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  • 1. Aoki, Koki
    et al.
    Benkö, Mária
    Davison, Andrew J
    De Jong, Jan C
    Echavarria, Marcela
    Erdman, Dean D
    Harrach, Balázs
    Kajon, Adriana E
    Schnurr, David
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Towards an integrated human adenovirus designation system that utilizes molecular and serological data and serves both clinical and fundamental virology2011Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, nr 11, s. 5703-5704Artikel i tidskrift (Refereegranskat)
  • 2.
    Arnberg, Niklas
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Edlund, Karin
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Kidd, Alistair H
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Adenovirus type 37 uses sialic acid as a cellular receptor2000Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 74, nr 1, s. 42-48Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) alpha2, have recently been identified. In the absence of CAR, MHC-I alpha2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expressing CHO cells and MHC-I alpha2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via alpha(2-->3)-linked sialic acid saccharides rather than alpha(2-->6)-linked ones, since (i) alpha(2-->3)-specific but not alpha(2-->6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with alpha(2-->3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of alpha(2-->3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I alpha2.

  • 3.
    Arnberg, Niklas
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Kidd, Alistair H
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Edlund, Karin
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Olfat, Farzad
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Initial interactions of subgenus D adenoviruses with A549 cellular receptors: sialic acid versus alpha(v) integrins2000Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 74, nr 16, s. 7691-3Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, alpha(v) integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.

  • 4.
    Arnberg, Niklas
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Pring-Akerblom, Patricia
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Adenovirus type 37 uses sialic acid as a cellular receptor on Chang C cells2002Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 76, nr 17, s. 8834-8841Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epidemic keratoconjunctivitis (EKC) is a severe eye infection caused mainly by adenovirus type 8 (Ad8), Ad19, and Ad37. We have shown that the EKC-causing adenoviruses use sialic acid as a cellular receptor on A549 cells instead of the coxsackie-adenovirus receptor, which is used by most adenoviruses. Recently, Wu et al. (Virology 279:78-89, 2001) proposed that Ad37 uses a 50-kDa protein as a receptor on Chang C conjunctival cells and that this interaction is independent of sialic acid. According to the American Type Culture Collection, this cell line carries HeLa cell markers and should be considered to be a genital cell line. This prompted us to investigate the function of sialic acid as a cellular receptor for Ad37 in Chang C cells. In this study, we demonstrate that enzymatic removal or lectin-mediated blocking of cell surface sialic acid inhibits the binding of Ad37 virions to Chang C cells, as does soluble, virion-interacting sialic acid-containing substances. The binding was Ca2+ or Mg2+ ion independent and mediated by the knob domain of the trimeric viral fiber polypeptide. Moreover, Ad37 virions infected Chang C cells and two other genital cell lines (HeLa and SiHa) as well as a corneal cell line in a strictly sialic acid-dependent manner. From these results, we conclude that Ad37 uses sialic acid as a major receptor in cell lines derived from both genital and corneal tissues.

  • 5. Burmeister, Wim P
    et al.
    Guilligay, Delphine
    Cusack, Stephen
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites2004Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 78, nr 14, s. 7727-7736Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both alpha(2,3) and alpha(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose.

  • 6. Cupelli, Karolina
    et al.
    Müller, Steffen
    Persson, David B
    Jost, Marco
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Stehle, Thilo
    Structure of adenovirus type 21 knob in complex with CD46 reveals key differences in receptor contacts among species B adenoviruses2010Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 84, nr 7, s. 3189-3200Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The complement regulation protein CD46 is the primary attachment receptor for most species B adenoviruses (Ads). However, significant variability exists in sequence and structure among species B Ads in the CD46-binding regions, correlating with differences in affinity. Here, we report a structure-function analysis of the interaction of the species B Ad21 knob with the two N-terminal repeats SCR1 and SCR2 of CD46, CD46-D2. We have determined the structures of the Ad21 knob in its unliganded form as well as in complex with CD46-D2, and we compare the interactions with those observed for the Ad11 knob-CD46-D2 complex. Surface plasmon resonance measurements demonstrate that the affinity of Ad21 knobs for CD46-D2 is 22-fold lower than that of the Ad11 knob. The superposition of the Ad21 and Ad11 knob structures in complex with CD46-D2 reveals a substantially different binding mode, providing an explanation for the weaker binding affinity of the Ad21 knob for its receptor. A critical difference in both complex structures is that a key interaction point, the DG loop, protrudes more in the Ad21 knob than in the Ad11 knob. Therefore, the protruding DG loop does not allow CD46-D2 to approach the core of the Ad21 knob as closely as in the Ad11 knob-CD46-D2 complex. In addition, the engagement of CD46-D2 induces a conformational change in the DG loop in the Ad21 knob but not in the Ad11 knob. Our results contribute to a more profound understanding of the CD46-binding mechanism of species B Ads and have relevance for the design of more efficient gene delivery vectors.

