The biological functions of mouse twinfilin isoforms

The actin cytoskeleton is essential for many cellular processes, including motility, morphogenesis, endocytosis and signal transduction. Actin can exist in monomeric (G-actin) or filamentous (F-actin) form. Actin filaments are considered to be the functional form of actin, generating the protrusive forces characteristic for the actin cytoskeleton. The structure and dynamics of the actin filament and monomer pools are regulated by a large number of actin-binding proteins in eukaryotic cells. Twinfilin is an evolutionarily conserved small actin monomer binding protein. Twinfilin is composed of two ADF/cofilin-like domains, separated by a short linker and followed by a C-terminal tail. Twinfilin forms a stable, high affinity complex with ADP-G-actin, inhibits the nucleotide exchange on actin monomers, and prevents their assembly into filament ends. Twinfilin was originally identified from yeast and has since then been found from all organisms studied except plants. Not much was known about the role of twinfilin in the actin dynamics in mammalian cells before this study. We set out to unravel the mysteries still covering twinfilins functions using biochemistry, cell biology, and genetics. We identified and characterized two mouse isoforms for the previously identified mouse twinfilin-1. The new isoforms, twinfilin-2a and -2b, are generated from the same gene through alternative promoter usage. The three isoforms have distinctive expression patterns, but are similar biochemically. Twinfilin-1 is the major isoform during development and is expressed in high levels in almost all tissues examined. Twinfilin-2a is also expressed almost ubiquitously, but at lower levels. Twinfilin-2b turned out to be a muscle-specific isoform, with very high expression in heart and skeletal muscle. It seems all mouse tissues express at least two twinfilin isoforms, indicating that twinfilins are important regulators of actin dynamics in all cell and tissue types. A knockout mouse line was generated for twinfilin-2a. The mice homozygous for this knockout were viable and developed normally, indicating that twinfilin-2a is dispensable for mouse development. However, it is important to note that twinfilin-2a shows similar expression pattern to twinfilin-1, suggesting that these proteins play redundant roles in mice. All mouse isoforms were shown to be able to sequester actin filaments and have higher affinity for ADP-G-actin than ATP-G-actin. They are also able to directly interact with heterodimeric capping protein and PI(4,5)P2 similar to yeast twinfilin. In this study we also uncovered a novel function for mouse twinfilins; capping actin filament barbed ends. All mouse twinfilin isoforms were shown to possess this function, while yeast and Drosophila twinfilin were not able to cap filament barbed ends. Twinfilins localize to the cytoplasm but also to actin-rich regions in mammalian cells. The subcellular localizations of the isoforms are regulated differently, indicating that even though twinfilins biochemical functions in vitro are very similar, in vivo they can play different roles through different regulatory pathways. Together, this study show that twinfilins regulate actin filament assembly both by sequestering actin monomers and by capping filament barbed ends, and that mammals have three biochemically similar twinfilin isoforms with partially overlapping expression patterns.

Functions of Alphavirus Macrodomain-containing protein nsP3

Alphaviruses are positive strand RNA viruses that replicate in association with cellular membranes. The viral RNA replication complex consists of four non-structural proteins nsP1-nsP4 which are essential for viral replication. The functions of nsP1, nsP2 and nsP4 are well established, but the roles of nsP3 are mainly unknown. In this work I have clarified some of the functions of nsP3 in order to better understand the importance of this protein in virus replication. Semliki Forest virus (SFV) has been mostly used as a model alphavirus during this work, but some experiments have also been conducted with Sindbis and Chikungunya viruses. NsP3 is composed of three different protein domains. The N-terminus of nsP3 contains an evolutionarily conserved macrodomain, the central part of nsP3 contains a domain that is only found in alphaviruses, and the C-terminus of the protein is hypervariable and predicted to be unstructured. In this work I have analyzed the functions of nsP3 macrodomain, and shown that viral macrodomains bind poly(ADP-ribose) and that they do not resemble cellular macrodomains in their properties. Furthermore, I have shown that some macrodomains, including viral macrodomains of SFV and hepatitis E virus, also bind poly(A). Mutations in the ligand binding pocket of SFV macrodomain hamper virus replication but do not confer lethality, indicating that macrodomain function is beneficial but not mandatory for virus replication. The hypervariable C-terminus of nsP3 is heavily phosphorylated and is enriched in proline residues. In this work it is shown that this region harbors an SH3 domain binding motif (Sh3BM) PxRxPR through which cellular amphiphysin is recruited to viral replication sites and to nsP3 containing cytoplasmic aggregate structures. The function of Sh3BM was destroyed by a single point mutation, which led to impaired viral RNA replication in HeLa cells, pointing out the functional importance of amphiphysin recruitment by the Sh3BM. In addition, evidence is provided tho show that the endosomal localization of alphavirus replication is mediated by nsP3 and that the phosphorylation of hypervariable region might be important for the endosomal targeting. Together these findings demonstrate that nsP3 contains multiple important host interaction motifs and domains, which facilitate successful viral propagation in host cells.

