derived from virus RNA. These were predominantly 21 nt long and mapped along the entire SFV genome and genome complementary RNAs. Generation of these viRNAs was not random. Some areas produced high frequencies and others no or very few; hot and cold spots respectively. There were no correlations between viRNA frequency and base pairing or secondary structures predictions. Cold spot-derived viRNAs were more effective than hot-spot viRNAs in inhibiting virus replication. Similar results were observed in Aedes aegypti-derived cells. Attempts were made to investigate the source of these viRNAs using a virus containing an IRES element which had been reported to prevent virus replication in insect cells but which did not efficiently do so in this study. A virus containing the RNAi inhibitor p19 was characterised and shown to increase virus production. Techniques for infecting mosquitoes via a blood meal feed were established. No infection was observed with virus replicon particles carrying a fluorescent marker gene. Infection was established using virus containing p19.

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.