  • 7. Fedeli, Chiara
    et al.
    Torriani, Giulia
    Galan-Navarro, Clara
    Moraz, Marie-Laurence
    Moreno, Hector
    Gerold, Gisa
    TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Kunz, Stefan
    Axl can serve as entry factor for lassa virus depending on the functional glycosylation of dystroglycan2018Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, nr 5, artikel-id e01613-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The highly pathogenic arenavirus Lassa virus (LASV) represents a serious public health problem in Africa. Although the principal LASV receptor, dystroglycan (DG), is ubiquitously expressed, virus binding critically depends on DG's posttranslational modification, which does not always correlate with tissue tropism. The broadly expressed phosphatidylserine receptor Axl was recently identified as an alternative LASV receptor candidate, but its role in LASV entry is unclear. Here, we investigate the exact role of Axl in LASV entry as a function of DG's posttranslational modification. We found that in the absence of functional DG, Axl can mediate LASV entry via apoptotic mimicry. Productive entry requires virus-induced receptor activation, involves macropinocytosis, and critically depends on LAMP-1. In endothelial cells that express low levels of glycosylated DG, both receptors can promote LASV entry. In sum, our study defines the roles of Axl in LASV entry and provides a rationale for targeting Axl in antiviral therapy.

  • 8. Flick, Kirsten
    et al.
    Katz, Anna
    Överby, Anna
    Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Stockholm, Sweden.
    Feldmann, Heinz
    Pettersson, Ralf F
    Flick, Ramon
    Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments2004Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 78, nr 21, s. 11726-11738Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5' and 3' ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.

  • 9. Flick, Ramon
    et al.
    Elgh, Fredrik
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Pettersson, Ralf F
    Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance2002Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 76, nr 21, s. 10849-10860Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75:1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5'- and 3'-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8, where underlined nucleotides refer to positions in the vRNA 3' end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5' and 3' ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5' and 3' ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.

  • 10. Flick, Ramon
    et al.
    Flick, Kirsten
    Feldmann, Heinz
    Elgh, Fredrik
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Reverse genetics for crimean-congo hemorrhagic fever virus.2003Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 77, nr 10, s. 5997-6006Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family BUNYAVIRIDAE: The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family BUNYAVIRIDAE: This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

  • 11. Forsell, Mattias N E
    et al.
    Li, Yuxing
    Sundbäck, Maria
    Svehla, Krisha
    Liljeström, Peter
    Mascola, John R
    Wyatt, Richard
    Karlsson Hedestam, Gunilla B
    Biochemical and immunogenic characterization of soluble human immunodeficiency virus type 1 envelope glycoprotein trimers expressed by semliki forest virus.2005Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 79, nr 17, s. 10902-14Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The current lack of envelope glycoprotein immunogens that elicit broadly neutralizing antibody responses remains a major challenge for human immunodeficiency virus type 1 (HIV-1) vaccine development. However, the recent design and construction of stable soluble gp140 trimers have shown that some neutralization breadth can be achieved by using immunogens that better mimic the functional viral spike complex. The use of genetic delivery systems to drive the in vivo expression of such immunogens for the stimulation of neutralizing antibodies against HIV-1 may offer advantages by maintaining the quaternary structure of the trimeric envelope glycoproteins. Here, we describe the biochemical and immunogenic properties of soluble HIV-1 envelope glycoprotein trimers expressed by recombinant Semliki Forest virus (rSFV). The results presented here demonstrate that rSFV supports the expression of stable soluble gp140 trimers that retain recognition by conformationally sensitive antibodies. Further, we show that rSFV particle immunizations efficiently primed immune responses as measured after a single boost with purified trimeric gp140 protein, resulting in a Th1-biased antibody response. This differed from the Th2-biased antibody response obtained after repeated immunizations with purified gp140 protein trimers. Despite this difference, both regimens stimulated neutralizing antibody responses of similar potency. This suggests that rSFV may be a useful component of a viral vector prime-protein boost regimen aimed at stimulating both cell-mediated immune responses and neutralizing antibodies against HIV-1.

