Research Group Keppler

From understanding fundamental principles of virus-host interaction to the development of an HIV-susceptible small animal model

Projects

Publications

Group members

Projects

Intrinsic immunity and control of retroviral infection
Besides classical innate and acquired immune responses, mammals have evolved a set of dominant genes that are capable of suppressing or preventing virus replication at the host cell level. Among these major cellular defences against retroviral infection are the Friend-virus-susceptibility-1 (Fv1) gene product and multiple members of the tripartite interaction motif (TRIM) family that both target incoming retroviral particles, with some TRIM proteins also interfering with transcription or late steps in the retroviral replication cycle. A second broadly-active cellular antiviral activity has been identified for members of the apolipoprotein editing complex (APOBEC) class of cytidine deaminases that hypermutate and/or destabilize the genomes of diverse viruses and retroelements. These restriction factors, which are frequently up-regulated by host cells in response to virus infection, appear to impose particularly effective barriers in the context of cross-species transmission of viruses. Accordingly, they have been classified as belonging to the “intrinsic” immunity, representing an innate cellular network for the front-line defence in an immunologically naïve host.

In 2008, human CD317 (BST-2, HM1.24) was identified as an additional, interferon-α-inducible restriction factor, the expression of which prevents the final step in the release of fully matured HIV particles from infected cells through surface-protein-based tethers. Accordingly, the molecule has also been referred to as “tetherin” (Neil et al., Nature 2008). The antiviral activity of human CD317 appears to be very broadly acting since all retroviruses and filoviruses tested thus far are restricted. However, the mode of action CD317 employs to induce virion tethering is not understood.

Electronmicroscopic images of cells producing HIV-1 in the absence (left panel) or presence of human CD317 (BST-2/tetherin) (right panel). (Goffinet et al., Cell Host Microbe 2009).

HIV-1 encodes a unique set of accessory gene products (Vif, Nef, Vpr, Vpu) to optimize its replication in the human host, and this is achieved, in part, by counteracting host restriction factors. As an important example, the HIV-1 Vpu protein, besides downregulating the surface receptor CD4 in the late phase of the replication cycle, couteracts the restriction imposed by human CD317 to ensure the release of infectious progeny from the plasma membrane (Neil et al., Nature 2008; van Damme et al., Cell Host Microbe 2008; Goffinet et al., Cell Host Microbe 2009).

Using techniques from molecular virology, cell biology, immunology and biochemistry, in this project our laboratory pursues the following goals:

1. Elucidate the mechanisms by which CD317 induces this potent restriction of virus replication for HIV-1 and other pathogenic viruses.
2. Decipher how the HIV-1 Vpu protein antagonizes the action of human CD317.
3. Define species-specific characteristics of CD317 and TRIM proteins to shed light on virus-host coevolution and the regulation of cross-species transmission.

Studies on HIV-1 pathogenesis in the human lymphoid ex vivo tissue histoculture

The human lymphoid ex vivo tissue histoculture is a biologically relevant experimental platform permissive for HIV-1 infection independent of exogenous stimulation by mitogens or interleukin-2. This histoculture maintains the tonsil’s natural cytokine milieu, cellular activation status, and cell-to-cell interactions. In collaboration with our colleagues from the Department of Otolaryngology, Head and Neck Surgery at the University Clinic Heidelberg, and the group of Oliver Fackler, we have established this valuable ex vivo model system in the Department of Virology.

Here, we are interested in the following questions:

1. Define differences in replication potential and pathogenesis of primary, patient-derived HIV-1 isolates in this ex vivo-model in light of their variable coreceptor usage, clinical behavior, and contribution to the HIV pandemic.
2. Exploit this ex vivo-model to probe the relevance of findings relating to viral pathogenesis, cellular cofactors and antiviral strategies made in HIV-infected cell lines.

