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  • 1.
    Assadian, Farzaneh
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandström, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Laurell, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Lidian, Adnan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Svensson, Catharina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Distribution and Molecular Characterization of Human Adenovirus and Epstein-Barr Virus Infections in Tonsillar Lymphocytes Isolated from Patients Diagnosed with Tonsillar Diseases2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 5, article id e0154814Article in journal (Refereed)
    Abstract [en]

    Surgically removed palatine tonsils provide a conveniently accessible source of T and B lymphocytes to study the interplay between foreign pathogens and the host immune system. In this study we have characterised the distribution of human adenovirus (HAdV), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) in purified tonsillar T and B cell-enriched fractions isolated from three patient age groups diagnosed with tonsillar hypertrophy and chronic/recurrent tonsillitis. HAdV DNA was detected in 93 out of 111 patients (84%), while EBV DNA was detected in 58 patients (52%). The most abundant adenovirus type was HAdV-5 (68%). None of the patients were positive for HCMV. Furthermore, 43 patients (39%) showed a co-infection of HAdV and EBV. The majority of young patients diagnosed with tonsillar hypertrophy were positive for HAdV, whereas all adult patients diagnosed with chronic/recurrent tonsillitis were positive for either HAdV or EBV. Most of the tonsils from patients diagnosed with either tonsillar hypertrophy or chronic/recurrent tonsillitis showed a higher HAdV DNA copy number in T compared to B cell-enriched fraction. Interestingly, in the majority of the tonsils from patients with chronic/recurrent tonsillitis HAdV DNA was detected in T cells only, whereas hypertrophic tonsils demonstrated HAdV DNA in both T and B cell-enriched fractions. In contrast, the majority of EBV positive tonsils revealed a preference for EBV DNA accumulation in the B cell-enriched fraction compared to T cell fraction irrespective of the patients' age.

  • 2.
    Bergfors, Assar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Leenheer, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology. Univ Tsukuba, PhD Program Human Biol, Sch Integrat & Global Majors, Tsukuba, Ibaraki 3058577, Japan..
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Ameur, Adam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing2016In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 126, p. 81-89Article in journal (Refereed)
    Abstract [en]

    Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naive-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naive patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual.

  • 3.
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Why does hepatitis C virus cause chronic infection2000In: Review Series. Hepatitis, ISSN 1402-4144, Vol. 1, no 2-6, p. 23-24Article in journal (Other academic)
  • 4.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.1990In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
  • 5.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology.
    Öhrmalm, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Strand, Mårten
    Lysvand, Hilde
    Teppor, Mona
    Kakkola, Laura
    Paavilainen, Henrik
    Laajala, Mira
    Kallio-Kokko, Hannimari
    Valkonen, Miia
    Kantele, Anu
    Telling, Kaidi
    Lutsar, Irja
    Letjuka, Pille
    Metelitsa, Natalja
    Oksenych, Valentyn
    Bjørås, Magnar
    Nordbø, Svein Arne
    Dumpis, Uga
    Vitkauskiene, Astra
    Öhrmalm, Christina
    Bondeson, Kåre
    Bergqvist, Anders
    Aittokallio, Tero
    Cox, Rebecca J
    Evander, Magnus
    Hukkanen, Veijo
    Marjomaki, Varpu
    Julkunen, Ilkka
    Vapalahti, Olli
    Tenson, Tanel
    Merits, Andres
    Kainov, Denis
    Novel activities of safe-in-human broad-spectrum antiviral agents.2018In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, article id S0166-3542(18)30098-6Article in journal (Refereed)
    Abstract [en]

    According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.

  • 6.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rice, Charles M.
    Washington University, St Louis.
    Transcriptional activation of the interleukin-2 promoter by hepatitis C virus core protein2001In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 75, no 2, p. 772-781Article in journal (Refereed)
    Abstract [en]

    Most patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56lck, and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections.

  • 7.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sundström, Sara
    Karolinska Institutet.
    Dimberg, Lina Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Masucci, Maria G.
    Karolinska Institutet.
    The Hepatitis C Virus Core Protein Modulates T Cell Responses by Inducing Spontaneous and Altering T-cell Receptor-triggered Ca2+ Oscillations2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 21, p. 18877-18883Article in journal (Refereed)
    Abstract [en]

    Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infection, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Expression of the HCV core (C) protein modulates transcription of the IL-2 promoter in T lymphocytes by activating the nuclear factor of activated T lymphocyte (NFAT) pathway. Here we report on the effect of HCV C on Ca2+ signaling, which is essential for activation of NFAT. Expression of HCV C correlated with increased levels of cytosolic Ca2+ and spontaneous Ca2+ oscillations in transfected Jurkat cells. Triggering of the T-cell receptor induced a prolonged Ca2+ response characterized by vigorous high frequent oscillations in a high proportion of the responding cells. This was associated with decreased sizes and accelerated emptying of the intracellular calcium stores. The effect of HCV C on calcium mobilization was not dependent on phospholipase C-1 (PLC-) activity or increased inositol 1,4,5-trisphosphate (IP3) production and did not require functional IP3 receptors, suggesting that insertion of the viral protein in the endoplasmic reticulum membrane may be sufficient to promote Ca2+ leakage with dramatic downstream consequences on the magnitude and duration of the response. Our data suggest that expression of HCV C in infected T lymphocytes may contribute to the establishment of persistent infections by inducing Ca2+ oscillations that regulate both the efficacy and information content of Ca2+ signals and are ultimately responsible for induction of gene expression and functional differentiation.

