962 resultados para Oncogenic viruses
Resumo:
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.
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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.
Resumo:
Context: Tumor-induced osteomalacia (TIO) is a rarely diagnosed disorder presenting with bone pain, fractures, muscle weakness, and moderate-to-severe hypophosphatemia resulting from fibroblast growth factor 23-mediated renal phosphate wasting. Tumors secreting fibroblast growth factor 23 are often small and difficult to find with conventional imaging. Objective: We studied the utility of 68Ga-DOTA-octreotate (DOTATATE) somatostatin receptor positron emission tomography (PET)/computed tomography (CT) imaging in the diagnosis of TIO. Design and Setting: A multicenter case series was conducted at tertiary referral hospitals. Patients and Methods: Six patients with TIO diagnosed between 2003 and 2012 in Australia were referred for DOTATATE PET imaging. We reviewed the clinical history, biochemistry, imaging characteristics, histopathology, and clinical outcome of each patient. Results: Each case demonstrated delayed diagnosis despite severe symptoms. DOTATATE PET/CT imaging demonstrated high uptake and localized the tumor with confidence in each case. After surgical excision, there was resolution of clinical symptoms and serum phosphate, except in one patient who demonstrated residual disease on PET/CT. All tumors demonstrated high somatostatin receptor subtype 2 cell surface receptor expression using immunohistochemistry. Conclusions: In patients with TIO, DOTATATE PET/CT can successfully localize phosphaturic mesenchymal tumors and may be a practical first step in functional imaging for this disorder. Serum phosphate should be measured routinely in patients with unexplained muscle weakness, bone pain, or stress fractures to allow earlier diagnosis of TIO. - See more at: http://press.endocrine.org/doi/abs/10.1210/jc.2012-3642#sthash.eXD0CopL.dpuf
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
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A randomised and population-based screening design with new technologies has been applied to the organised cervical cancer screening programme in Finland. In this experiment the women invited to routine five-yearly screening are individually randomised to be screened with automation-assisted cytology, human papillomavirus (HPV) test or conventional cytology. By using the randomised design, the ultimate aim is to assess and compare the long-term outcomes of the different screening regimens. The primary aim of the current study was to evaluate, based on the material collected during the implementation phase of the Finnish randomised screening experiment, the cross-sectional performance and validity of automation-assisted cytology (Papnet system) and primary HPV DNA testing (Hybrid Capture II assay for 13 oncogenic HPV types) within service screening, in comparison to conventional cytology. The parameters of interest were test positivity rate, histological detection rate, relative sensitivity, relative specificity and positive predictive value. Also, the effect of variation in performance by screening laboratory on age-adjusted cervical cancer incidence was assessed. Based on the cross-sectional results, almost no differences were observed in the performance of conventional and automation-assisted screening. Instead, primary HPV screening found 58% (95% confidence interval 19-109%) more cervical lesions than conventional screening. However, this was mainly due to overrepresentation of mild- and moderate-grade lesions and, thus, is likely to result in overtreatment since a great deal of these lesions would never progress to invasive cancer. Primary screening with an HPV DNA test alone caused substantial loss in specificity in comparison to cytological screening. With the use of cytology triage test, the specificity of HPV screening improved close to the level of conventional cytology. The specificity of primary HPV screening was also increased by increasing the test positivity cutoff from the level recommended for clinical use, but the increase was more modest than the one gained with the use of cytology triage. The performance of the cervical cancer screening programme varied widely between the screening laboratories, but the variation in overall programme effectiveness between respective populations was more marginal from the very beginning of the organised screening activity. Thus, conclusive interpretations on the quality or success of screening should not be based on performance parameters only. In the evaluation of cervical cancer screening the outcome should be selected as closely as possible to the true measure of programme effectiveness, which is the number of invasive cervical cancers and subsequent deaths prevented in the target population. The evaluation of benefits and adverse effects of each new suggested screening technology should be performed before the technology becomes an accepted routine in the existing screening programme. At best, the evaluation is performed randomised, within the population and screening programme in question, which makes the results directly applicable to routine use.