Development of a multi-transgenic rat model of HIV-1 infection

HIV/AIDS has become the most devastating pandemic in recorded history. AIDS is the fourth-biggest global killer and the leading cause of death in Africa. HIV/AIDS persists as a major cause of morbidity in Western societies, since currently available pharmacotherapies can only partly control, but not cure this immunodestructive viral infection. Furthermore, these drugs frequently cause severe side effects and HIV drug resistance development is rapidly emerging. Globally, the lack of effective treatment regimens causes immense human suffering and high cost for society. It is therefore of utmost importance to develop more potent and conceptually novel drugs and therapies for the treatment of HIV/AIDS.However, a major bottleneck for development of novel therapeutic strategies against HIV is the lack of a suitable and readily available small animal model, since normal rats and mice cannot be infected with HIV since they impose multiple cellular barriers to HIV replication. Recent scientific advances have demonstrated that specifically designed transgenic rodents can overcome individual species-specific barriers to HIV replication.

 Schematic representation of consecutive steps (1-8) in the HIV replication cycle. Also indicated are barriers to replication in primary rat cells as well as thus far introduced human transgenes (yellow) that successfully overcome the respective barriers (No. 1 and No. 2). Factors X and Y (blue) exemplify yet to be identified human proteins, required to overcome remaining barriers (No. 3 and No. 4, respectively) and achieve high-level replication of HIV.

We propose to develop a multi-transgenic rat model that is highly susceptible to infection by HIV-1 and that recapitulates key aspects of the disease in humans, thus enabling the development of improved pharmacotherapy and possibly vaccines to fight HIV.

Towards this goal, we have pioneered the generation of rats transgenically expressing human CD4, human CCR5 (together referred to as the HIV receptor complex), and human Cyclin T1 in relevant target cells (Keppler et al., J. Exp. Med. 2002; Michel et al., Retrovirology 2009). These transgenic Sprague-Dawley rats display a high proviral load in lymphatic organs early after intravenous HIV-1 challenge, while viremia in plasma is low and transient.  These animals allow a rapid and predictive preclinical testing of antiviral compounds targeting virus entry or reverse transcription (Goffinet et al., PNAS 2007) and, in collaboration with Jan Münch, Frank Kirchhoff and colleagues, have contributed to the in vivo-evaluation of a novel, semen-derived pathogenicity factor (Münch et al., Cell 2007). A recent in vivo-study with an HIV-1 integrase inhibitor, belonging to the highly potent class of new antiretrovirals, unmasked a drug-induced disturbance of the balance of major HIV-1 cDNA species that may help to explain the unexpectedly rapid decay of HIV RNA plasma loads in HIV/AIDS patients.

Our goals in this project are:

1. Exploit the multi-transgenic rat model for studies on viral transmission and pathogenesis.
2. Validate and mechanistically dissect novel antiviral strategies in vivo prior to advancement to phase I clinical trials.
3. Characterize and overcome remaining late-phase barriers to efficient HIV-1 replication in rats and other rodents.

 The reverse transcriptase inhibitor efavirenz blocks HIV-1 infection in hCD4/hCCR5-transgenic rats (for details see Goffinet et al., PNAS 2007).

Group members

Oliver T. Keppler, Prof. Dr. med.

Email: oliver.keppler@med.uni-heidelberg.de

Phone: +49 6221 56-5007, -1326

Fax: +49 6221 56-5003

 +49 6221 56-5003

 .

Ina Ambiel, technician

Email: ina.ambiel@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003

.

Hanna-Mari Baldauf, Dr. postdoctoral fellow

Email: hanna-mari.baldauf@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003

.

Malin Bigos, MD student

malin.bigos@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003

.

Elina Erikson, PhD student

elina.erikson@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003

.

Klara Kaaden, masters student

klara.kaaden@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003

.

Kristina Schenkova, Dr. postdoctoral fellow

Email: kristina.schenkova@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003

  .

Sarah Schmidt, PhD student 

Email: sarah.schmidt@med.uni-heidelberg.de

Phone: +49 6221 56-1326

Fax: +49 6221 56-5003 

Former lab members

Tarek Adam (visiting medical student)

Ina Ambiel (technician)

Julia Bitzegeio (Diploma student)

Paul Burda (Masters student)

Michael Färber (PhD student)

Kerstin Ganter (technician)

Silvia Geuenich (PhD student)

Christine Goffinet (PhD student, postdoc)

Ebrahim Hassan, Masters student

Dr. Nico Michel (postdoctoral fellow)

Lena Oberbremer (technician)

Claudia Rückert (master student)

Daniel Rupp, MD student

Lismarie Schüller (technician)

Stephanie Venzke (PhD student)

Awards

AIDS Forschungspreis 2007 (Deutsche Gesellschaft für Infektiologie): Christine Goffinet und Oliver Keppler