  • 12.
    Gustafsson, Dan J
    et al.
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Segerman, Anna
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Lindman, Kristina
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Mei, Ya-Fang
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Wadell, Göran
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    The Arg279Gln [corrected] substitution in the adenovirus type 11p (Ad11p) fiber knob abolishes EDTA-resistant binding to A549 and CHO-CD46 cells, converting the phenotype to that of Ad7p.2006Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 80, nr 4, s. 1897-905Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The major determinant of adenovirus (Ad) attachment to host cells is the C-terminal knob domain of the trimeric fiber protein. Ad type 11p (Ad11p; species B2) in contrast to Ad7p (species B1) utilizes at least two different cellular attachment receptors, designated sBAR (species B adenovirus receptor) and sB2AR (species B2 adenovirus receptor). CD46 has recently been identified as one of the Ad11p attachment receptors. However, CD46 did not seem to constitute a functional receptor for Ad7p. Although Ad7p shares high knob amino acid identity with Ad11p, Ad7p is deficient in binding to both sB2AR and CD46. To determine what regions of the Ad11p fiber knob are necessary for sB2AR-CD46 interaction, we constructed recombinant fiber knobs (rFK) with Ad11p/Ad7p chimeras and Ad11p sequences having a single amino acid substitution from Ad7p. Binding of the constructs to A549 and CHO-CD46 BC1 isoform-expressing cells was analyzed by flow cytometry. Our results indicate that an Arg279Gln [corrected] substitution is sufficient to convert the Ad11p receptor-interaction phenotype to that of Ad7p and abolish sB2AR and CD46 interaction. Also a Glu279Arg substitution in Ad7p rFKs increases CD46 binding. Thus, the lateral HI loop of the Ad11p fiber knob seems to be the key determinant for Ad11p sB2AR-CD46 interaction. This result is comparable to another non-coxsackie-adenovirus receptor binding Ad (Ad37p), where substitution of one amino acid abolishes virus-cell interaction. In conjunction with previous results, our findings also strongly suggest that sB2AR is equivalent to CD46.

  • 13. Habjan, Matthias
    et al.
    Pichlmair, Andreas
    Elliott, Richard M
    Överby, Anna K
    Department of Virology, University of Freiburg, Freiburg, Germany.
    Glatter, Timo
    Gstaiger, Matthias
    Superti-Furga, Giulio
    Unger, Hermann
    Weber, Friedemann
    NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase2009Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 83, nr 9, s. 4365-4375Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

  • 14. Huiskonen, Juha T
    et al.
    Överby, Anna K
    Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79008 Freiburg, Germany.
    Weber, Friedemann
    Grünewald, Kay
    Electron cryo-microscopy and single-particle averaging of Rift Valley fever virus: evidence for GN-GC glycoprotein heterodimers2009Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 83, nr 8, s. 3762-3769Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G(N) and G(C), organized on a T=12 icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G(N) and G(C) homodimers or G(N)-G(C) heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five- and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G(N)-G(C) heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.

  • 15.
    Johansson, Cecilia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Jonsson, Mari
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Marttila, Marko
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Persson, David
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Fan, Xiao-Long
    Skog, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Frängsmyr, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB), Anatomi.
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells2007Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 81, nr 2, s. 954-963Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.