Integrated semantic processing of complex pictures and spoken sentences - Evidence from event-related potentials

It has been suggested that semantic information processing is modularized according to the input form (e.g., visual, verbal, non-verbal sound). A great deal of research has concentrated on detecting a separate verbal module. Also, it has traditionally been assumed in linguistics that the meaning of a single clause is computed before integration to a wider context. Recent research has called these views into question. The present study explored whether it is reasonable to assume separate verbal and nonverbal semantic systems in the light of the evidence from event-related potentials (ERPs). The study also provided information on whether the context influences processing of a single clause before the local meaning is computed. The focus was on an ERP called N400. Its amplitude is assumed to reflect the effort required to integrate an item to the preceding context. For instance, if a word is anomalous in its context, it will elicit a larger N400. N400 has been observed in experiments using both verbal and nonverbal stimuli. Contents of a single sentence were not hypothesized to influence the N400 amplitude. Only the combined contents of the sentence and the picture were hypothesized to influence the N400. The subjects (n = 17) viewed pictures on a computer screen while hearing sentences through headphones. Their task was to judge the congruency of the picture and the sentence. There were four conditions: 1) the picture and the sentence were congruent and sensible, 2) the sentence and the picture were congruent, but the sentence ended anomalously, 3) the picture and the sentence were incongruent but sensible, 4) the picture and the sentence were incongruent and anomalous. Stimuli from the four conditions were presented in a semi-randomized sequence. Their electroencephalography was simultaneously recorded. ERPs were computed for the four conditions. The amplitude of the N400 effect was largest in the incongruent sentence-picture -pairs. The anomalously ending sentences did not elicit a larger N400 than the sensible sentences. The results suggest that there is no separate verbal semantic system, and that the meaning of a single clause is not processed independent of the context.