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Innovationspreis 2007 der BioRegionen Deutschlands: Oliver Keppler

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Hygiene-Preis 2007 der Rudolf Schülke Stiftung: Christine Goffinet, Ina

.Allespach, Oliver Keppler

Loeffler-Frosch-Preis 2008 der Gesellschaft für Virologie: Oliver Keppler

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Nachwuchsforscherpreis 2009 der Deutschen AIDS Gesellschaft: Hanna-Mari Tervo

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Wolfgang-Stille Preis 2010 der Paul-Ehrlich-Gesellschaft f. Chemotherapie: Oliver Keppler und Christine Goffinet

Selected publications

Complete Publication List (PubMed)

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Erikson E, Adam T, Lehmann-Koch J, Schmidt S, Over B, Goffinet C, Bekeredjian-Ding I, Sertel S, Lasitschka F, Keppler OT  (2011)  In vivo-expression profile of the antiviral restriction factor and tumor-targeting antigen CD317/BST-2/HM1.24/Tetherin in humans.   Proceedings of the National Academy of Sciences USA, in press

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Welsch S, Groot H, Kräusslich HG, Keppler OT, Sattentau Q (2011) Architecture and regulation of the HIV-1 assembly and holding compartment in macrophages. Journal of Virology, in press.

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Schmidt S, Fritz J, Bitzegeio J, Fackler OT, Keppler OT (2011) HIV-1 Vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor CD317/Tetherin to overcome the virion release restriction. mBio, in press.

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Tervo HM, Homann S, Ambiel I, Fritz JV, Fackler OT, Keppler OT (2011) Beta-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release. Retrovirology 8:9.

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Tervo HM, Keppler OT (2010) High Natural Permissivity of Primary Rabbit Cells for HIV-1 with a Virion Infectivity Defect in Macrophages as the Final Replication Barrier. Journal of Virology 84:12300

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Goffinet C, Schmidt S, Kern C, Oberbremer L, Keppler OT (2010) Endogenous CD317/Tetherin limits replication of HIV-1 and MLV in rodent cells and is resistant to antagonists from primate viruses. Journal of Virology Aug 11. [Epub ahead of print]

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Rupp D, Geuenich S, Keppler OT (2010) Poor performance of bioinformatics programs for genotypic prediction of coreceptor usage of HIV-1 group O isolates. Journal of Acquired Immune Deficiency Syndrome 53:412-413.

Goffinet C, Schmidt S, Kern C, Oberbremer L, Keppler OT (2010) Endogenous CD317/Tetherin limits replication of HIV-1 and MLV in rodent cells and is resistant to antagonists from primate viruses. Journal of Virology 84, 4089-94

Goffinet C, Homann S, Tibroni N, Rupp D, Keppler OT, Oliver T. Fackler (2010) Antagonism of CD317 restriction of HIV-1 particle release and depletion of CD317 are separable activities of HIV-1 Vpu. Journal of Virology 84, 4089-94.

Müller B, Anders M, Akiyama H, Welsch S, Glass B, Nikovics K, Clavel F, Tervo H-M, Keppler OT, Kräusslich H-G (2009) HIV-1 Gag processing intermediates trans-dominantly interfere with HIV-1 infectivity. Journal of Biological Chemistry 284:29692-29703.

Geuenich S, Kaderali L, Allespach I, Sertel S, Keppler OT (2009) Biological signature characteristics of primary isolates from HIV‑1 group O in the ex vivo human tonsil histoculture. Journal of Virology 83:10494-10503.

Goffinet C, Allespach I, Homann S, Tervo H-M, Habermann A, Rupp D, Oberbremer L, Kern C, Tibroni N, Welsch S, Krijnse-Locker J, Banting G, Kräusslich H-G, Fackler OT, Keppler OT (2009) HIV-1 antagonism of CD317 is species-specific and involves Vpu-mediated proteasomal degradation of the restriction factor. Cell Host Microbe 5:285-97.

Bosch V, Pfeiffer T, Devitt G, Allespach I, Ebensen T, Emerson V, Guzman C.A. und  Keppler OT. (2009). HIV pseudovirion vaccine exposing Env “fusion intermediates” – Response to immunisation in human CD4/CCR5-transgenic rats. Vaccine 27:2202-12.