  • 16.
    Jonsson, Mari
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Lenman, Annasara E
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Frängsmyr, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Nyberg, Cecilia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Abdullahi, Mohamed
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells2009Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 83, nr 8, s. 3816-3825Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most adenoviruses bind directly to the coxsackie and adenovirus receptor (CAR) on target cells in vitro, but recent research has shown that adenoviruses can also use soluble components in body fluids for indirect binding to target cells. These mechanisms have been identified upon addressing the questions of how to de- and retarget adenovirus-based vectors for human gene and cancer therapy, but the newly identified mechanisms also suggest that the role of body fluids and their components may also be of importance for natural, primary infections. Here we demonstrate that plasma, saliva, and tear fluid promote binding and infection of adenovirus type 5 (Ad5) in respiratory and ocular epithelial cells, which corresponds to the natural tropism of most adenoviruses, and that plasma promotes infection by Ad31. By using a set of binding and infection experiments, we also found that Ad5 and Ad31 require coagulation factors IX (FIX) or X (FX) or just FIX, respectively, for efficient binding and infection. The concentrations of these factors that were required for maximum binding were 1/100th of the physiological concentrations. Preincubation of virions with heparin or pretreatment of cells with heparinase I indicated that the role of cell surface heparan sulfate during FIX- and FX-mediated adenovirus binding and infection is mechanistically serotype specific. We conclude that the use of coagulation factors by adenoviruses may be of importance not only for the liver tropism seen when administering adenovirus vectors to the circulation but also during primary infections by wild-type viruses of their natural target cell types.

  • 17.
    Lenman, Annasara
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Mueller, Steffen
    Nygren, Mari I
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Frängsmyr, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Stehle, Thilo
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Coagulation factor IX mediates serotype-specific binding of species A adenoviruses to host cells2011Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, nr 24, s. 13420-13431Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Human species A adenoviruses (HAdVs) comprise three serotypes: HAdV-12, -18, and -31. These viruses are common pathogens and cause systemic infections that usually involve the airways and/or intestine. In immunocompromised individuals, species A adenoviruses in general, and HAdV-31 in particular, cause life-threatening infections. By combining binding and infection experiments, we demonstrate that coagulation factor IX (FIX) efficiently enhances binding and infection by HAdV-18 and HAdV-31, but not by HAdV-12, in epithelial cells originating from the airways or intestine. This is markedly different from the mechanism for HAdV-5 and other human adenoviruses, which utilize coagulation factor X (FX) for infection of host cells. Surface plasmon resonance experiments revealed that the affinity of the HAdV-31 hexon-FIX interaction is higher than that of the HAdV-5 hexon-FX interaction and that the half-lives of these interactions are profoundly different. Moreover, both HAdV-31-FIX and HAdV-5-FX complexes bind to heparan sulfate-containing glycosaminoglycans (GAGs) on target cells, but binding studies utilizing cells expressing specific GAGs and GAG-cleaving enzymes revealed differences in GAG dependence and specificity between these two complexes. These findings add to our understanding of the intricate infection pathways used by human adenoviruses, and they may contribute to better design of HAdV-based vectors for gene and cancer therapy. Furthermore, the interaction between the HAdV-31 hexon and FIX may also serve as a target for antiviral treatment.

  • 18. Lindgren, Therese
    et al.
    Ahlm, Clas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Mohamed, Nahla
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Evander, Magnus
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Ljunggren, Hans-Gustaf
    Björkström, Niklas K
    Longitudinal analysis of the human T cell response during acute hantavirus infection2011Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, nr 19, s. 10252-10260Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Longitudinal studies of T cell immune responses during viral infections in humans are essential for our understanding of how effector T cell responses develop, clear infection, and provide long-lasting immunity. Here, following an outbreak of a Puumala hantavirus infection in the human population, we longitudinally analyzed the primary CD8 T cell response in infected individuals from the first onset of clinical symptoms until viral clearance. A vigorous CD8 T cell response was observed early following the onset of clinical symptoms, determined by the presence of high numbers of Ki67(+)CD38(+)HLA-DR(+) effector CD8 T cells. This response encompassed up to 50% of total blood CD8 T cells, and it subsequently contracted in parallel with a decrease in viral load. Expression levels of perforin and granzyme B were high throughout the initial T cell response and likewise normalized following viral clearance. When monitoring regulatory components, no induction of regulatory CD4 or CD8 T cells was observed in the patients during the infection. However, CD8 as well as CD4 T cells exhibited a distinct expression profile of inhibitory PD-1 and CTLA-4 molecules. The present results provide insight into the development of the T cell response in humans, from the very onset of clinical symptoms following a viral infection to resolution of the disease.

  • 19.
    Marttila, Marko
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Persson, David
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Gustafsson, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Liszewski, M Kathryn
    Atkinson, John P
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    CD46 is a cellular receptor for all species B adenoviruses except types 3 and 72005Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 79, nr 22, s. 14429-14436Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.

