Molecular details of phage phi6 RNA-dependent RNA synthesis

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.

Lipid-Containing Icosahedral dsDNA Bacteriophages: Entry, Exit and Structure

In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.

Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins

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.

The viral coat protein is regulated by HSP70 and HSP40 in Potato virus A infection

Plus-stranded (plus) RNA viruses multiply within a cellular environment as tightly integrated units and rely on the genetic information carried within their genomes for multiplication and, hence, persistence. The minimal genomes of plus RNA viruses are unable to encode the molecular machineries that are required for virus multiplication. This sets requisites for the virus, which must form compatible interactions with host components during multiplication to successfully utilize primary metabolites as building blocks or metabolic energy, and to divert the protein synthesis machinery for production of viral proteins. In fact, the emerging picture of a virus-infected cell displays tight integration with the virus, from simple host and virus protein interactions through to major changes in the physiological state of the host cell. This study set out to develop a method for the identification of host components, mainly host proteins, that interact with proteins of Potato virus A (PVA; Potyvirus) during infection. This goal was approached by developing affinity-tag based methods for the purification of viral proteins complexed with associated host proteins from infected plants. Using this method, host membrane-associated viral ribonucleoprotein (RNP) complexes were obtained, and several host and viral proteins could be identified as components of these complexes. One of the host proteins identified using this strategy was a member of the heat shock protein 70 (HSP70) family, and this protein was chosen for further analysis. To enable the analysis of viral gene expression, a second method was developed based on Agrobacterium-mediated virus genome delivery into plant cells, and detection of virally expressed Renilla luciferase (RLUC) as a quantitative measure of viral gene expression. Using this method, it was observed that down-regulation of HSP70 caused a PVA coat protein (CP)-mediated defect associated with replication. Further experimentation suggested that CP can inhibit viral gene expression and that a distinct translational activity coupled to replication, referred to as replication-associated translation (RAT), exists. Unlike translation of replication-deficient viral RNA, RAT was dependent on HSP70 and its co-chaperone CPIP. HSP70 and CPIP together regulated CP turnover by promoting its modification by ubiquitin. Based on these results, an HSP70 and CPIP-driven mechanism that functions to regulate CP during viral RNA replication and/or translation is proposed, possibly to prevent premature particle assembly caused by CP association with viral RNA.

Phospholipids of lipid-containing bacteriophages and their transbilayer distribution

In this study we used electro-spray ionization mass-spectrometry to determine phospholipid class and molecular species compositions in bacteriophages PM2, PRD1, Bam35 and phi6 as well as their hosts. To obtain compositional data of the individual leaflets, phospholipid transbilayer distribution in the viral membranes was studied. We found that 1) the membranes of all studied bacteriophage are enriched in PG as compared to the host membranes, 2) molecular species compositions in the phage and host membranes are similar, and 3) phospholipids in the viral membranes are distributed asymmetrically with phosphatidylglycerol enriched in the outer leaflet and phosphatidylethanolamine in the inner one (except Bam35). Alternative models for selective incorporation of phospholipids to phages and for the origins of the asymmetric phospholipid transbilayer distribution are discussed. Notably, the present data are also useful when constructing high resolution structural models of bacteriophages, since diffraction methods cannot provide a detailed structure of the membrane due to high motility of the lipids and lack of symmetric organization of membrane proteins.