  • 8.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Söderbärg, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Magnusson, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Altered susceptibility to tumor necrosis factor alpha-induced apoptosis of mouse cells expressing polyomavirus middle and small T antigens1997In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 71, no 1, p. 276-283Article in journal (Refereed)
    Abstract [en]

    Infection with some virus types induces susceptibility to the cytotoxic effect of tumor necrosis factor alpha (TNF-alpha). To investigate whether expression of polyomavirus proteins has this effect on cells, the TNF-alpha sensitivity of C127 and L929 mouse cells transfected with viral DNA was analyzed. Expression of all three polyomavirus early proteins, the tumor (T) antigens, had no apparent effect. In contrast, middle T antigen by itself induced hypersensitivity to TNF-alpha. This effect was reversed by retransfection of the cells with DNA encoding small T antigen. Expression of this polypeptide also decreased the sensitivity of bovine papillomavirus type 1-transformed cells to TNF- alpha, showing that the protective function of the polyomavirus small T antigen was not strictly linked to a middle-T-antigen-induced event. Mouse and human TNF-alpha had the same effect on normal and transformed mouse cells, suggesting that this effect was mediated by TNF receptor 1. Consistent with this conclusion, all cell clones used in the experiments expressed TNF receptor 1 at similar levels, while we failed to detect TNF receptor 2. The amount of receptor on the cells was not influenced by binding of the ligand. Addition of TNF-alpha at cytotoxic concentrations to cells expressing middle T antigen by itself resulted in significant fragmentation of chromosomal DNA after only a few hours, indicating induction of apoptosis. Addition of the cytokine to these cells also leads to release of arachidonic acid, showing that phospholipase A2 was activated. However, production of arachidonic acid did not appear to significantly precede loss of cell viability.

  • 9.
    Houshmand, Hamid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Interaction of hepatitis C virus NS5A with La protein revealed by T7 phage display2003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 309, no 3, p. 695-701Article in journal (Refereed)
    Abstract [en]

    Although the hepatitis C virus (HCV) genome is synthesized by the virus-encoded RNA-dependent RNA polymerase NS5B, other viral and cellular factors are assumed to be required for template-specific initiation and regulation of RNA-synthesis. The cellular protein La, which normally associates with RNA polymerase III transcripts, also interacts with the 5'- and 3'-untranslated regions of several RNA viruses, including HCV. To investigate whether other viral gene products may be involved in this interaction, we constructed an HCV cDNA expression library in bacteriophage T7 allowing portions of the HCV polyprotein to be displayed on the phage surface. Screening of the phage library against La resulted in selection of clones displaying the N-terminal region of HCV NS5A. Co-precipitation of full-length and truncated forms of recombinant NS5A with La revealed that the N-terminal region of NS5A was both necessary and sufficient for binding to La. Although this region of NS5A is essential for HCV replication, the role of the NS5A-La interaction in the infected cell remains to be established.

  • 10. Lemm, Julie A
    et al.
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Read, Carol M
    Rice, Charles M
    Template-dependent initiation of Sindbis virus RNA replication in vitro1998In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 72, no 8, p. 6546-6553Article in journal (Refereed)
    Abstract [en]

    Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

  • 11. Ruediger, R
    et al.
    Roeckel, D
    Fait, J
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Magnusson, G
    Walter, G
    Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.1992In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 12, no 11, p. 4872-82Article in journal (Refereed)
    Abstract [en]

    Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.

  • 12.
    Sundström, Sara
    et al.
    Karolinska Institutet.
    Ota, Seisuke
    Karolinska Institutet.
    Dimberg, Lina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Masucci, Maria
    Karolinska Institutet.
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Hepatitis C virus core protein induces an anergic state characterized by decreased interleukin-2 production and perturbation of mitogen-activated protein kinase responses2005In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 79, no 4, p. 2230-2239Article in journal (Refereed)
    Abstract [en]

    Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infections, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Here we demonstrate that the expression of the HCV core (C) protein in stably transfected T cells correlates with a selective reduction of interleukin-2 (IL-2) promoter activity and IL-2 production in response to T-cell receptor triggering, whereas the activation of IL-4, IL-10, gamma interferon, and tumor necrosis factor alpha was moderately increased. This altered cytokine expression profile was associated with a perturbation of mitogen-activated protein (MAP) kinase responses. Extracellular regulated kinase and p38 were constitutively phosphorylated in C-expressing cells, while triggering of the costimulatory c-Jun N-terminal kinase (JNK) signaling cascade and activation of the CD28 response element within the IL-2 promoter appeared to be impaired. The perturbations of MAP kinase phosphorylation could be eliminated by cyclosporine A-mediated inhibition of nuclear factor of activated T cells, suggesting that the inactivation of JNK signaling and hyporesponsiveness to IL-2 induction were downstream consequences of C-induced Ca2+ flux in a manner that mimics the induction of clonal anergy.

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