Semliki Forest viiruse replikatsiooni ja patogeensuse uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Alfaviirused on maailmas väga laialt levinud selgroogsete organismide patogeenid ja nad põhjustavad erinevate sümptomitega haigusi, sealhulgas ka artriiti (liigesepõletik) ja vahel surmaga lõppevat entsefaliiti (ajupõletik). Alfaviiruste nakkuse ennetamist võimaldavad efektiivsed vaktsiinid ja nakkuse ravimiseks vajalikud spetsiifilised ravimid hetkel puuduvad. Selleks, et viiruste poolt põhjustatud haiguseid oleks võimalik ennetada või ravida, on esmalt vaja viiruse nakkust põhjalikult uurida ning detailselt mõista. Üheks kõige põhjalikumalt uuritud alfaviiruseks on Semliki Forest viirus (SFV), millele keskendus ka minu uurimistöö. SFV põhjustab hiirtes entsefaliiti ning SFV nakkust kasutatakse ühe võimaliku mudelina viirusliku entsefaliidi uurimisel. Hõlbustamaks viiruse paljunemise jälgimist organismis, disainisin ma SFV vormid, mis tootsid kergesti visualiseeritavaid või kvantiteeritavaid valke, nn markervalke. Loodud markerviirused võimaldasid detailselt jälgida SFV levikut täskasvanud hiire kesknärvisüsteemis. Minu uurimustöö näitas, et SFV nakkuse dünaamika on erinevat tüüpi rakkudes erinev – neuronid surusid viiruse valkude tootmise kiiresti maha, aga oligodendrotsüütid ei saanud sellega hakkama ning viiruse valkude tootmine toimus ka hilises nakkuse staadiumis. Leidsin ka, et nakkuse kulg neuronites sõltub nende rakkude küpsusastmest. Noored diferentseerumata neuronid ei suutnud viiruse valkude sünteesi efektiivselt maha suruda ja seetõttu toimus SFV paljunemine noortes rakkudes palju kiiremini kui küpsetes diferentseerunud neuronites. Saadud tulemused aitavad paremini mõista nii SFV kui teiste alfaviiruste infektsiooni ja patogeneesi selgroogsete, kaasa arvatud inimese, organismis.Alphaviruses are widely spread vertebrate pathogens, which cause different diseases, including arthritis (inflammation of joints) and encephalitis (inflammation of brain). To date, there are no available efficient vaccines for alphaviruses nor specific drugs to cure the disease. To protect or cure an individual form infection, one must first study the virus infection and understand it in detail. One of the best studied alphaviruses, Semliki Forest virus (SFV), has been the focus of my research. In mice, SFV infection leads to the development of encephalitis and SFV-infected mice serve as a good model for studying the disease. I designed recombinant SFVs, which produce marker proteins that are easy to visualise and quantify. These viruses enabled me to monitor the SFV infection in the central nervous system of adult mice and led to the discovery that the dynamics of SFV infection depend on the cell type infected. In neurons, the synthesis of viral proteins was rapidly downregulated. Oligodendrocytes, on the other hand, were unable to efficiently control virus propagation and the synthesis of viral proteins continued also during later stages of infection. I also found that the course of SFV infection depends on the differentiation state of neurons. Immature neurons were unable to control the synthesis of viral proteins and the virus was propagating in these cells more rapidly than in mature neurons. These data help us better understand the infection and pathogenesis of SFV and other alphaviruses in vertebrates, including humans

Properties of non-structural protein 1 of Semliki Forest virus and its interference with virus replication

Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5–10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis

Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications

Satb1 Ablation Alters Temporal Expression of Immediate Early Genes and Reduces Dendritic Spine Density during Postnatal Brain Development

Complex behaviors, such as learning and memory, are associated with rapid changes in gene expression of neurons and subsequent formation of new synaptic connections. However, how external signals are processed to drive specific changes in gene expression is largely unknown. We found that the genome organizer protein Satb1 is highly expressed in mature neurons, primarily in the cerebral cortex, dentate hilus, and amygdala. In Satb1-null mice, cortical layer morphology was normal. However, in postnatal Satb1-null cortical pyramidal neurons, we found a substantial decrease in the density of dendritic spines, which play critical roles in synaptic transmission and plasticity. Further, we found that in the cerebral cortex, Satb1 binds to genomic loci of multiple immediate early genes (IEGs) (Fos, Fosb, Egr1, Egr2, Arc, and Bdnf) and other key neuronal genes, many of which have been implicated in synaptic plasticity. Loss of Satb1 resulted in greatly alters timing and expression levels of these IEGs during early postnatal cerebral cortical development and also upon stimulation in cortical organotypic cultures. These data indicate that Satb1 is required for proper temporal dynamics of IEG expression. Based on these findings, we propose that Satb1 plays a critical role in cortical neurons to facilitate neuronal plasticity