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Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Well-known risk factors include tobacco smoking and alcohol consumption. Overall survival has improved, but is still low especially in developing countries. One reason for this is the often advanced stage of the disease at the time of diagnosis, but also lack of reliable prognostic tools to enable individualized patient treatment to improve outcome. To date, the TNM classification still serves as the best disease evaluation criterion, although it does not take into account the molecular basis of the tumor. The need for surrogate molecular markers for more accurate disease prediction has increased research interests in this field. We investigated the prevalence, physical status, and viral load of human papillomavirus (HPV) in HNSCC to determine the impact of HPV on head and neck carcinogenesis. The prevalence and genotyping of HPV were assessed with an SPF10 PCR microtiter plate-based hybridization assay (DEIA), followed by a line probe-based genotyping assay. More than half of the patients had HPV DNA in their tumor specimens. Oncogenic HPV-16 was the most common type, and coinfections with other oncogenic and benign associated types also existed. HPV-16 viral load was unevenly distributed among different tumor sites; the tonsils harbored significantly greater amounts of virus than other sites. Episomal location of HPV-16 was associated with large tumors, and both integrated and mixed forms of viral DNA were detected. In this series, we could not show that the presence of HPV DNA correlated with survival. In addition, we investigated the prevalence and genotype of HPV in laryngeal carcinoma patients in a prospective Nordic multicenter study based on fresh-frozen laryngeal tumor samples to determine whether the tumors were HPV-associated. These patients were also examined and interviewed at diagnosis for known risk factors, such as tobacco smoking and alcohol consumption, and for several other habituations to elucidate their effects on patient survival. HPV analysis was performed with the same protocols as in the first study. Only 4% of the specimens harbored HPV DNA. Heavy drinking was associated with poor survival. Heavy drinking patients were also younger than nonheavy drinkers and had a more advanced stage of disease at diagnosis. Heavy drinkers had worse oral hygiene than nonheavy drinkers; however, poor oral hygiene did not have prognostic significance. History of chronic laryngitis, gastroesophageal reflux disease, and orogenital sex contacts were rare in this series. To clarify why vocal cord carcinomas seldom metastasize, we determined tumor lymph vessel (LVD) and blood vessel (BVD) densities in HNSCC patients. We used a novel lymphatic vessel endothelial marker (LYVE-1 antibody) to locate the lymphatic vessels in HNSCC samples and CD31 to detect the blood microvessels. We found carcinomas of the vocal cords to harbor less lymphatic and blood microvessels than carcinomas arising from sites other than vocal cords. The lymphatic and blood microvessel densities did not correlate with tumor size. High BVD was strongly correlated with high LVD. Neither BVD nor LVD showed any association with survival in our series. The immune system plays an important role in tumorigenesis, as neoplastic cells have to escape the cytotoxic lymphocytes in order to survive. Several candidate HLA class II alleles have been reported to be prognostic in cervical carcinomas, an epithelial malignancy resembling HNSCC. These alleles may have an impact on head and neck carcinomas as well. We determined HLA-DRB1* and -DQB1* alleles in HNSCC patients. Healthy organ donors served as controls. The Inno-LiPA reverse dot-blot kit was used to identify alleles in patient samples. No single haplotype was found to be predictive of either the risk for head and neck cancer, or the clinical course of the disease. However, alleles observed to be prognostic in cervical carcinomas showed a similar tendency in our series. DRB1*03 was associated with node-negative disease at diagnosis. DRB1*08 and DRB1*13 were associated with early-stage disease; DRB1*04 had a lower risk for tumor relapse; and DQB1*03 and DQB1*0502 were more frequent in controls than in patients. However, these associations reached only borderline significance in our HNSCC patients.
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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.
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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.
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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.
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Although prevention and early detection of the disease greatly improved over the past few years, lung cancer remains the leading cause of cancer deaths. In order to be able to treat a larger population, we are in urgent need for novel treatments. While it is known that DNA repair genes play a major role in the oncogenic transformation, they also represent a weakness of cancers that constitute a therapeutic opportunity. To identify novel DNA repair genes implicated in Lung cancers, we conducted an in silico investigation to identify genes co-regulated with two DNA repair factors, BRCA2 and hSSB1. This approach allowed for the identification of EXOSC4, a component of the RNA Exosome machinery, as a potential factor involved in the maintenance of genome stability and that is deregulated in lung cancer.
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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.