Goffinet C, Allespach I, Oberbremer L, Golden PL, Foster SA, Johns BA, Weatherhead JG, Novick SJ, Chiswell KE, Garvey EP, Keppler OT. Pharmacovirological Impact of an Integrase Inhibitor on HIV-1 cDNA Species in vivo.Journal of Virology 2009 May 20. [Epub ahead of print]

Michel N, Goffinet C, Ganter K, Allespach I, Kewalramani VN, Saifuddin M, Littman DR, Greene WC, Goldsmith MA, Keppler OT (2009) Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo. Retrovirology. 2009 Jan 13;6(1):2

Homann S, Tibroni N, Baumann I, Sertel S, Keppler OT, Fackler OT (2009) Determinants in HIV-1 Nef for enhancement of virus replication and depletion of CD4+ T lymphocytes in human lymphoid tissue ex vivo. Retrovirology. 2009 Jan 15;6(1):6

Tervo HM, Goffinet C, Keppler OT (2008) Mouse T-cells restrict replication of human immunodeficiency virus at the level of integration. Retrovirology. 2008 Jul 8;5:58

Tervo HM, Allespach I, Keppler OT (2008) High-level transfection of primary rabbit T lymphocytes.J Immunol Methods. 2008 Jul 20;336(1):85-9

Geuenich S, Goffinet C, Venzke S, Nolkemper S, Baumann I, Plinkert P, Reichling J, Keppler OT (2008) Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density. Retrovirology. 2008 Mar 20;5:27

Münch J, Rücker E, Ständker L, Adermann K, Goffinet C, Schindler M, Wildum S, Chinnadurai R, Rajan D, Specht A, Giménez-Gallego G, Sánchez PC, Fowler DM, Koulov A, Kelly JW, Mothes W, Grivel JC, Margolis L, Keppler OT, Forssmann WG, Kirchhoff F (2007) Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131(6):1059-71

Goffinet C, Allespach I, Tervo H-M, Hermann V, Kräusslich H-G, Greene WC, Keppler OT (2007) Primary T-cells from human CD4/CCR5-transgenic rats efficiently support all early steps of HIV-1 replication including integration, but display impaired viral gene expression. Retrovirology, 2007 Jul 26;4(1):53.

Welsch S, Keppler OT, Habermann A, Allespach I, Krijnse-Locker J, Kräusslich H-G (2007) The primary site of HIV-1 budding in infected macrophages is the plasma membrane, PLoS Pathogens 3:e36.

Goffinet C, Allespach I, Keppler OT (2007) HIV-susceptible transgenic rats allow rapid preclinical evaluation of antiviral compounds targeting virus entry or reverse transcription, Proc Natl Acad Sci USA 104:1015-1020
 .Venzke S, Keppler OT (2006) The role of macrophages in HIV infection and persistence, Exp Review Clin Immunol 2:613-626.

Michel N, Ganter K, Venzke S, Bitzegeio J, Fackler OT, Keppler OT (2006) Chemokine receptor cell surface levels are efficiently reduced by lentiviral Nef proteins independent of agonist-engagement and classical motifs for receptor endocytosis, Mol Biol Cell 17: 3578-3590

Haller CS, Rauch S, Michel N, Hannemann S, Lehmann MJ, Keppler OT, Fackler OT (2006) The HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-lymphocyte contacts by modulation of N-WASP activity, J Biol Chem 281: 19618-19630.

Keppler OT, Welte FJ, Ngo TA, Chin PS, Patton KS, Tsou CL, Abbey NW, Sharkey ME, Grant RM, You Y, Scarborough JD, Ellmeier W, Littman DR, Stevenson M, Charo IF, Herndier BG, Speck RF, Goldsmith MA (2002) Progress toward a human CD4/CCR5 transgenic rat model for de novo infection by human immunodeficiency virus type 1. J Exp Med 195:719-736.

Keppler OT, Yonemoto W, Welte FJ, Patton KS, Iacovides D, Atchison RE, Ngo T, Hirschberg DL, Speck RF, Goldsmith MA (2001) Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1. J Virol 75:8063-8073.

Keppler OT, Hinderlich S, Langner J, Schwartz-Albiez R, Reutter W, Pawlita M (1999) UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation. Science 284:1372-1376