Hepatiit C viiruse ja Chikungunya viiruse vastased lähenemised

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Tänapäeval on võimalik ennast erinevate viiruste vastu vaktsineerida ning ka viirushaiguste ravi on muutunud oluliselt tõhusamaks. Samas leidub endiselt meditsiiniliselt olulisi viiruseid, mille vastu puudub vaktsiin ja/või mille poolt põhjustatud haigustele pole siiani adekvaatset ravi. Viirus-vastaste ühendite ja vaktsiinide väljatöötamist raskendavad nii viiruste suur mitmekesisus kui ka nende keeruline elutsükkel. Üheks selliseks viiruseks on C hepatiidi viirus (HCV), mis on kroonilise maksahaiguse levinuimaks tekkepõhjuseks. Hinnanguliselt on selle viirusega krooniliselt nakatunud ~3% inimkonnast. Kuigi HCV infektsiooni ravis on toimunud suur läbimurre, on viiruse geneetilise mitmekesisuse, ravimresistentsete vormide tekkimise ning patsientide ravile mitteallumise tõttu endiselt väga oluline uute HCV vastaste ravimite väljatöötamine. Antud uurimustöö üheks eesmärgiks oli analüüsida erinevaid tehnoloogilisi lahendusi HCV vastaste ühendite loomiseks. Ühe lähenemisena valiti FQSAR arvutiprogrammi põhiselt välja madal-molekulaarsed ühendid, mis seondudes HCV NS3/4A proteaasiga inhibeerivad HCV replikatsiooni, ja iseloomustati nende mõju viiruse infektsioonile. Kõik analüüsitud seitse ühendit omasid HCV-vastast efekti, kuid ainult üks ühend (23332) oli kasutatavas kontsentratsioonis mitte-toksiline. Teine lähenemisviis seisnes looduslikult esineva modifikatsiooni (8-oxo-dG) mõju analüüsimises oligonukleotiidsete (ON) inhibiitorite efektiivsusele. Kombineerides erinevaid modifikatsioone leiti ON ühend, mis inhibeeris HCV replikatsiooni nanomolaarsetel kontsentratsioonidel. Lisaks HCV uurimisele on võimalik käsitletud lähenemise kasutada ka teiste viiruste vastu suunatud ühendite väljatöötamisel. Chikungunya viirus (CHIKV, perekond Alfaviirus) on troopilistes piirkondades leviv arboviirus, mis on viimasel aastakümnel korduvalt väljunud oma tavalisest levialast ja põhjustanud epideemiaid erinevates maailmajagudes. Antud töö kolmandaks eesmärgiks oli analüüsida uudsete CHIKV-vastaste vaktsiinikandidaatide geneetilist stabiilsust ning uurida nendes sisalduvate viirust nõrgestavate mutatsioonide mõju CHIKV elutsüklile. Leiti, et viirustel CHIKVΔ5nsP3 ja CHIKVΔ6K on nõrgestatud fenotüüp ka pärast mitmekordset passeerimist koekultuuri rakkudes. Mitmetest analüüsitud CHIKV-vastastest vaktsiini kandidaatidest osutus kõige efektiivsemaks CHIKVΔ5nsP3. See nõrgestatud viirus sisaldab suurt deletsiooni nsP3 valgu C-terminaalses regioonis. Katsetest selgus, et nimetatud regioon interakteerub sama valgu keskmise domeeni ning nsP2 valgu C-terminaalse osaga ja need kontaktid on olulised viiruse replikatsioonil. Need avastused võimaldavad edaspidi välja selgitada CHIKVΔ5nsP3 mitte-patogeense fenotüübi põhjused. CHIKV Δ5nsP3 vaktsiini tüvi on kasutusele võetud edasiseks arendamiseks farmatseutilise firma poolt.Viruses have been and will be an important part of every ecosystem. In the past, viral outbreaks have left painful marks on mankind. Using vaccines and antivirals has greatly reduced the number of infections and virus-caused pathology. Despite extensive research, some viruses and viral diseases are still lacking any good vaccine or treatment. Viral features like high mutation rate, complexity of viral lifecycle and genome diversity are only some of the obstacles needed to overcome for antivirals and vaccines to be safe and efficient. Hepatitis C virus (HCV) is associated with different liver pathologies and it is estimated that approximately 3% of the world population is chronically infected with HCV. It is lacking efficient vaccine and the options for combating HCV infection, HCV-induced pathology, spread and persistence are limited to the use of antiviral drugs. One part of this dissertation is focused on the development of anti-HCV inhibitors using two different technological approaches. Firstly, a new FQSAR method based approach allowed rapid prediction of hit compounds targeting the NS3/4A protease of HCV. Seven compounds analysed in this project displayed some anti-HCV properties but only the effect caused by the non-cytotoxic compound 23332 can be considered to be direct. Secondly, a novel technology – incorporation of naturally occurring minimally modified nucleobases into ASOs – was evaluated using ASOs binding to the HCV non-structural region. This approach led to the development of ASO compounds with high anti-HCV activity. The technology based on the use of novel modified ONs is promising as well for the development antivirals for other viruses and diseases. Chikungunya virus (CHIKV) re-emerged in the past decade and is currently spreading around the world, affecting millions of people. The second part of this study is focused on the analysis of a laboratory-developed attenuated CHIKV vaccine strain. CHIKVΔ5nsP3 and CHIKVΔ6K viruses were found to have a stably attenuated phenotype and the introduced molecular changes were maintained during serial passages. From all studied vaccine candidates the CHIKVΔ5nsP3 was the most potent. Further studies revealed that the region removed from CHIKVΔ5nsP3 vaccine candidate, is apparently involved in interactions with another domain of nsP3 as well as with the C-terminal region of nsP2. These findings provide a platform for further analysis of biological reasons for the attenuation of CHIKVΔ5nsP3 vaccine candidate

Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes.

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50) : 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50 : 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide: RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.Peer reviewe

The methyltransferase N6AMT1 participates in the cell cycle by regulating cyclin E levels.

The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1

N6AMT1 impacts the cyclin E levels.

(A) Unsynchronized cells and cells that were synchronized with serum depletion, thymidine treatment and nocodazole treatment were analysed via Western blotting with antibodies against Cyclin A, Cyclin B1, Cyclin E, N6AMT1, TRMT112 and GAPDH. 10 μg of total protein was loaded per well. (B-E) Cyclin A, cyclin B, cyclin E and TRMT112 levels in U2OS and ΔN6AMT1#1 cells. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests. (F) Untreated cells and cells that were treated with the indicated siRNAs were analysed via Western blotting with antibodies against Cyclin E, N6AMT1 and GAPDH. (G) Cyclin E levels in U2OS cells after siRNA treatment. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests. (H) qRT‒PCR verification of cyclin A, cyclin B and cyclin E levels in ΔN6AMT1#1 cells. The average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests.</p

N6AMT is important for cell proliferation and cyclin E regulation in HEK293 cells.

(A) The proliferation rate of the U2OS, ΔN6AMT1#1 and ΔN6AMT1#2 cell lines were determined using an xCELLigence Real-Time cell analyser, and data were recorded for a total of 7 days. (B) qRT‒PCR verification of cyclin A, cyclin B and cyclin E levels in HEK293ΔN6AMT1 cells. The average of two biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. (C) Unsynchronized cells and cells that were synchronized with serum depletion and thymidine treatment were analysed via Western blotting with antibodies against Cyclin A, Cyclin B1, Cyclin E, N6AMT1 and GAPDH. 10 μg of total protein was loaded per well. (D-F) Cyclin A, cyclin B and cyclin E levels in U2OS and ΔN6AMT1#1 cells. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P (PDF)</p

N6AMT1 impacts the cell proliferation rate.

(A) The U2OS and N6AMT1-depleted cell lines (ΔN6AMT1#1 and ΔN6AMT1#2) were analysed via Western blotting with antibodies against N6AMT1 and α-tubulin. (B) The proliferation rate of the U2OS, ΔN6AMT1#1 and ΔN6AMT1#2 cell lines was determined using an xCELLigence Real-Time cell analyser, and data were recorded for a total of 7 days. (C) Doubling time of the U2OS, ΔN6AMT1#1 and ΔN6AMT1#2 cell lines. The average of three biologically independent samples is shown.</p

Changes in the cyclin levels after serum depletion.

(A) The population density of the U2OS and ΔN6AMT1#1 cell lines in serum-depleted media was determined using an xCELLigence Real-Time cell analyser over a period of 120 h. (B) Schematic representation of the experimental setup for cell synchronization in the G1 phase. The cyclin A, B1 and E levels in (C) U2OS and (D) ΔN6AMT1#1 cells were analysed via Western blotting at the indicated timepoints. Cyclin A, B and E images were obtained after exposure time of 10 min for U2OS and 30 min ΔN6AMT1#1 samples. (E-G) Graphical representation of changes in the cyclin levels. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown.</p