Institute of Molecular Virology

Neu-Ulm, Germany

Institute of Molecular Virology

Neu-Ulm, Germany
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Blazejewska P.,Helmholtz Center for Infection Research | Koscinski L.,Adam Mickiewicz University | Viegas N.,Helmholtz Center for Infection Research | Anhlan D.,Institute of Molecular Virology | And 2 more authors.
Virology | Year: 2011

Experimental mouse models were used to compare virulence and reproduction rate of three mouse-adapted variants of the PR8 influenza A virus strain. We observed large differences in pathogenicity in two mouse strains. The PR8M variant was lethal in DBA/2J mice but not in C57BL/6J mice, whereas PR8F and hvPR8 variants were lethal in both mouse strains. High lethality of PR8M in DBA/2J correlated with high viral load at early time points after infection and spread of the virus into alveolar regions. Also, higher viral loads and mortality in mice infected with PR8F resulted in a higher number of infiltrating leukocytes. 3D-protein structure predictions of the HA indicated amino acid sequence alterations which may render the HA cleavage site in PR8F more accessible to host proteases. Infection of C57BL/6J mice with a re-assorted PR8 virus revealed that the HA gene is the main determinant of virulence of the PR8F variant. © 2010 Elsevier Inc.


Sonntag F.,German Cancer Research Center | Kother K.,German Cancer Research Center | Kother K.,Institute of Molecular Virology | Schmidt K.,German Cancer Research Center | And 11 more authors.
Journal of Virology | Year: 2011

Adeno-associated virus type 2 (AAV2) capsid assembly requires the expression of a virally encoded assembly- activating protein (AAP). By providing AAP together with the capsid protein VP3, capsids are formed that are composed of VP3 only. Electron cryomicroscopy analysis of assembled VP3-only capsids revealed all charac- teristics of the wild-type AAV2 capsids. However, in contrast to capsids assembled from VP1, VP2, and VP3, the pores of VP3-only capsids were more restricted at the inside of the 5-fold symmetry axes, and globules could not be detected below the 2-fold symmetry axes. By comparing the capsid assembly of several AAV serotypes with AAP protein from AAV2 (AAP-2), we show that AAP-2 is able to efficiently stimulate capsid formation of VP3 derived from several serotypes, as demonstrated for AAV1, AAV2, AAV8, and AAV9. Capsid formation, by coexpressing AAV1-, AAV2-, or AAV5-VP3 with AAP-1, AAP-2, or AAP-5 revealed the ability of AAP-1 and AAP-2 to complement each other in AAV1 and AAV2 assembly, whereas for AAV5 assembly more specific conditions are required. Sequence alignment of predicted AAP proteins from the known AAV serotypes indicates a high degree of homology of all serotypes to AAP-2 with some divergence for AAP-4, AAP-5, AAP-11, and AAP-12. Immunolocalization of assembled capsids from different serotypes confirmed the preferred nucleolar localization of capsids, as observed for AAV2; however, AAV8 and AAV9 capsids could also be detected throughout the nucleus. Taken together, the data show that AAV capsid assembly of different AAV serotypes also requires the assistance of AAP proteins. © 2011, American Society for Microbiology.


Dudek S.E.,Institute of Molecular Virology | Dudek S.E.,ViroLogik GmbH | Luig C.,Institute of Molecular Virology | Pauli E.-K.,ViroLogik GmbH | And 2 more authors.
Journal of Virology | Year: 2010