  • 20.
    Mistry, Nitesh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Inoue, Hirotoshi
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Jamshidi, Fariba
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Storm, Rickard J.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Oberste, M. Steven
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Coxsackievirus A24 variant uses aialic acid-containing O-Linked glycoconjugates as cellular receptors on human ocular cells2011Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, nr 21, s. 11283-11290Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Coxsackievirus A24 variant (CVA24v) is a main causative agent of acute hemorrhagic conjunctivitis (AHC), which is a highly contagious eye infection. Previously it has been suggested that CVA24v uses sialic acid-containing glycoconjugates as attachment receptors on corneal cells, but the nature of these receptors is poorly described. Here, we set out to characterize and identify the cellular components serving as receptors for CVA24v. Binding and infection experiments using corneal cells treated with deglycosylating enzymes or metabolic inhibitors of de novo glycosylation suggested that the receptor(s) used by CVA24v are constituted by sialylated O-linked glycans that are linked to one or more cell surface proteins but not to lipids. CVA24v bound better to mouse L929 cells overexpressing human P-selectin glycoprotein ligand-1 (PSGL-1) than to mock-transfected cells, suggesting that PSGL-1 is a candidate receptor for CVA24v. Finally, binding competition experiments using a library of mono- and oligosaccharides mimicking known PSGL-1 glycans suggested that CVA24v binds to Neu5Ac alpha 2,3Gal disaccharides (Neu5Ac is N-acetylneuraminic acid). These results provide further insights into the early steps of the CVA24v life cycle.

  • 21.
    Nilsson, Emma C
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Jamshidi, Fariba
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Johansson, Susanne M. C.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Oberste, M Steven
    Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential2008Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 82, nr 6, s. 3061-3068Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Binding to target cell receptors is a critical step in the virus life cycle. Coxsackievirus A24 variant (CVA24v) has pandemic potential and is a major cause of acute hemorrhagic conjunctivitis, but its cellular receptor has hitherto been unknown. Here we show that CVA24v fails to bind to and infect CHO cells defective in sialic acid expression. Binding of CVA24v to and infection of corneal epithelial cells are efficiently inhibited by treating cells with a sialic acid-cleaving enzyme or sialic acid-binding lectins and by treatment of the virus with soluble, multivalent sialic acid. Protease treatment of cells efficiently inhibited virus binding, suggesting that the receptor is a sialylated glycoprotein. Like enterovirus type 70 and influenza A virus, CVA24v can cause pandemics. Remarkably, all three viruses use the same receptor. Since several unrelated viruses with tropism for the eye use this receptor, sialic acid-based antiviral drugs that prevent virus entry may be useful for topical treatment of such infections.

  • 22. Nystrom, Kristina
    et al.
    Wanrooij, Paulina H.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Waldenstrom, Jesper
    Adamek, Ludmila
    Brunet, Sofia
    Said, Joanna
    Nilsson, Staffan
    Wind-Rotolo, Megan
    Hellstrand, Kristoffer
    Norder, Helene
    Tang, Ka-Wei
    Lagging, Martin
    Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus2018Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, nr 19, artikel-id e01087-18Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies. IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.

  • 23.
    Panayiotou, Christakis
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lindqvist, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kurhade, Chaitanya
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Vonderstein, Kirstin
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Pasto, Jenny
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Edlund, Karin
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Upadhyay, Arunkumar S.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Överby, Anna K.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation2018Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, nr 7, artikel-id e02054-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus, affording the possible availability of new drug targets that can be used for therapeutic intervention.

    IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses, but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism employed by viperin to inhibit the replication of two flaviviruses: tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV non-structural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's antiflavivirus activity. Taking the results together, our work shows important similarities and differences among the members of the genus Flavivirus and could lead to the possibility of therapeutic intervention.