Molecular epidemiology of human rhinoviruses

The first part of this work investigates the molecular epidemiology of a human enterovirus (HEV), echovirus 30 (E-30). This project is part of a series of studies performed in our research team analyzing the molecular epidemiology of HEV-B viruses. A total of 129 virus strains had been isolated in different parts of Europe. The sequence analysis was performed in three different genomic regions: 420 nucleotides (nt) in the VP4/VP2 capsid protein coding region, the entire VP1 capsid protein coding gene of 876 nt, and 150 nt in the VP1/2A junction region. The analysis revealed a succession of dominant sublineages within a major genotype. The temporally earlier genotypes had been replaced by a genetically homogenous lineage that has been circulating in Europe since the late 1970s. The same genotype was found by other research groups in North America and Australia. Globally, other cocirculating genetic lineages also exist. The prevalence of a dominant genotype makes E-30 different from other previously studied HEVs, such as polioviruses and coxsackieviruses B4 and B5, for which several coexisting genetic lineages have been reported. The second part of this work deals with molecular epidemiology of human rhinoviruses (HRVs). A total of 61 field isolates were studied in the 420-nt stretch in the capsid coding region of VP4/VP2. The isolates were collected from children under two years of age in Tampere, Finland. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades. Seasonal clustering was found. Also, several distinct serotype-like clusters were found to co-circulate during the same epidemic season. Reappearance of a cluster after disappearing for a season was observed. The molecular epidemiology of the analyzed strains turned out to be complex, and we decided to continue our studies of HRV. Only five previously published complete genome sequences of HRV prototype strains were available for analysis. Therefore, all designated HRV prototype strains (n=102) were sequenced in the VP4/VP2 region, and the possibility of genetic typing of HRV was evaluated. Seventy-six of the 102 prototype strains clustered in HRV genetic group A (HRV-A) and 25 in group B (HRV-B). Serotype 87 clustered separately from other HRVs with HEV species D. The field strains of HRV represented as many as 19 different genotypes, as judged with an approximate demarcation of a 20% nt difference in the VP4/VP2 region. The interserotypic differences of HRV were generally similar to those reported between different HEV serotypes (i.e. about 20%), but smaller differences, less than 10%, were also observed. Because some HRV serotypes are genetically so closely related, we suggest that the genetic typing be performed using the criterion "the closest prototype strain". This study is the first systematic genetic characterization of all known HRV prototype strains, providing a further taxonomic proposal for classification of HRV. We proposed to divide the genus Human rhinoviruses into HRV-A and HRV-B. The final part of the work comprises a phylogenetic analysis of a subset (48) of HRV prototype strains and field isolates (12) in the nonstructural part of the genome coding for the RNA-dependent RNA polymerase (3D). The proposed division of the HRV strains in the species HRV-A and HRV-B was also supported by 3D region. HRV-B clustered closer to HEV species B, C, and also to polioviruses than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D coding region than in the VP4/VP2 coding region, in contrast to HEV. Moreover, the diversity of HRV in 3D exceeded that of HEV. One group of HRV-A, designated HRV-A', formed a separate cluster outside other HRV-A in the 3D region. It formed a cluster also in the capsid region, but located within HRV-A. This may reflect a different evolutionary history of distinct genomic regions among HRV-A. Furthermore, the tree topology within HRV-A in the 3D region differed from that in the VP4/VP2, suggesting possible recombination events in the evolution of the strains. No conflicting phylogenies were observed in any of the 12 field isolates. Possible recombination was further studied using the Similarity and Bootscanning analyses of the complete genome sequences of HRV available in public databases. Evidence for recombination among HRV-A was found, as HRV2 and HRV39 showed higher similarity in the nonstructural part of the genome. Whether HRV2 and HRV39 strains - and perhaps also some other HRV-A strains not yet completely sequenced - are recombinants remains to be determined.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.

Human herpesvirus-6 infection in liver transplantation

Rejection and infections are the two most common complications after liver transplantation. Human herpesvirus-6 (HHV-6) belongs to the betaherpesviruses, together with its close relatives cytomegalovirus (CMV) and human herpesvirus-7 (HHV-7). The impact of CMV in liver transplantation is well characterized, but the roles of the other two betaherpesviruses have been acknowledged only recently. Although, HHV-6 reactivation after transplantation is usually asymptomatic, the virus may infect the liver transplant, cause an intragraft lymphocyte dominated inflammatory reaction and graft dysfunction. HHV-6 is also suggested to be associated with liver allograft rejection but the mechanisms are unclear. The aim of this study was to investigate the intragraft immunological processes associated with HHV-6, the involvement of HHV-6 in acute liver failure (ALF) and the hepatic HHV-6 infection of the same patients after transplantation. In addition, the occurrence of HHV-6 and HHV-7 was investigated in liver transplant patients with symptomatic CMV infection. HHV-6 infection of the liver graft was associated with portal lymphocyte infiltration and with a significant increase of adhesion molecules (ICAM-1 and VCAM-1) and the number of cells expressing their ligand molecules (LFA-1, VLA-4) and class II antigens. HHV-6 infection was associated with significant immunological changes, but the immune response was limited to lymphocyte infiltration and the adhesion molecule level. However, one third of these patients developed chronic rejection during the follow-up. Of the patients with ALF of unknown origin, most patients demonstrated HHV-6 antigens in the liver, whereas the opposite was seen in ALF patients with a known disease. After transplantation, HHV-6 recurrence was found in the liver transplant in half of these patients with pre-transplant HHV-6 infection of the liver, whereas no post-transplant HHV-6 infection of the liver was seen in patients without pre-transplant HHV-6. Our studies further demonstrated that both HHV-6 and HHV-7 antigenemia often appeared in association with CMV disease in liver transplant patients. The time-related occurrence of the viruses differed, as HHV-6 appeared early after transplantation and regularly preceded CMV whereas HHV-7 often appeared concurrently with CMV. In conclusion, these results indicate that all three betaherpesviruses are common after liver transplantation, often associated with each other. The immunological events caused by HHV-6 in the liver transplant may be involved in, or trigger mechanisms of allograft rejection. In addition, HHV-6 could be one of the causes of ALF, and pre-transplant HHV-6 infection in ALF patients is a risk factor for post-transplant HHV-6 infection of the graft. These results strongly support the clinical significance of HHV-6 in liver transplantation. Even though the reactivation is usually asymptomatic, in some individuals HHV-6 infection may lead to severe manifestations, such as liver failure or in transplant patients, graft dysfunction and rejection.

