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Viruses of Archaea are the least studied group of viruses. Fewer than 50 archaeal viruses have been reported which constitutes less than one percent of all the isolated prokaryotic viruses. Only about one third of the isolated archaeal viruses infect halophiles. The diversity of haloviruses, virus ecology in highly saline environments and the interactions of haloviruses with their hosts have been little studied. The exiguous knowledge available on halophilic systems is not only due to inadequate sampling but also reflects the extra challenge highly saline systems set on biochemical studies. In this study six new haloviruses were isolated and characterized. Viruses included four archaeal viruses and two bacteriophages. All of the other isolates exhibited head-tail morphology, except SH1 which was the first tailless icosahedral virus isolated from a high salt environment. Production and purification procedures were set up for all of these viruses and they were subjected to stability determinations. Archaeal virus SH1 was studied in more detail. Biochemical studies revealed an internal membrane underneath the protein capsid and a linear dsDNA genome. The overall structure of SH1 resembles phages PRD1, PM2 and Bam35 as well as an archaeal virus STIV. SH1 possesses about 15 structural proteins that form complexes under non-reducing conditions. Quantitative dissociation provided information about the positions of these proteins in the virion. The life cycle of SH1 was also studied. This lytic virus infects Haloarcula hispanica. Adsorption to the host cells is fairly inefficient and the life cycle rather long. Finally, virus responses in a variety of ionic conditions were studied. It was discovered that all of the studied viruses from low salt, marine and high salt environments tolerated larger range of salinities than their bacterial or archaeal hosts. The adsorption efficiency was not determined by the natural environment of a virus. Even though viruses with the slowest binding kinetics were among the haloviruses, fast binders were observed in viruses from all environments. When the salinity was altered, the virus adsorption responses were diverse. Four different behavioral patterns were observed: virus binding increased or decreased in increasing salinity, adsorption maximum was at a particular salt concentration or the salinity did not affect the binding. The way the virus binding was affected did not correlate with the environment, virus morphology or the organism the virus infects. Solulliset eliöt jaetaan kolmeen domeeniin: eukaryootit, bakteerit ja arkit. Virukset infektoivat eliöitä näistä kaikista ryhmistä. Vähiten tutkittu virusryhmä on arkkeja infektoivat virukset. Alle 50 arkkivirusta on eristetty ja karakterisoitu, mikä on vähemmän kuin yksi prosentti tunnetuista bakteereita infektoivista viruksista. Korkean suolapitoisuuden virukset, halovirukset, ovat suurelta osin arkkiviruksia, ja eristetyistä arkkiviruksista näitä on noin kolmannes. Väitöskirjatyössä eristettiin ja karakterisoittin kuusi halovirusta. Neljä näistä viruksista infektoi arkkeja ja kaksi bakteereita. Morfologialtaan ne olivat viruksia, joilla oli pää ja häntä, paitsi yksi joka oli ikosahdraalinen hännätön virus ja morfologialtaan uusi halovirusten joukossa. Tästä ikosahdraalisesta viruksesta tehtiin muita tarkempi karakterisaatio. Lisäksi väitöskirjatyössä tutkittiin myös laajemmin eristettyjen halovirusten ja muiden, sekä korkean että matalan suolan, virusten sopeutumista vaihtelevaan suolapitoisuuteen. Tämä tehtiin seuraamalla viruksen tarttumista isäntäsoluihin erilaisissa ioniolosuhteissa sekä tutkimalla viruksen suolankestävyyttä.