  • 24.
    Persson, B David
    et al.
    Interfaculty Institute for Biochemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Müller, Steffen
    Interfaculty Institute for Biochemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Reiter, Dirk M
    Interfaculty Institute for Biochemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Schmitt, Benedikt B T
    Institute for Physical and Theoretical Chemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Marttila, Marko
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Sumowski, Chris Vanessa
    Institute for Physical and Theoretical Chemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Schweizer, Sabine
    Institute for Physical and Theoretical Chemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Scheu, Ulrike
    Interfaculty Institute for Biochemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Ochsenfeld, Christian
    Institute for Physical and Theoretical Chemistry, University of Tübingen, D-72076 Tübingen, Germany.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Stehle, Thilo
    Interfaculty Institute for Biochemistry, University of Tübingen, D-72076 Tübingen, Germany.
    An arginine switch in the species B adenovirus knob determines high-affinity engagement of cellular receptor CD46.2009Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 83, nr 2, s. 673-686Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.

  • 25. Rezelj, Veronica V
    et al.
    Överby, Anna K
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Elliott, Richard M
    Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.2015Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 89, nr 9, s. 4849-4856Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in man. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with GFP, allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells, but it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in man.

    IMPORTANCE: Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for man and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the US, that are highly pathogenic, and UUKV will now serve as a comparison to aid understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses.

  • 26.
    Segerman, A
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Mei, Y F
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Wadell, G
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Adenovirus types 11p and 35p show high binding efficiencies for committed hematopoietic cell lines and are infective to these cell lines.2000Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 74, nr 3, s. 1457-67Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hematopoietic cells are attractive targets for gene therapy. However, no satisfactory vectors are currently available. A major problem with the most commonly used adenovirus vectors, based on adenovirus type 2 (Ad2) or Ad5, is their low binding efficiency for hematopoietic cells. In this study we identify two adenovirus serotypes with high affinity for hematopoietic cells. The binding efficiency of prototype serotypes Ad4p, Ad11p, and Ad35p for different committed hematopoietic cell lines representing T cells (Jurkat), B cells (DG75), monocytes (U937-2), myeloblasts (K562), and granulocytes (HL-60) was evaluated and compared to that of Ad5v, the commonly used adenovirus vector, using flow cytometry. In contrast to Ad5v, which bound to less than 10% of the cells in all experiments, Ad11p and Ad35p showed high binding efficiency for all of the different hematopoietic cell lines. Ad4p bound to the lymphocytic cell lines to some extent but less well to the myelomonocytic cell lines. The abilities of the different serotypes to infect, replicate, and form complete infectious particles in the hematopoietic cell lines were also investigated by immunostaining, (35)S labeling of viral proteins, and titrations of cell lysates. Ad11p and Ad35p infected the highest proportion of cells, and Ad11p infected all of the cell lines investigated. The Ad11p hexon was expressed equally well in K562 and A549 cells. Jurkat cells also showed high levels of expression of Ad11p hexons, but the production of infectious particles was low. The binding properties of virions were correlated to their ability to infect and be expressed.

  • 27.
    Segerman, Anna
    et al.
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Atkinson, John P.
    Marttila, Marko
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Dennerquist, Veronica
    Wadell, Göran
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Arnberg, Niklas
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Virologi.
    Adenovirus type 11 uses CD46 as a cellular receptor2003Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 77, nr 17, s. 9183-9191Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.

  • 28.
    Segerman, Anna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Mei, Ya-Fang
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Adenovirus types 11p and 35p show high binding efficiencies for committed hematopoietic cell lines and are infective to these cell lines: ad serotypes with tropism for hematopoietic cells2000Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 74, nr 3, s. 1457-1467Artikel i tidskrift (Refereegranskat)
  • 29. Shirinian, Margret
    et al.
    Kambris, Zakaria
    Hamadeh, Lama
    Grabbe, Caroline
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Journo, Chloe
    Mahieux, Renaud
    Bazarbachi, Ali
    A Transgenic Drosophila melanogaster Model To Study Human T-Lymphotropic Virus Oncoprotein Tax-1-Driven Transformation In Vivo2015Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 89, nr 15, s. 8092-8095Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Human T-cell lymphotropic virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma is an aggressive malignancy. HTLV-2 is genetically related to HTLV-1 but does not cause any malignant disease. HTLV-1 Tax transactivator (Tax-1) contributes to leukemogenesis via NF-kappa B. We describe transgenic Drosophila models expressing Tax in the compound eye and plasmatocytes. We demonstrate that Tax-1 but not Tax-2 induces ommatidial perturbation and increased plasmatocyte proliferation and that the eye phenotype is dependent on Kenny (IKK gamma/NEMO), thus validating this new in vivo model.