Androgen receptor-dependent and independent functions of ARIP4

Androgen receptor (AR) is necessary for normal male phenotype development and essential for spermatogenesis. AR is a classical steroid receptor mediating actions of male sex steroids testosterone and 5-alpha-dihydrotestosterone. Numerous coregulators interact with the receptor and regulate AR activity on target genes. This study deals with the characterization of androgen receptor-interacting protein 4 (ARIP4). ARIP4 binds DNA, interacts with AR in vitro and in cultured yeast and mammalian cells, and modulates AR-dependent transactivation. ARIP4 is an active DNA-dependent ATPase, and this enzymatic activity is essential for the ability of ARIP4 to modulate AR function. On the basis of sequence homology in its ATPase domain, ARIP4 belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA repair, and homologous recombination. Similar to its closest homologs ATRX and Rad54, ARIP4 does not seem to be a classical chromatin remodeling protein in that it does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. However, ARIP4 is able to generate superhelical torsion on linear DNA fragments. ARIP4 is covalently modified by SUMO-1, and mutation of six potential SUMO attachment sites abolishes the ability of ARIP4 to bind DNA, hydrolyze ATP, and activate AR function. ARIP4 expression starts in early embryonic development. In mouse embryo ARIP4 is present mainly in the neural tube and limb buds. In adult mouse tissues ARIP4 expression is virtually ubiquitous. In mouse testis ARIP4 is expressed in the nuclei of Sertoli cells in a stage-dependent manner. ARIP4 is also present in the nuclei of Leydig cells, spermatogonia, pachytene and diplotene spermatocytes. Testicular expression pattern of ARIP4 does not differ significantly in wild-type, FSHRKO, and LuRKO mice. In the testis of hpg mice, ARIP4 is found mainly in interstitial cells and has very low, if any, expression in Sertoli and germ cells. Heterozygous Arip4+/ mice are fertile and appear normal; however, they are haploinsufficient with regard to androgen action in Sertoli cells. In contrast, Arip4 / embryos are not viable. They have significantly reduced body size at E9.5 and die by E11.5. Compared to wild-type littermates, Arip4 / embryos possess a higher percentage of apoptotic cells at E9.5 and E10.5. Fibroblasts derived from Arip4 / embryos cease growing after 2-3 passages and exhibit a significantly increased apoptosis and decreased proliferation rate than cells from wild-type embryos. Our findings demonstrate that ARIP4 plays an essential role in mouse embryonic development. In addition, testicular expression and AR coregulatory activity of ARIP4 suggest a role of ARIP4-AR interaction in the somatic cells of the testis.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja sydänlihaksen supistumisyksikkö, sarkomeeri, koostuu tarkoin järjestyneistä aktiini- ja myosiinisäikeistä. Rakenne eroaa muista solutyypeistä, joissa aktiinisäikeistö muovautuu jatkuvasti ja sen järjestyminen säätelee solun muotoa, solujakautumista, soluliikettä ja solunsisäisten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteenä ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyvät aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementteinä ja säätelijöinä. Myotilinia ja myopalladinia ilmennetään poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeissä kuten hermostossa, ja eri muodoilla saattaa olla solutyypistä riippuvia tehtäviä. Poikkijuovaisessa lihaksessa kaikki perheen jäsenet sijaitsevat aktiinisäikeitä yhdistävässä Z-levyssä ja ne sitovat Z-levyn rakenneproteiinia, -aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyvän periytyvään haimasyöpään ja lisääntyneeseen sydäninfarktin riskiin. Tässä tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa löydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niistä on aktiinidynamiikkaa säätelevä profilin. Profilinilla on kahdenlaisia tehtäviä; se edesauttaa aktiinisäikeiden muodostumista, mutta se voi myös eristää yksittäisiä aktiinimolekyylejä ja edistää säikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenevä runsaasti aktiinia sisältävillä solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin tehtävään aktiinisäideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinisäikeitä yhteensitova -aktiniini. -Aktiniini liittää solutukirangan solukalvon proteiineihin ja ankkuroi solunsisäisiä viestintämolekyylejä. Sitoutumista välittävä alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttejä sisältäviä tiivistymiä ja lopulta uusia sarkomeereja. Löydöstemme perusteella myotilinin uudelleenjärjestyminen noudattaa aktiinin muutoksia. Tämä viittaa siihen, että myotilin liittää yhteen uudismuodostuvia aktiinisäikeitä ja vakauttaa niitä. Myotilin saattaa myös ankkuroida viesti- tai rakennemolekyylejä, joiden tehtävänä on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, että myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinisäikeiden säätelijänä. Palladinin puute johtaa sikiöaikaiseen kuolemaan hiirillä, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyvän ja kehittyvän normaalisti eikä niillä esiintynyt rakenteellisia tai toiminnallisia häiriöitä. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, nähdään samankaltaisia kuin sairailla ihmisillä. Näin ollen muuntunut myotilin näyttä olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvittääksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilmentää puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilmentävien hiirten sydänlihaksessa todettiin vähäisiä hienorakenteen muutoksia, mutta risteytetyillä hiirillä tavattiin rakenteellisia ja toiminnallisia muutoksia myös luurankolihaksessa. Tulosten perusteella voidaan todeta, että palladinin 200 kDa muoto säätelee sydänlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla näyttäisi olevan päällekkäisiä tehtäviä.

