| Abstract | Herpes simplex virus 1 (HSV-1) is a dsDNA virus, which causes many different pathological conditions in the human population. The virus has the ability to modulate host immune response, which enables its replication. Adenosine deaminase acting on RNAs (ADAR) proteins are enzymes, whose main function is deamination of adenosine to inosine on dsRNA molecules, which consequently alters their structure and function. ADAR1 is the member of ADAR family, which is a known modulator of the immune response. Therefore, ADAR1 can exhibit proviral and antiviral effects, during viral infections. ADAR1 possesses two functionally active isoforms: p110 (constitutively expressed) and p150 (expression induced with the interferons (IFNs)). To add new insights to the HSV-1 field of research, we investigated whether ADAR1 proteins exhibit the ability to change their subcellular localisation during the early phase of HSV-1 productive infection. Firstly, by using the immunofluorescence and subcellular fractionation methods, we determined the ADAR1 expression profile of MOCK infected RPE1 cells. Results show that both p110 and p150 isoforms localise mainly in the nucleus of uninfected and unstimulated RPE1 cells. We also used immunofluorescence and subcellular fractionation to examine whether the translocation of ADAR1 proteins happens during first 24 hours after the infection. These results show that ADAR1 p110 isoform translocates from the nucleus to the cytoplasm, at a time point between 7 and 24 hours after infection. On the other hand, p150 isoform depletes in both the nucleus and the cytoplasm during the first day of the infection. Afterwards, we tried to compare cellular expression of ADAR1 isoforms between MOCK and HSV-1 infected RPE1 cells. Also, we analysed expression profile of ADAR1 proteins, during first 24 hours after infection, in several different cell lines. These data, together with the ADAR1 transcriptome analysis, show that the expression of ADAR1 proteins does not elevate during the early phase of HSV-1 productive infection. Nonetheless, increased expression of both ADAR1 isoforms, induced by the IFN-β, does not contribute to increased amounts of ADAR1 proteins in the cytoplasm. This was confirmed by the immunoflurescence and Western blot method. Overall, described results indicate that ADAR1 p110 translocates from the nucleus to the cytoplasm of RPE1 cells, and that this phenotype is not caused by the increase in protein's expression. |
| Abstract (croatian) | Herpes simpleks virus 1 (HSV-1) je dvolančani DNA virus, koji uzrokuje više različitih patofizioloških stanja u ljudskoj populaciji. Virus ima sposobnost moduliranja imunosnog odgovora domaćina, što omogućuje njegovu replikaciju. Adenozin deaminaza koja djeluje na RNA (engl. Adenosine deaminase acting on RNAs, ADAR) je grupa proteina s enzimatskom funkcijom deaminacije adenozina u inozin, na dvolančanim RNA molekulama, što posljedično mijenja njihovu strukturu i funkciju. ADAR1 je član ADAR obitelji te znani modulator imunosnog odgovora. Stoga, ADAR1 može pokazivati proviralne i antiviralne učinke, tijekom virusnih infekcija. ADAR1 posjeduje dvije funkcionalno aktivne izoforme: p110 (konstitutivno eksprimiran u stanicama) te p150 (ekspresija potaknuta interferonima (IFN)). Kako bi dodali nova saznanja u područje istraživanja koje se bavi s HSV-1 virusom, istražili smo mogu li ADAR1 proteini pokazivati mogućnost promjene njihovog unutarstaničnog položaja, tijekom rane produktivne faze HSV-1 infekcije. Najprije smo, korištenjem imunofluorescencije te metode unutastaničnoga frakcioniranja , odredili ADAR1 ekspresijski profil u RPE1 stanica, koje nisu bile niti tretirane niti inficrane (engl. „MOCK“). Rezultati ukazuju na to da su obje izoforme ADAR1 proteina ponajviše eksprimirane u jezgri MOCK inficiranih RPE1 stanica. Metode imunofluorescnecije te unutarstaničnoga frakcioniranja koristili smo i za ispitivanje moguće translokacije ADAR1 proteina, unutar prva 24 sata nakon infekcije. Dobiveni rezultati pokazuju da ADAR1 p110 izoforma prelazi iz jezgre u citoplazmu inficiranih stanica, u vremenskom intervalu između 7 i 24 sata nakon infekcije. Nasuprot tome, ekspresija p150 izoforme se smanjuje tijekom prvoga dana infekcije, u jezgri kao i u citoplazmi. Nakon toga, pokušali smo usporediti staničnu ekspresiju ADAR1 proteina između MOCK te inficiranih RPE1 stanica. Također smo analizirali razinu ekspresije proteina, tijekom prva 24 sata infekcije, u različitim staničnim linijama. Svi dobiveni rezultati, uz analizu ADAR1 transkripta, ukazuju na to da se ekspresija ADAR1 proteina ne povećava tijekom rane, produktivne HSV-1 infekcije. No ipak, povećana ekspresija obje ADAR1 izoforme, potaknuta s IFN-β, ne doprinosi povećanoj količini ADAR1 proteina u citoplazmi. Taj rezultat je potvrđen preko imunofluorescencije i „Western blot“ metode. Generano, opisani rezultati ukazuju na translokaciju ADAR1 p110 proteina iz jezgre u citoplazmu RPE1 stanica te da navedeni fenotip nije uzrokovan povećanom ekspresijom proteina. |