Approaches for improving the safety and efficacy of adenoviral gene therapy

Cancer is a devastating disease with poor prognosis and no curative treatment, when widely metastatic. Conventional therapies, such as chemotherapy and radiotherapy, have efficacy but are not curative and systemic toxicity can be considerable. Almost all cancers are caused due to changes in the genetic material of the transformed cells. Cancer gene therapy has emerged as a new treatment option, and past decades brought new insights in developing new therapeutic drugs for curing cancer. Oncolytic viruses constitute a novel therapeutic approach given their capacity to replicate in and kill specifically tumor cells as well as reaching tumor distant metastasis. Adenoviral gene therapy has been suggested to cause liver toxicity. This study shows that new developed adenoviruses, in particular Ad5/19p-HIT, can be redirected towards kidney while adenovirus uptake by liver is minimal. Moreover, low liver transduction resulted in a favorable tumor to liver ratio of virus load. Further, we established a new immunocompetent animal model Syrian hamsters. Wild type adenovirus 5 was found to replicate in Hap-T1 hamster tumors and normal tissues. There are no antiviral drugs available to inhibit adenovirus replication. In our study, chlorpromazine and cidofovir efficiently abrogated virus replication in vitro and showed significant reduction in vivo in tumors and liver. Once safety concerns were addressed together with the new given antiviral treatment options, we further improved oncolytic adenoviruses for better tumor penetration, local amplification and host system modulation. Further, we created Ad5/3-9HIF-Δ24-VEGFR-1-Ig, oncolytic adenovirus for improved infectivity and antiangiogenic effect for treatment of renal cancer. This virus exhibited increased anti-tumor effect and specific replication in kidney cancer cells. The key player for good efficacy of oncolytic virotherapy is the host immune response. Thus, we engineered a triple targeted adenovirus Ad5/3-hTERT-E1A-hCD40L, which would lead to tumor elimination due to tumor-specific oncolysis and apoptosis together with an anti-tumor immune response prompted by the immunomodulatory molecule. In conclusion, the results presented in this thesis constitute advances in our understanding of oncolytic virotherapy by successful tumor targeting, antiviral treatment options as a safety switch in case of replication associated side-effects, and modulation of the host immune system towards tumor elimination.  

Complement factor C4 and immunoglobulins in recurrent or chronic mucosal infections