Structural characteristics affecting functions of two actin regulating proteins

Structural biology is a branch of science that concentrates on the relationship between the structure and function of biological macromolecules. The prevalence of a large number of three dimensional structures offers effective tools for bio-scientists to understand the living world. Actin is the most abundant cellular protein and one of its main functions is to produce movement in living cells. Actin forms filaments that are dynamic and which are regulated by a number of different proteins. A class of these regulatory proteins contains actin depolymerizing factor homology (ADF-H) domains. These directly interact with actin through their ADF-H domains. Although ADF-H domains possess very similar three dimensional structures to one another, they vary in their functional properties. One example of this is the ability to bind to actin monomers or filaments. During the work for this thesis two structures of ADF-H domains were solved by nuclear magnetic resonance spectroscopy (NMR). The elucidated structures help us understand the binding specificities of the ADF-H family members.

Augustine and the functions of concupiscence

This study analyses Augustine s concept of concupiscentia, or evil desire (together with two cognate terms, libido and cupiditas) in the context of his entire oeuvre. By the aid of systematic analysis, the concept and its development is explored in four distinct ways. It is claimed that Augustine used the concept of concupiscentia for several theological purposes, and the task of the study is to represent these distinct functions, and their connections to Augustine s general theological and philosophical convictions. The study opens with a survey on terminology. A general overview of the occurrences of the negatively connoted words for desire in Latin literature precedes a corresponding examination of Augustine s own works. In this introductory chapter it is shown that, despite certain preferences in the uses of the words, a sufficient degree of synonymy reigns so as to allow an analysis of the concept without tightly discriminating between the terms. The theological functions of concupiscentia with its distinct contexts are analysed in separate chapters. The function of concupiscentia as a divine punishment is explored first (Ch 3). It is seen how Augustine links together concupiscentia and ideas about divine justice, and finally suggests that in the inordinate, psychologically experienced sexual desire, the original theological disobedience of Adam and Eve can be perceived. Augustine was criticized for this solution already in his own times, and the analysis of the function of concupiscentia as a divine punishment ends in a discussion on the critical response of punitive concupiscentia by Julian of Aeclanum. Augustine also attached to concupiscentia another central theological function by viewing evil desire as an inward originating cause for all external evil actions. In the study, this function is analysed by surveying two formally distinct images of evil desire, i.e. as the root (radix) of all evil, and as a threefold (triplex) matrix of evil actions (Ch 4). Both of these images were based on a single verse of the Bible (1 Jn 2, 16 and 1 Tim 6, 10). This function of concupiscentia was formed both parallel to, and in answer to, Manichaean insights into concupiscentia. Being familiar with the traditional philosophical discussions on the nature and therapy of emotions, Augustine situated concupiscentia also into this context. It is acknowledged that these philosophical traditions had an obvious impact into his way of explaining psychological processes in connection with concupiscentia. Not only did Augustine implicitly receive and exploit these traditions, but he also explicitly moulded and criticized them in connection with concupiscentia. Eventually, Augustine conceives the philosophical traditions of emotions as partly useful but also partly inadequate to deal with concupiscentia (Ch 5). The role of concupiscentia in connection to divine grace and Christian renewal is analysed in the final chapter of the study. Augustine s gradual development in internalizing the effects of concupiscentia also into the life of a baptized Christian are elucidated, as are the strong limitations and mitigations Augustine makes to the concept when attaching it into the life under grace (sub gratia). A crucial part in the development of this function is played by Augustine s changing interpretation of Rom 7, and the way concupiscentia appears in Augustine s readings of this text is therefore also analysed. As a result of the analysis of these four distinct functions and contexts of concupiscentia, it is concluded that Augustine s concept of concupiscentia is fairly tightly and coherently connected to his views of central theological importance. Especially the functions of concupiscentia as a punishment and the function of concupiscentia in Christian renewal were both tightly interwoven into Augustine s view of God s being and God s grace. The study shows the importance of reading Augustine s discussions on evil desire with a constant awareness of their role in their larger context, that is, of their function in each situation. The study warns against too simplistic and unifying readings of Augustine s concupiscentia, emphasizing the need to acknowledge both the necessitating, sinful aspects of concupiscentia, and the domesticated features of concupiscentia during Christian renewal.

