The first part of this thesis (I) concentrated on the regulation of the Uukuniemi virus (UUKV) genome expression; the non‐coding regions (NCRs) of UUKV RNA segments were analyzed in order to understand how transcription and replication machineries of the virus function. A comparison of three RNA segments of UUKV using the minigenome system showed that NCRs carry all the necessary signals for the transcription, replication and packaging of the virus. The intergenic region (IGR), located between the N and NSs protein ORFs, was also analyzed in the minigenome system and it was demonstrated that this region is essential for transcription termination.
When this study was carried out, the minigenome system was the only reverse genetics tool to study the transcription and replication of UUKV. However, since then the techniques have developed, and the VLP‐system was subsequently developed for UUKV, enabling a more sophisticated approach to study the molecular characteristics of UUKV and other bunyaviruses. Many questions about bunyavirus transcription, replication, and encapsidation of the RNAs and co‐packaging the RNPs to the virions remain still open. Moreover, the role(s) of the signaling sequences and NCRs in these processes requires further exploration. The discovery of new, pathogenic phleboviruses makes the development of reverse genetics systems for phleboviruses even more topical. The rescue of infectious UUKV would be an interesting step in developing UUKV reverse genetics systems, and all the tools for this are already at hand. The unique ambisense coding strategy for the UUKV S segment provides also an possibility to develope e.g. live‐attenuated vaccines; it was shown for RVFV, a pathogenic phlebovirus related to UUKV, that the NSs gene can be replaced and a recombinant virus containing only two genomic segments was generated. A similar technique could be adopted for UUKV, which as a non‐pathogenic member of the
Phlebovirus genus could serve as an safe alternative to the pathogenic RVFV.
The second part of the study focused on the UUKV N protein (II, III), particularly on its oligomerization (i.e. N‐N interactions) and RNA‐binding (i.e. N‐RNA interactions). The mutational analyses of the N protein showed that both the N‐ and C‐ termini of the UUKV N protein are needed for oligomerization, and especially the two α‐helices in the N‐terminus are important for the N‐N interactions (II). The amino acids, which were likely to be involved in RNA‐binding were analyzed for their functional competence. Some of the mutations affected the N protein functionality severely. The predicted model for the UUKV N protein supported these findings:
hydrophobic residues in the N‐terminal part of the protein were important for oligomerization, and the positively charged residues in the proposed central cavity of the protein may be involved in the RNA‐binding. Although the N protein structure for the RVFV was already solved, the N protein structure of UUKV N protein would clarify many open questions in oligomerization and RNA‐binding of UUKV and other phleboviruses. The solved N protein structure would be of help to design potential antivirals for pathogenic phleboviruses.
ACKNOWLEDGEMENTS
The work for this thesis was carried out at the Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, and Smittskyddsinstitutet, Stockholm, Sweden (publication I), and Department of Virology, Haartman Institute, University of Helsinki, Finland (publications II and III).
I warmly thank my thesis supervisors, Docent Alexander Plyusnin and Professor emeritus Antti Vaheri for guiding me in this study. Alexander Plyusnin is thanked for having the focus on finalizing the projects and patience in this long process, and Antti Vaheri is thanked for inspiring discussions and enthusiastic attitude towards science. Thank you both for this opportunity. The late Professor Ralf Pettersson is thanked for guiding me to the world of the Uukuniemi virus, and for the kind supervision in the first publication. The Head of the Department, Professor Kalle Saksela is thanked for providing the facilities to carry out this work. Thesis committee members, Docent Maarit Suomalainen and Docent Maria Söderlund‐Venermo are thanked for their guidance. I owe sincere gratitude to the reviewers of this thesis, Professor Sarah Butcher and Docent Petri Susi. You really helped me to improve this work, and I thank you for the support and good discussions during this process.
Collaborators and co‐authors are thanked for their contributions: Dr. Ramon Flick is thanked also for co‐supervising me in the first publication and Alexander Freiberg for great help: it was a pleasure to work with you both. Professor Liisa Holm and Vera Backström are thanked for their expertise in the bioinformatics, Vera also for the good company in the office back in the old days.
Maurice Forget is thanked for the language revision of this thesis.
There are many people in the Department who helped me with this study, and I am especially thankful for all the people in the zoonosis lab during these years of research. Kirsi Aaltonen, Leena Kostamovaara, Tytti Manni, Pirjo Sarjakivi, and Irina Suomalainen have always been there ready to help in the lab. I have learned a lot from you. All the "iloiset virologit": it has been a great joy to work with all of you. There has always been an expert on hand whom I could ask, whatever the matter concerns, in between the heaven and the earth. And Hadanka and the lab. Thank you all! I want to mention some fellow PhD students escpecially – most of you already PhDs for a longer time – Agne Alminaite, Eili Huhtamo, Anu Jääskeläinen, Paula Kinnunen, Suvi Kuivanen, Satu Kurkela, Niina Putkuri, Maria Razzauti Sanfeliu, Satu Saraheimo, Tarja Sironen, and Liina Voutilainen, few of you to mention. You have been great company and support during these years.
The “Microbes”, Anu Jääskeläinen, Samuel Myllykangas, Tiina Partanen, Hanna Sinkko, and Ville Veckman are thanked for the friendship for all these years. Somehow the majority of this microbial colony has moved from the fields of Viikki to Meilahti, it has been good to have you around here. My heartwelt thanks goes to my dear old friends, who have always been there: Lumi, Maarit, Lulu, Jonna, Meri, Paula, Nora, Aaro, Esko, Janne, Tuomas... and all the other good old “Töölö people” and their families: thank you for your friendship and support. Seeing you feels like coming home, always.
I am extremely lucky to have a magnificent family around me. My parents, Harry and Marja; your love and support and interest towards my work has been very important to me. I'm deeply grateful to you for everything. My dear siblings Otto and Eeva and their families are also thanked, just for being there. My parents‐in‐law, Marja‐Liisa and Veikko, thank you for the support and taking care of the children, and all the relaxing times in Porvoo with you and Tuomo's family.
And then last but not the least: our lively, lovely children, Saara and Eero, and my love, Velkka. I am so fortunate to have you in my life. Velkka is thanked for the endless love, support, patience, and understanding, everything. The book is done and mom is back. This study was financially supported by the Helsinki Biomedical Graduate Program, the Finnish Cultural Foundation, and the Sigrid Jusélius Foundation. Helsinki, May 2012 Anna Katz
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