Recently it has been shown that the proinflammatory NF-κB pathway promotes efficient influenza virus propagation. Based on these findings, it was suggested that NF-κB blockade may be a promising approach for antiviral intervention. The classical virus-induced activation of the NF-κB pathway requires proteasomal degradation of the inhibitor of NF-κB, IκB. Therefore, we hypothesized that inhibition of proteasomal IκB degradation should impair influenza A virus (IAV) replication. We chose the specific proteasome inhibitor PS-341, which is a clinically approved anticancer drug also known as Bortezomib or Velcade. As expected, PS-341 treatment of infected A549 cells in a concentration range that was not toxic resulted in a significant reduction of progeny virus titers. However, we could not observe the proposed suppression of NF-κB-signaling in vitro. Rather, PS-341 treatment resulted in an induction of IκB degradation and activation of NF-κB as well as the JNK/AP-1 pathway. This coincides with enhanced expression of antiviral genes, such as interleukin-6 and, most importantly, MxA, which is a strong interferon (IFN)-induced suppressor of influenza virus replication. This suggests that PS-341 may act as an antiviral agent via induction of the type I IFN response. Accordingly, PS-341 did not affect virus titers in Vero cells, which lack type I IFN genes, but strongly inhibited replication of vesicular stomatitis virus (VSV), a highly IFN-sensitive pathogen. Thus, we conclude that PS-341 blocks IAV and VSV replication by inducing an antiviral state mediated by the NF-κB-dependent expression of antivirus-acting gene products. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Zirafi O.,Institute of Molecular Virology | Munch J.,Institute of Molecular Virology | Munch J.,University of Ulm
Journal of Leukocyte Biology | Year: 2016

The chemokine receptor CXCR4 is an important G protein-coupled receptor. Signaling via CXCL12 regulates a number of important biologic processes, including immune responses, organogenesis, or hematopoiesis. Dysregulation of CXCR4 signaling is associated with a variety of diseases, such as cancer development and metastasis, immunodeficiencies, or chronic inflammation. Here, we review our findings on endogenous peptide inhibitor of CXCR4 as a novel antagonist of CXCR4. This peptide is a 16-residue fragment of human serum albumin and was isolated as an inhibitor of CXCR4-tropic human immunodeficiency virus type 1 from a blood-derived peptide library. Endogenous peptide inhibitor of CXCR4 binds the second extracellular loop of CXCR4, thereby preventing engagement of CXCL12 and antagonizing the receptor. Consequently, endogenous peptide inhibitor of CXCR4 inhibits CXCL12-mediated migration of CXCR4-expressing cells in vitro, mobilizes hematopoietic stem cells, and suppresses inflammatory responses in vivo. We discuss the generation of endogenous peptide inhibitor of CXCR4, its relevance as biomarker for disease, and its role in human immunodeficiency virus/acquired immunodeficiency syndrome pathogenesis and cancer. Furthermore, we discuss why optimized endogenous peptide inhibitor of CXCR4 derivatives might have advantages over other CXCR4 antagonists. © Society for Leukocyte Biology.


Lackey L.,Institute of Molecular Virology | Lackey L.,University of Minnesota | Law E.K.,Institute of Molecular Virology | Law E.K.,University of Minnesota | And 4 more authors.
Cell Cycle | Year: 2013

Humans have seven APO BEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APO BEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APO BEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APO BEC3 proteins have access to genomic DNA, we analyzed the localization of the APO BEC3 proteins during mitosis. We show that APO BEC3A, APO BEC3C and APO BEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APO BEC3B, APO BEC3D, APO BEC3F and APO BEC3G are excluded from chromatin throughout mitosis. After mitosis, APO BEC3B becomes nuclear, and APO BEC3D, APO BEC3F and APO BEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APO BEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APO BEC3A and APO BEC3B altered the cell cycle profile, and, unexpectedly, APO BEC3D also caused changes. We conclude that several APO BEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APO BEC3B in breast cancers. Copyright © 2013 Landes Bioscience.


Roan N.R.,Gladstone | Muller J.A.,Institute of Molecular Virology | Liu H.,University of California at San Francisco | Chu S.,Gladstone | And 9 more authors.
Cell Host and Microbe | Year: 2011

Semen serves as a vehicle for HIV and promotes sexual transmission of the virus, which accounts for the majority of new HIV cases. The major component of semen is the coagulum, a viscous structure composed predominantly of spermatozoa and semenogelin proteins. Due to the activity of the semen protease PSA, the coagulum is liquefied and semenogelins are cleaved into smaller fragments. Here, we report that a subset of these semenogelin fragments form amyloid fibrils that greatly enhance HIV infection. Like SEVI, another amyloid fibril previously identified in semen, the semenogelin fibrils exhibit a cationic surface and enhance HIV virion attachment and entry. Whereas semen samples from healthy individuals greatly enhance HIV infection, semenogelin-deficient semen samples from patients with ejaculatory duct obstruction are completely deficient in enhancing activity. Semen thus harbors distinct amyloidogenic peptides derived from different precursor proteins that commonly enhance HIV infection and likely contribute to HIV transmission. © 2011 Elsevier Inc.