Currently, there are nine known human herpesviruses and these viruses appear to have been a very common companion of humans throughout the millenia. Of human herpesviruses, herpes simplex viruses 1 and 2 (HSV-1, HSV-2), causative agents of herpes labialis and genital herpes, and varicella-zoster virus (VZV), causative agent of chicken pox, are also common causes of central nervous system (CNS) infections. In addition, human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), all members of the herpesvirus family, can also be associated with encephalitis and meningitis. Accurate diagnostics and fast treatment are essential for patient recovery in CNS infections and therefore sensitive and effective diagnostic methods are needed. The aim of this thesis was to develop new potential detection methods for diagnosing of human herpesvirus infections, especially in immunocompetent patients, using the microarray technique. Therefore, methods based on microarrays were developed for simultaneous detection of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6A, HHV-6B, and HHV-7 nucleic acids, and for HSV-1, HSV-2, VZV, and CMV antibodies from various clinical samples. The microarray methods developed showed potential for efficiently and accurately detecting human herpesvirus DNAs, especially in CNS infections, and for simultaneous detection of DNAs or antibodies for multiple different human herpesviruses from clinical samples. In fact, the microarray method revealed several previously unrecognized co-infections. The microarray methods developed were sensitive and provided rapid detection of human herpesvirus DNA, and therefore the method could be applied to routine diagnostics. The microarrays might also be considered as an economical tool for diagnosing human herpesvirus infections. Herpesvirukset ovat kulkeneet ihmisen kumppaneina vuosituhat toisensa jälkeen ja tällä hetkellä tunnetaan yhdeksän ihmiselle patogeenista herpesvirusta. Huuli- ja genitaaliherpestä aiheuttavat herpes simplex virus 1 ja 2 (HSV-1, HSV-2) sekä vesirokkoa aiheuttava varicalla-zoster virus (VZV) ovat ihmisen herpesviruksia, jotka voivat aiheuttaa myös vakavia keskushermostoperäisiä infektioita. Ihmisen herpesviruksista myös ihmisen cytomegalovirus (CMV), Epstein-Barr virus (EBV), ihmisen herpesvirukset 6 ja 7 (HHV-6, HHV-7), voidaan liittää aivokuumeeseen ja aivokalvontulehdukseen. Näiden virusten nopea ja tarkka diagnostiikka on oleellista oikean hoidon aloittamiselle sekä potilaan toipumiselle varsinkin keskushermostoperäisissä infektioissa. Taudinaiheuttajien diagnosoimiseksi tarvitaan herkkiä ja tehokkaita menetelmiä. Tämän väitöskirjan tavoitteina oli kehittää uusia mikrosirupohjaisia menetelmiä ihmisen herpesvirusten aiheuttamien infektioiden diagnostiikkaan. Kehitetyillä mikrosiruilla tunnistettiin yhtä aikaa kahdeksan eri herpesviruksen nukleiinihappoa erilaisista kliinisistä näytteistä. Toisella serologisella mikrosirulla detektoitiin HSV-1, HSV-2, VZV ja CMV:lle spesifisiä vasta-aineita. Herpesvirusten nukleiinihappojen ja vasta-aineiden tunnistamiseen kehitetyt mikrosirumenetelmät osoittautuivat soveltuviksi usean herpesviruksen samanaikaiseen diagnosointiin. Varsinkin keskushermostopohjaisten infektioiden tunnistamisessa mikrosiru osoittautui tehokkaaksi. Mikrosirujen avulla tunnistettiin myös mahdollisia useamman viruksen samanaikaisia infektioita.