The Structure and Functions of Hantavirus Nucleocapsid Protein

Hantaviruses, members of the genus Hantavirus in the Bunyaviridae family, are enveloped single-stranded RNA viruses with tri-segmented genome of negative polarity. In humans, hantaviruses cause two diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which vary in severity depending on the causative agent. Each hantavirus is carried by a specific rodent host and is transmitted to humans through excreta of infected rodents. The genome of hantaviruses encodes four structural proteins: the nucleocapsid protein (N), the glycoproteins (Gn and Gc), and the polymerase (L) and also the nonstructural protein (NSs). This thesis deals with the functional characterization of hantavirus N protein with regard to its structure. Structural studies of the N protein have progressed slowly and the crystal structure of the whole protein is still not available, therefore biochemical assays coupled with bioinformatical modeling proved essential for studying N protein structure and functions. Presumably, during RNA encapsidation, the N protein first forms intermediate trimers and then oligomers. First, we investigated the role of N-terminal domain in the N protein oligomerization. The results suggested that the N-terminal region of the N protein forms a coiled-coil, in which two antiparallel alpha helices interact via their hydrophobic seams. Hydrophobic residues L4, I11, L18, L25 and V32 in the first helix and L44, V51, L58 and L65 in the second helix were crucial for stabilizing the structure. The results were consistent with the head-to-head, tail-to-tail model for hantavirus N protein trimerization. We demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein. We also added new details to the head-to-head, tail-to-tail model of trimerization by suggesting that the initial step is based on interaction(s) between intact intra-molecular coiled-coils of the monomers. We further analyzed the importance of charged aa residues located within the coiled-coil for the N protein oligomerization. To predict the interacting surfaces of the monomers we used an upgraded in silico model of the coiled-coil domain that was docked into a trimer. Next the predicted target residues were mutated. The results obtained using the mammalian two-hybrid assay suggested that conserved charged aa residues within the coiled-coil make a substantial contribution to the N protein oligomerization. This contribution probably involves the formation of interacting surfaces of the N monomers and also stabilization of the coiled-coil via intramolecular ionic bridging. We proposed that the tips of the coiled-coils are the first to come into direct contact and thus initiate tight packing of the three monomers into a compact structure. This was in agreement with the previous results showing that an increase in ionic strength abolished the interaction between N protein molecules. We also showed that residues having the strongest effect on the N protein oligomerization are not scattered randomly throughout the coiled-coil 3D model structure, but form clusters. Next we found evidence for the hantaviral N protein interaction with the cytoplasmic tail of the glycoprotein Gn. In order to study this interaction we used the GST pull-down assay in combination with mutagenesis technique. The results demonstrated that intact, properly folded zinc fingers of the Gn protein cytoplasmic tail as well as the middle domain of the N protein (that includes aa residues 80 248 and supposedly carries the RNA-binding domain) are essential for the interaction. Since hantaviruses do not have a matrix protein that mediates the packaging of the viral RNA in other negatve stranded viruses (NSRV), hantaviral RNPs should be involved in a direct interaction with the intraviral domains of the envelope-embedded glycoproteins. By showing the N-Gn interaction we provided the evidence for one of the crucial steps in the virus replication at which RNPs are directed to the site of the virus assembly. Finally we started analysis of the N protein RNA-binding region, which is supposedly located in the middle domain of the N protein molecule. We developed a model for the initial step of RNA-binding by the hantaviral N protein. We hypothesized that the hantaviral N protein possesses two secondary structure elements that initiate the RNA encapsidation. The results suggest that amino acid residues (172-176) presumably act as a hook to catch vRNA and that the positively charged interaction surface (aa residues 144-160) enhances the initial N-RNA interacation. In conclusion, we elucidated new functions of hantavirus N protein. Using in silico modeling we predicted the domain structure of the protein and using experimental techniques showed that each domain is responsible for executing certain function(s). We showed that intact N terminal coiled-coil domain is crucial for oligomerization and charged residues located on its surface form a interaction surface for the N monomers. The middle domain is essential for interaction with the cytoplasmic tail of the Gn protein and RNA binding.