  • 30. Stoltz, Malin
    et al.
    Ahlm, Clas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Lundkvist, Ake
    Smittskyddsinstitutet.
    Klingström, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Lambda interferon (IFN-lambda) in serum is decreased in hantavirus-infected patients, and in vitro-established infection is insensitive to treatment with all IFNs and inhibits IFN-gamma-induced nitric oxide production.2007Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 81, nr 16, s. 8685-91Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-alpha]/IFN-beta and IFN-gamma, respectively). Elevated serum levels of NO and IFN-gamma have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-alpha and IFN-beta are similar, whereas the level of IFN-lambda (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-lambda on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-lambda alone inhibited HTNV replication, and IFN-lambda combined with IFN-gamma induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-alpha, -beta, -gamma, and -lambda stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-gamma and IL-1beta. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis.

  • 31.
    Storm, Rickard J
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Persson, David B
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Skalman, Lars Nygård
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Frängsmyr, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Lindström, Mona
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Rankin, Greg
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Domellöf, Fatima Pedrosa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Oftalmiatrik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells2017Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, nr 5, artikel-id e02019-16Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVβ1 and α3β1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.

  • 32.
    Vonderstein, Kirstin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nilsson, Emma
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hubel, Philipp
    Nygård Skalman, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Upadhyay, Arunkumar
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Pasto, Jenny
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Pichlmair, Andreas
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Överby, Anna K.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles2018Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, nr 1, artikel-id e01751-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention.Importance The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.

  • 33.
    Weber, Elvira
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Finsterbusch, Katja
    Innate Immunity and Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Lindquist, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Nair, Sharmila
    Innate Immunity and Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Lienenklaus, Stefan
    Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Gekara, Nelson O
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Janik, Dirk
    Institute of Pathology, Helmholtz Center Munich, Neuherberg, Germany.
    Weiss, Siegfried
    Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Kalinke, Ulrich
    Institute for Experimental Infection Research, TWINCORE, Hannover, Germany.
    Överby, Anna K
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Kröger, Andrea
    Innate Immunity and Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses2014Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 89, nr 21, s. 12202-12212Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

  • 34.
    Westerberg, Sonja
    et al.
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Hagbom, Marie
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Rajan, Anandi
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Loitto, Vesa
    Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Persson, David
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Allard, Annika
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nordgren, Johan
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Sharma, Sumit
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Magnusson, Karl-Eric
    Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Svensson, Lennart
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Medicine, Karolinska Institute, Stockholm, Sweden.
    Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells2018Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, nr 7, artikel-id e00026-18Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells.

  • 35.
    Överby, Anna K
    et al.
    Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Box 240, SE-17177 Stockholm, Sweden.
    Pettersson, Ralf F
    Neve, Etienne P A
    The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging2007Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 81, nr 7, s. 3198-3205Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G(N) for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G(N)/G(C) glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein G(N) cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

  • 36.
    Överby, Anna K
    et al.
    Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, D-79008 Freiburg, Germany.
    Popov, Vsevolod L
    Niedrig, Matthias
    Weber, Friedemann
    Tick-borne encephalitis virus delays interferon induction and hides its double-stranded RNA in intracellular membrane vesicles2010Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 84, nr 17, s. 8470-8483Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tick-borne encephalitis virus (TBEV) (family Flaviviridae, genus Flavivirus) accounts for approximately 10,000 annual cases of severe encephalitis in Europe and Asia. Here, we investigated the induction of the antiviral type I interferons (IFNs) (alpha/beta IFN [IFN-alpha/beta]) by TBEV. Using strains Neudörfl, Hypr, and Absettarov, we demonstrate that levels of IFN-beta transcripts and viral RNA are strictly correlated. Moreover, IFN induction by TBEV was dependent on the transcription factor IFN regulatory factor 3 (IRF-3). However, even strain Hypr, which displayed the strongest IFN-inducing activity and the highest RNA levels, substantially delayed the activation of IRF-3. As a consequence, TBEV can keep the level of IFN transcripts below the threshold value that would permit the release of IFN by the cell. Only after 24 h of infection have cells accumulated sufficient IFN transcripts to produce detectable amounts of secreted IFNs. The delay in IFN induction appears not to be caused by a specific viral protein, since the individual expressions of TBEV C, E, NS2A, NS2B, NS3, NS4A, NS4B, NS5, and NS2B-NS3, as well as TBEV infection itself, had no apparent influence on specific IFN-beta induction. We noted, however, that viral double-stranded RNA (dsRNA), an important trigger of the IFN response, is immunodetectable only inside intracellular membrane compartments. Nonetheless, the dependency of IFN induction on IFN promoter stimulator 1 (IPS-1) as well as the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) suggest the cytoplasmic exposure of some viral dsRNA late in infection. Using ultrathin-section electron microscopy, we demonstrate that, similar to other flaviviruses, TBEV rearranges intracellular membranes. Virus particles and membrane-connected vesicles (which most likely represent sites of virus RNA synthesis) were observed inside the endoplasmic reticulum. Thus, apparently, TBEV rearranges internal cell membranes to provide a compartment for its dsRNA, which is largely inaccessible for detection by cytoplasmic pathogen receptors. This delays the onset of IFN induction sufficiently to give progeny particle production a head start of approximately 24 h.