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype. Parvorokkovirus eli B19 on hyvin yleinen ihmisiä infektoiva virus joka aiheuttaa ihmiselle monenlaisia sairauksia. Tavallisin on lapsilla ja nuorilla esiintyvä ihottumatauti, parvorokko. Raskauden aikana virus voi siirtyä äidistä sikiöön aiheuttaen jopa sikiön kuoleman. B19-virus lisääntyy luuytimessä, punasolujen esiasteissa, joten se voi tiettyjä veritauteja sairastaville henkilöille aiheuttaa rajun lyhytkestoisen anemian, joka hoitamattomana voi olla kohtalokas. Immuunipuutteiset henkilöt voivat sairastua vuosienkin mittaiseen anemiaan. Parvorokkoviruksen aiheuttama niveltulehdus on yleinen etenkin naisilla ja kestoltaan sekä vaikeusasteeltaan vaihteleva, vaikeimmat tapaukset jopa nivelreuman kaltaisia. Tässä työssä löysimme uuden, ennestään tuntemattoman parvovirusvariantin (LaLi), joka perimäainekseltaan eroaa sekä B19-prototyypistä että vuonna 1998 löydetystä V9-variantista. Nykyisin parvorokkovirukset jaetaankin kolmeen tyyppiin: perinteiset isolaatit kuuluvat tyyppiin 1, LaLi:n kaltaiset tyyppiin 2 ja V9:n kaltaiset tyyppiin 3. Herkkiä geenimonistusmenetelmiä käyttämällä parvorokkoviruksen DNA:n on osoitettu säilyvän perusterveiden, oireettomien ihmisten kudoksissa vieläpä vuosia infektion jälkeenkin. Näin ollen kudosperäistä B19-DNA-löydöstä sellaisenaan ei voida käyttää taudinmääritykseen. Osoitimme myös tyypin 2 DNA:n varastoituvan ihmisten kudoksiin, jopa prototyyppiä yleisemmin. Kudosten parvorokkovirus-DNA:n varastointimekanismia ei vielä tunneta, mutta osoitimme virusgeenien säilyvän pilkkoutumatta ja muuntumatta. Vielä ei tiedetä kykenevätkö nämä varastoidut genomit joissain tilanteissa tuottamaan esim. RNA:ta, proteiineja tai jopa infektiivistä virusta, ja siten aiheuttamaan ihmisille myöhäiskomplikaatioita. Parvorokkoviruksen on osoitettu leviävän myös verituotteiden välityksellä. Evaluoimme verituotteiden seulontaan tarjolla olevat kaupalliset geenimonistusmenetelmät uusien virustyyppien suhteen ja osoitimme, etteivät kaikki käytössä olevat testit löydä uusia parvorokkovirustyyppejä. Lisäksi tarkastelimme uusien virustyyppien esiintyvyyttä suomalaisten verenluovuttajien keskuudessa seulomalla n. 140 000 luovuttajan näytteet: tästä joukosta löytyi ainoastaan tyyppiä 1. Toisin sanoen, vaikka tyypin 2 DNA:ta löytyi yleisesti terveiden henkilöiden ihonäytteistä, tyypin 2 virusta ei suomalaisten verenluovuttajien keskuudessa havaittu. Vertaillessamme B19-prototyypin ja uusien B19-tyyppien biologisia ominaisuuksia sekä infektiokykyä soluviljelmissä osoitimme, että myös uudet parvorokkovirustyypit ovat biologisesti aktiivisia, infektiokykyisiä viruksia. Lisäksi vertailimme tyyppien 1 ja 2 immunologiaa ja vasta-ainediagnostiikkaa: osoitimme että kumpaa tahansa virustyyppiä kohtaan muodostuneet vasta-aineet tunnistavat myös toisen virustyypin, ja näyttäisivät suojaavan kummankin virustyypin uusintainfektioilta.

Viral infections caused by herpesviruses are common complications after organ transplantation and they are associated with substantial morbidity and even mortality. Herpesviruses remain in a latent state in a host after primary infection and may reactivate later. CMV infection is the most important viral infection after liver transplantation. Less is known about the significance of human herpesvirus-6 (HHV-6). EBV is believed to play a major role in the development of post-transplant lymphoproliferative disorders (PTLD). The aim of this study was to investigate the CMV-, EBV- and HHV-6 DNAemia after liver transplantation by frequent monitoring of adult liver transplant patients. The presence of CMV, EBV and HHV-6 DNA were demonstrated by in situ hybridization assays and by real-time PCR methods from peripheral blood specimens. CMV and HHV-6 antigens were demonstrated by antigenemia assays and compared to the viral DNAemia. The response to antiviral therapy was also investigated. CMV-DNAemia appeared earlier than CMV pp65-antigenemia after liver transplantation. CMV infections were treated