PubMed | Institute of Molecular Virology
Type: Journal Article | Journal: The Journal of infectious diseases | Year: 2011

Influenza impressively reflects the paradigm of a viral disease in which continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics in humans. Because of the continuous threat of novel influenza outbreaks, it is essential to gather further knowledge about viral pathogenicity determinants. Here, we explored the adaptive potential of the influenza A virus subtype H1N1 variant isolate A/Hamburg/04/09 (HH/04) by sequential passaging in mice lungs. Three passages in mice lungs were sufficient to dramatically enhance pathogenicity of HH/04. Sequence analysis identified 4 nonsynonymous mutations in the third passage virus. Using reverse genetics, 3 synergistically acting mutations were defined as pathogenicity determinants, comprising 2 mutations in the hemagglutinin (HA[D222G] and HA[K163E]), whereby the HA(D222G) mutation was shown to determine receptor binding specificity and the polymerase acidic (PA) protein F35L mutation increasing polymerase activity. In conclusion, synergistic action of all 3 mutations results in a mice lethal pandemic H1N1 virus.


PubMed | Institute of Molecular Virology
Type: Journal Article | Journal: Cellular microbiology | Year: 2010

The non-structural protein 1 (A/NS1) of influenza A viruses (IAV) harbours several src-homology domain (SH) binding motifs that are required for interaction with cellular proteins. The SH3 binding motif at aa212-217 [PPLPPK] of A/NS1 was shown to be essential for binding to the cellular adaptor proteins CRK and CRKL. Both regulate diverse cellular effector pathways, including activation of the MAP-kinase JNK that in turn mediates antiviral responses to IAV infection. By studying functional consequences of A/NS1-CRK interaction we show here that A/NS1 binding to CRK contributes to suppression of the antiviral-acting JNK-ATF2 pathway. However, only IAV that encode an A/NS1-protein harbouring the CRK/CRKL SH3 binding motif PPLPPK were attenuated upon downregulation of CRKI/II and CRKL, but not of CRKII alone. The PPLPPK site-harbouring candidate strains could be discriminated from other strains by a pronounced viral activation of the JNK-ATF2 signalling module that was even further boosted upon knock-down of CRKI/II. Interestingly, this enhanced JNK activation did not alter type-I IFN-expression, but rather resulted in increased levels of virus-induced cell death. Our results imply that binding capacity of A/NS1 to CRK/CRKL has evolved in virus strains that over-induce the antiviral acting JNK-ATF2 signalling module and helps to suppress the detrimental apoptosis promoting action of this pathway.


PubMed | Institute of Molecular Virology
Type: Journal Article | Journal: Cellular microbiology | Year: 2011

The phosphatidylinositol-3-kinase (PI3K) was identified to be activated upon influenza A virus (IAV) infection. An early and transient induction of PI3K signalling is caused by viral attachment to cells and promotes virus entry. In later phases of infection the kinase is activated by the viral NS1 protein to prevent premature apoptosis. Besides these virus supporting functions, it was suggested that PI3K signalling is involved in dsRNA and IAV induced antiviral responses by enhancing the activity of interferon regulatory factor-3 (IRF-3). However, molecular mechanisms of activation remained obscure. Here we show that accumulation of vRNA in cells infected with influenza A or B viruses results in PI3K activation. Furthermore, expression of the RNA receptors Rig-I and MDA5 was increased upon stimulation with virion extracted vRNA or IAV infection. Using siRNA approaches, Rig-I was identified as pathogen receptor necessary for influenza virus vRNA sensing and subsequent PI3K activation in a TRIM25 and MAVS signalling dependent manner. Rig-I induced PI3K signalling was further shown to be essential for complete IRF-3 activation and consequently induction of the type I interferon response. These data identify PI3K as factor that is activated as part of the Rig-I mediated anti-pathogen response to enhance expression of type I interferons.