  • 37.
    Överby, Anna K
    et al.
    Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Box 240, SE-17177 Stockholm, Sweden.
    Popov, Vsevolod L
    Pettersson, Ralf F
    Neve, Etienne P A
    The cytoplasmic tails of Uukuniemi Virus (Bunyaviridae) G(N) and G(C) glycoproteins are important for intracellular targeting and the budding of virus-like particles2007Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 81, nr 20, s. 11381-11391Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Functional motifs within the cytoplasmic tails of the two glycoproteins G(N) and G(C) of Uukuniemi virus (UUK) (Bunyaviridae family) were identified with the help of our recently developed virus-like particle (VLP) system for UUK virus (A. K. Overby, V. Popov, E. P. Neve, and R. F. Pettersson, J. Virol. 80:10428-10435, 2006). We previously reported that information necessary for the packaging of ribonucleoproteins into VLPs is located within the G(N) cytoplasmic tail (A. K. Overby, R. F. Pettersson, and E. P. Neve, J. Virol. 81:3198-3205, 2007). The G(N) glycoprotein cytoplasmic tail specifically interacts with the ribonucleoproteins and is critical for genome packaging. In addition, two other regions in the G(N) cytoplasmic tail, encompassing residues 21 to 25 and 46 to 50, were shown to be important for particle generation and release. By the introduction of point mutations within these two regions, we demonstrate that leucines at positions 23 and 24 are crucial for the initiation of VLP budding, while leucine 46, glutamate 47, and leucine 50 are important for efficient exit from the endoplasmic reticulum and subsequent transport to the Golgi complex. We found that budding and particle generation are highly dependent on the intracellular localization of both glycoproteins. The short cytoplasmic tail of UUK G(C) contains a lysine at position -3 from the C terminus that is highly conserved among members of the Phlebovirus, Hantavirus, and Orthobunyavirus genera. Mutating this single amino acid residue in G(C) resulted in the mislocalization of not only G(C) but also G(N) to the plasma membrane, and VLP generation was compromised in cells expressing this mutant. Together, these results demonstrate that the cytoplasmic tails of both G(N) and G(C) contain specific information necessary for efficient virus particle generation.

  • 38.
    Överby, Anna K
    et al.
    Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Stockholm, Sweden.
    Popov, Vsevolod
    Neve, Etienne P A
    Pettersson, Ralf F
    Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding2006Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 80, nr 21, s. 10428-10435Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant. The particles are able to infect new cells, and reporter gene expression can be monitored if the trans-acting viral proteins (RNA polymerase and nucleoprotein) are also expressed in these cells. No minigenome transfer occurred in the absence of glycoproteins, demonstrating that the glycoproteins are absolutely required for the generation of infectious particles. Moreover, expression of glycoproteins alone was sufficient to produce and release VLPs. We show that the ribonucleoproteins (RNPs) are incorporated into VLPs but are not required for the generation of particles. Morphological analysis of the particles by electron microscopy revealed that VLPs, either with or without minigenomes, display a surface morphology indistinguishable from that of the authentic UUK virus and that they bud into Golgi vesicles in the same way as UUK virus does. This infectious VLP system will be very useful for studying the bunyaviral structural components required for budding and packaging of RNPs and receptor binding and may also be useful for the development of new vaccines for the human pathogens from this family.

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