with ganciclovir. However, most of the treated patients demonstrated persistence of CMV-DNA for up to several months. Continuous CMV-DNA expression of peripheral blood leukocytes showed that the virus is not eliminated by ganciclovir and recurrences can be expected during several months after liver transplantation. HHV-6 DNAemia / antigenemia was common and occurred usually within the first three months after liver transplantation together with CMV. The HHV-6 DNA expression in peripheral blood mononuclear cells correlated well with HHV-6 antigenemia. Antiviral treatment significantly decreased the number of HHV-6 DNA positive cells, demonstrating the response to ganciclovir treatment. Clinically silent EBV reactivations with low viral loads were relatively common after liver transplantation. These EBV-DNAemias usually appeared within the first three months after liver transplantation together with betaherpesviruses (CMV, HHV-6, HHV-7). One patient developed high EBV viral loads and developed PTLD. These results indicate that frequent monitoring of EBV-DNA levels can be useful to detect liver transplant patients at risk of developing PTLD. Herpesvirusinfektiot aiheuttavat merkittäviä komplikaatioita ja jopa kuolleisuutta elinsiirtopotilailla. Herpesviruksille tyypillinen piirre on, että ne jäävät primaari-infektion jälkeen elimistöön piilevinä ja reaktivoituvat myöhemmin. Sytomegalovirus (CMV) infektio on kliinisesti tärkein maksansiirron jälkeen esiintyvä virusinfektio. Vähemmän tiedetään human herpesvirus-6:n (HHV-6) uusintainfektioista ja niiden merkityksestä elinsiirtojen jälkeen. Epstein-Barrin virus (EBV) voi aiheuttaa siirron jälkeisen lymfoproliferatiivisen tilan (PTLD). Tämän väitöskirjatutkimuksen tarkoituksena oli tutkia CMV-, EBV- ja HHV-6- DNA:n esiintymistä potilaan perifeerisessä veressä maksansiirron jälkeen. Tutkimuksen aineisto koostui aikuisista maksansiirtopotilaista, joita monitoroitiin säännöllisesti näiden virusten suhteen. CMV-, HHV-6- ja EBV- DNA osoitettiin in situ hybridisaatio-tekniikoiden ja reaaliaikaisten PCR-menetelmien avulla. CMV- ja HHV-6- antigeenit osoitettiin immunohistokemiallisin värjäyksin veren soluista ja antigenemiaa verrattiin viruksen DNAemiaan. Samalla tutkittiin myös viruslääkityksen tehoa. CMV DNA:n todettiin ilmaantuvan selvästi ennen CMV- pp65 antigeenia. Maksansiirtopotilaiden CMV infektiot hoidettiin gansikloviirillä. Kuitenkin useimmilla hoidetuilla potilailla CMV DNA:ta esiintyi perifeerisen veren soluissa jopa useita kuukausia. Jatkuva CMV-DNA ekspressio osoittaa, ettei virusta kyetä kokonaan eliminoimaan viruslääkkeellä ja näin uusintainfektiot ovat mahdollisia ensimmäisten kuukausien aikana maksansiirron jälkeen. HHV-6-DNA:ta /antigenemiaa esiintyi yleisesti maksansiirtopotilailla ensimmäisen kolmen kuukauden aikana maksansiirron jälkeen ja usein samanaikaisesti CMV infektion kanssa. Kun HHV-6 DNA osoitettiin maksansiirtopotilaiden perifeerisen veren mononukleaarisista soluista in situ-hybridisaation avulla, todettiin sen korreloivan hyvin HHV-6 antigenemiaan. Gansikloviiri hoidon todettiin laskevan merkitsevästi HHV-6 DNA-positiivisten solujen määrää näiden potilaiden veressä. Gansikloviiri hoidolla on täten mahdollisesti tehoa myös HHV-6 infektioon. EBV reaktivaatiot olivat melko yleisiä maksansiirron jälkeen. Nämä olivat EBV-DNAemia tasoltaan matalia ja esiintyivät yleensä ensimmäisen kolmen kuukauden aikana maksansiirron jälkeen yhdessä muiden herpesvirusten kanssa (CMV, HHV-6 ja HHV-7). Yhdellä potilaalla todettiin maksansiirron jälkeen korkeatasoinen EBV-DNAemia ja hänelle kehittyi PTLD. Säännöllinen EBV-DNA tasojen seuranta auttaa havaitsemaan ne potilaat, joille on vaarana kehittyä PTLD.