News Article | December 19, 2016
Site: www.eurekalert.org

Unprecedented images of cancer genome-mutating enzymes acting on DNA provide vital clues into how the enzymes work to promote tumor evolution and drive poor disease outcomes. These images, revealed by University of Minnesota researchers, provide the first ever high-resolution pictures of molecular complexes formed between DNA and the human APOBEC3A and APOBEC3B enzymes. The research is published today online in Nature Structural and Molecular Biology. The DNA mutating enzymes called APOBECs are a major source of mutation in breast, lung, cervical, head/neck and many other cancer types. "These enzymes normally function to protect us from viral infections," said Reuben Harris, Ph.D., investigator of the Howard Hughes Medical Institute and professor of Biochemistry, Molecular Biology, and Biophysics, member of the Masonic Cancer Center, and associate director of the Institute of Molecular Virology, University of Minnesota. "However, these enzymes can become misregulated in cancer cells and cause mutations in our own genomic DNA. These mutations fuel tumor evolution and promote poor clinical outcomes such as drug resistance and metastasis." With an imaging technique called x-ray crystallography, which uses a high energy x-ray beam to visualize the atomic details of molecules, researchers were able to see an unexpected mode of DNA binding activity. A unique U-shaped DNA conformation and defined pockets for the target cytosine and the adjacent thymine base explains the specific mutation signature left behind by the enzyme interacting with tumor DNA. "Our crystal structures and corroborating biochemical experiments show how APOBEC3A and APOBEC3B engage DNA substrates," said Hideki Aihara, Ph.D., associate professor in the department of Biochemistry, Molecular Biology and Biophysics and member of the Institute of Molecular Virology and the Masonic Cancer Center at the University of Minnesota. "These structures show an unexpected mode of DNA engagement with a sharply bent DNA strand and flipped-out nucleotides. Our findings were surprising, but actually make a lot of sense and explain many previous observations about this family of proteins." The DNA-binding mechanism suggests ways to block enzyme activity in cancer, which could slow the rate at which tumors evolve. Inhibiting APOBEC activity could make current anti-cancer therapies more effective. Work continues to take and analyze additional portraits of these enzymes with different DNA substrates and to devise strategies for enzyme inhibition. Funding for this work is provided by grants from the National Institutes of Health (R01-GM118000, R35-GM118047, R01-GM110129, R21-CA206309, DP2-OD007237, P41-GM103426), the National Science Foundation (CHE060073N), the Prospect Creek Foundation and the Masonic Cancer Center, University of Minnesota. Harris is the Margaret Harvey Schering Land Grant Chair for Cancer Research at the University of Minnesota, and an Investigator of the Howard Hughes Medical Institute. College of Biological Sciences faculty conduct research in all areas of biology from molecules to ecosystems with applications in medicine, environment, and biotechnology. The college offers seven undergraduate majors and five graduate programs. Our highly competitive undergraduate program attracts some of the brightest students at the University of Minnesota. Visit cbs.umn.edub to learn more. Masonic Cancer Center, University of Minnesota is a Comprehensive Cancer Center designated by the National Cancer Institute. For more than 25 years, researchers, educators, and care providers have worked to discover the causes, prevention, detection, and treatment of cancer and cancer-related disease. Learn more about the Masonic Cancer Center at cancer.umn.edu. The University of Minnesota Medical School, with its two campuses in the Twin Cities and Duluth, is a leading educator of the next generation of physicians. Our graduates and the school's 3,370 faculty and affiliate physicians and scientists advance patient care, discover biomedical research breakthroughs with more than $177 million in sponsored research annually, and enhance health through world-class patient care for the state of Minnesota and beyond. Visit http://www. to learn more.

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