The study assessed whether plasma concentrations of complement factors C3, C4, or immunoglobulins, serum classical pathway hemolytyic activity, or polymorphisms in the class I and II HLA genes, isotypes and gene numbers of C4, or allotypes of IgG1 and IgG3 heavy chain genes were associated with severe frequently recurring or chronic mucosal infections. According to strict clinical criteria, 188 consecutive voluntary patients without a known immunodeficiency and 198 control subjects were recruited. Frequencies of low levels in IgG1, IgG2, IgG3 and IgG4 were for the first time tested from adult general population and patients with acute rhinosinusitis. Frequently recurring intraoral herpes simplex type 1 infections, a rare form of the disease, was associated with homozygosity in HLA -A*, -B*, -C*, and -DR* genes. Frequently recurrent genital HSV-2 infections were associated with low levels of IgG1 and IgG3, present in 54% of the recruited patients. This association was partly allotype-dependent. The G3mg,G1ma/ax haplotype, together with low IgG3, was more common in patients than in control subjects who lacked antibodies against herpes simplex viruses. This is the first found immunogenetic deficiency in otherwise healthy adults that predisposes to highly frequent mucosal herpes recurrences. According to previous studies, HSV effectively evades the allotype G1ma/ax of IgG1, whereas G3mg is associated with low IgG3. Certain HLA genes were more common in patients than in control subjects. Having more than one C4A or C4B gene was associated with neuralgias caused by the virus. Low levels of IgA, IgG1, IgG2, IgG3, and IgG4 were common in the general adult population, but even more frequent in patients with chronic sinusitis. Only low IgG1 was more common chronic than in acute rhinosinusitis. Clinically, nasal polyposis and bronchial asthma were associated with complicated disease forms. The best differentiating immunologic parameters were C4A deficiency and the combination of low plasma IgG4 together with low IgG1 or IgG2, performing almost equally. The lack of C4A, IgA, and IgG4, all known to possess anti-inflammatory activity, together with a concurrently impaired immunity caused by low subclass levels, may predispose to chronic disease forms. In severe chronic adult periodontitis, any C4A or C4B deficiency combined was associated with the disease. The new quantitative analysis of C4 genes and the conventional C4 allotyping method complemented each other. Lowered levels of plasma C3 or C4 or both, and serum CH50 were found in herpes and periodontitis patients. In rhinosinusitis, there was a linear trend with the highest levels found in the order: acute > chronic rhinosinusitis > general population > blood donors with no self-reported history of rhinosinusitis. Complement is involved in the defense against the tested mucosal infections. Seemingly immunocompetent patients with chronic or recurrent mucosal infections frequently have subtle weaknesses in different arms of immunity. Their susceptibility to chronic disease forms may be caused by these. Host s subtly impaired immunity often coincides with effective immune evasion from the same arms of immunity by the disease-causing pathogens. The interpretation of low subclass levels, if no additional predisposing immunologic factors are tested, is difficult and of limited value in early diagnosis and treatment.

Phosphorylation status of the phosphoprotein P of rinderpest virus modulates transcription and replication of the genome

The phosphoprotein P of paramyxoviruses is known to play more than one role in genome transcription and replication. Phosphorylation of P at the NH2 terminus by cellular casein kinase II has been shown to be necessary for transcription of the genome in some of the viruses, while it is dispensable for replication. The phosphorylation null mutant of rinderpest virus P protein, in which three serine residues have been mutated, has been shown earlier to be non-functional in an in vivo minigenome replication/transcription system. In this work, we have shown that the phosphorylation of P protein is essential for transcription, whereas the null mutant is active in replication of the genome in vivo. The null mutant P acts as a transdominant repressor of transcriptional activity of wild-type P and as an activator of replication carried out by wild-type P protein. These results suggest the phosphorylation status of P may act as a replication switch during virus replication. We also show that the phosphorylation null mutant P is capable of interacting with L and N proteins and is able to form a tripartite complex of L-(N-P) when expressed in insect cells, similar to wild-type P protein.

Unusual RNA plant virus integration in the soybean genome leads to the production of small RNAs

Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants.

Preparation and Characterization of Glycolipid-Bearing Multilamellar and Unilamellar Liposomes

The carbohydrate residues of glycosphingolipids were implicated in many biologic processes such as cell-to-cell interactions; and as receptors for some viruses, bacterial and plant toxins, hormones, and so forth, and invariably for all the lectins (1). However, their receptor functions remained poorly defined for a long time as they form micelles even at very low concentrations in aqueous medium. In micelles, the oligosaccharide chains are not expected to have a well defined orientation suitable for recognition by macromolecular ligands. This problem was overcome by incorporating them in model membranes, namely, the liposomes. The demonstration of lectin-glycolipid interaction using liposomal model membranes was a crucial development that established glycolipids as biological receptors. Moreover, glycolipid-bearing liposomes provide a convenient system for investigating the role of glycolipid density, orientation, and exposure of their oligosaccharide chains at the membrane interface relevant to their receptor function (2–4).

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.

Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.