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis. Polymeraasit ovat entsyymejä, jotka vastaavat eliöiden geneettisen materiaalin kopioimisesta jälkipolville. Tämä perinnöllinen aines voi koostua joko deoksiribonukleiini- tai ribonukleiinihaposta (DNA tai RNA), joiden emäsjärjestykseen on kirjattu geneettinen koodi. Väitöskirjatyössä kuvataan phi6 viruksen polymeraasin toimintaa ja rakennetta sen entsymaattisen toiminnan eri vaiheissa. Kyseisen viruksen RNA:sta riippuvainen RNA-polymeraasi pystyy käyttämään substraattinaan yksijuosteista RNA:ta tuottaen sille RNA-vastinjuosteen.Väitöskirjassa tutkittiin, miten eristetty RNA:sta riippuvainen RNA-polymeraasi aloittaa RNA-synteesin käyttäen geneettisesti muunneltuja polymeraasivariantteja. Lisäksi tutkittiin, miten viruksen RNA:sta riippuvainen RNA-polymeraasi toimii substraatista riippumattomana entsyyminä. Virukset infektoivat lähes kaikkia maapallolla esiintyviä organismeja. Bakteereita infektoivat virukset ovat nimeltään bakteriofaageja.Phi6 virusta on tutkittu pitkään ja se on kaksijuosteisten RNA-virusten malliorganismi. Phi6 virus infektoi kasvipatogeenisia Pseudomonas syringae bakteereita ja sen geneettinen materiaali koostuu kaksijuosteisista RNA-juosteista, joita ympäröi kaksi virusproteiineista koostuvaa vaippaa sekä uloimpana isäntäsolun fosfolipideistä koostuva kalvo. Yksi sisimmän vaipan virusproteiineista on tässä työssä tutkittu RNA:sta riippuvainen RNA-polymeraasi. Tällaisilla polymeraaseilla on tärkeä rooli RNA-virusten geneettisen materiaalin kopioinnissa ja elinkierrossa, mutta niiden toimintamekanismi on vielä epäselvä. Phi6:n polymeraasia voidaan tuottaa viruksesta erillään ja entsyymiä voidaan tutkia koeputkessa erossa muista viruksen tai solun osista. Väitöskirjatyön tuloksena phi6 viruksen RNA:sta riippuvaisen RNA-polymeraasin RNA-synteesin aloitusmekanismi on saatu selvitettyä hyvin yksityiskohtaisesti. Polymeraasin aktiivisen keskuksen lähellä sijaitseva alue on välttämätön osatekijä, jotta polymeraasi voi onnistuneesti aloittaa RNA-synteesin ja kopioida tarkasti viruksen RNA-juosteet seuraaville virussukupolville. Väitöskirjatyön tulokset osoittavat ja kuvaavat myös tarkasti miten phi6 viruksen RNA:sta riippuvainen RNA-polymeraasi voi lisätä epäspesifisesti nukleotideja vain viruksen nukleiinihappoketjun päähän. Työn tuloksia voidaan soveltaa suunniteltaessa RNA:ta kopioivia entsyymejä bioteknisiin sovelluksiin molekyylibiologian alalla.

Viruses are submicroscopic, infectious agents that are obligate intracellular parasites. They adopt various types of strategies for their parasitic replication and proliferation in infected cells. The nucleic acid genome of a virus contains information that redirects molecular machinery of the cell to the replication and production of new virions. Viruses that replicate in the cytoplasm and are unable to use the nuclear transcription machinery of the host cell have developed their own transcription and capping systems. This thesis describes replication strategies of two distantly related viruses, hepatitis E virus (HEV) and Semliki Forest virus (SFV), which belong to the alphavirus-like superfamily of positive-strand RNA viruses. We have demonstrated that HEV and SFV share a unique cap formation pathway specific for alphavirus-like superfamily. The capping enzyme first acts as a methyltransferase, catalyzing the transfer of a methyl group from S-adenosylmethionine to GTP to yield m7GTP. It then transfers the methylated guanosine to the end of viral mRNA. Both reactions are virus-specific and differ from those described for the host cell. Therefore, these capping reactions offer attractive targets for the development of antiviral drugs. Additionally, it has been shown that replication of SFV and HEV takes place in association with cellular membranes. The origin of these membranes and the intracellular localization of the components of the replication complex were studied by modern microscopy techniques. It was demonstrated that SFV replicates in cytoplasmic membranes that are derived from endosomes and lysosomes. According to our studies, site for HEV replication seems to be the intermediate compartment which mediates the traffic between endoplasmic reticulum and the Golgi complex. As a result of this work, a unique mechanism of cap formation for hepatitis E virus replicase has been characterized. It represents a novel target for the development of specific inhibitors against viral replication.