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«Die Bedeutung fehlerhafter ribosomaler Produkte für die MHC Klasse I Antigenpräsentation des humanen Immundefizienzvirus-1 Strukturproteins Gag Der ...»

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Targeting the human immunodeficiency virus type-1

Gag protein into the defective ribosomal product

pathway enhances its MHC class I

antigen presentation

Die Bedeutung fehlerhafter ribosomaler Produkte

für die MHC Klasse I Antigenpräsentation

des humanen Immundefizienzvirus-1

Strukturproteins Gag

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität

Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Sabine Hahn

aus Regensburg Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg Tag der mündlichen Prüfung:

Vorsitzender der Promotionskommission:.... Prof. Dr. Rainer Fink…........

Erstberichterstatter:

Zweitberichterstatter:

Table of contents 1

Abstract

2 Zusammenfassung

3 List of abbreviations

4 Introduction

4.1 The human immunodeficiency virus type 1

4.2 Gag proteins and their role in late processes of HIV-1 replication

4.3 The ubiquitin proteasome system

4.4 Role of the UPS in late steps of HIV-1 replication

4.5 Role of the UPS and defective ribosomal products (DRiPs) in MHC-I antigen processing

4.6 Regulation of UPS-mediated proteolysis by degradation signals

4.7 Correlation between metabolic half-life and MHC-I antigen presentation.......24 5 Results

5.1 Targeting HIV-1 Gag into the DRiP-pathway enhances MHC-I antigen presentation and CD8+ T-cell activation

5.1.1 Construction of Gag variants containing degradation signals

5.1.2 Introduction of the OVA-derived SL epitope as indicator for Ag processing of Gag

5.1.3 Generation and characterization of GagSL-expressing EL4 cell lines.........27 5.1.4 Half-life and DRiP-rate of UbRGagSL and UbMGagSL proteins...............28 5.1.5 Correlation of DRiP-rate with the MHC-I presentation of Gag-derived SL.31 In vitro activation of the SL-H2-Kb specific T-cell hybridoma B3Z............33 5.1.6 In vivo activation of SL-H2-Kb-specific OT-1 cells and induction of SLspecific CD8+ T cells in naïve mice

5.1.8 In human cells, Gag is targeted into the MHC-I pathway by the N-end rule, but even more efficiently by stable N-terminal fusion to Ub

5.1.9 N-end rule and UFD degradation signals do not influence the synthesis or metabolic half-life of Gag in HeLa cells

5.1.10 N-end rule and UFD degradation signals interfere with the release of VLPs

5.1.11 N-end rule and UFD degradation signals disturb the membrane localization of Gag

5.2 The PTAP Late domain regulates ubiquitination and MHC-I antigen presentation of HIV-1 Gag

5.2.1 The PTAP L-domain in the p6 region regulates budding of GagSL-derived VLPs.

5.2.2 The PTAP L-domain regulates ubiquitination of GagSL

5.2.3 The PTAP, but not the YP(X)nL L-domain regulates MHC-I antigen presentation of a Gag-derived epitope

5.2.4 Induction of the immunoproteasome enhances presentation of the SL-epitope derived from GagSL-GFP

5.2.5 The PTAP L-domain regulates MHC-I antigen presentation of the SL epitope derived from processed Gag

5.2.6 Enhanced SL-presentation of the PTAP-mutant is not a result of the budding defect and not entirely dependent on membrane association of Gag

5.2.7 The interaction with Tsg101 or ALIX is not essential for the regulation of MHC-I presentation of a Gag-derived epitope by the PTAP L-domain

5.2.8 Lys48-linked polyubiquitination is essential for the preferred entry of the PTAP-mutant into the MHC-I pathway

5.2.9 The PTAP-mutant displays a slightly decreased metabolic half-life and an increased DRiP-rate when compared to wt Gag

6 Discussion

7 Material and methods

8 References

9 Acknowledgements

Abstract 5 1 Abstract The major source for endogenous peptides presented via the major histocompatibility complex class-I (MHC-I) pathway are de novo synthesized, dysfunctional proteins, named defective ribosomal products (DRiPs), which are degraded in concert with or shortly after their synthesis by the ubiquitin proteasome system (UPS).

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, a bona fide substrate of the DRiP-pathway, was chosen as a model antigen to more precisely understand the relevance of erroneous protein synthesis for the generation of MHC-Ipresented peptides. To target Gag into the DRiP-pathway, various degradation signals have been introduced into Gag, and their effects on its protein synthesis, metabolic halflife, DRiP-formation as well as subcellular localization and the release of virus like particles have been investigated. As an indicator for antigen processing, the ovalbuminderived SIINFEKL (SL) epitope was introduced into Gag expressed from a codonoptimized gag gene (syngag). It was demonstrated that exchange of the N-terminal Met residue for Arg (RGag), a destabilizing amino acid according to the N-end rule, directed Gag more efficiently into the DRiP-pathway in murine EL4 cell lines. This correlated with enhanced MHC-I antigen presentation as well as more efficient CD8+ T-cell activation in vitro and in vivo. The enhanced MHC-I presentation of SL derived from RGag in murine cells could be reproduced in a human cell line. Furthermore, stable fusion to ubiquitin (Ub), converting Gag into a substrate for the Ub fusion degradation (UFD) pathway, was even more efficient in targeting Gag into the MHC-I pathway.





The PTAP late (L)-domain motif in the p6 domain of HIV-1 Gag plays an essential role during late stages of budding and has been recently implicated in the control of Gag ubiquitination. Mutations of PTAP in the context of syngag- or HIV-1-encoded Gag increased the ubiquitination as well as the DRiP-rate of Gag and enhanced the MHC-I presentation of the Gag-derived SL epitope. This novel function of the PTAP L-domain as a naturally occurring motif that regulates the DRiP-rate of Gag might be mediated by the sequence-specific recruitment of cellular factors, most likely components of the UPS.

Altogether, the results presented in this study further underline the role of the DRiPpathway in adaptive immunity and provide strategies to enhance the MHC-I antigen presentation of HIV-1 Gag and other antigens. It remains to be elucidated by studies performed in vivo whether such approaches may help to improve vaccination strategies.

Zusammenfassung 6

2 Zusammenfassung

Die Hauptquelle für endogene Peptide, die von MHC Klasse I (MHC-I) Molekülen präsentiert werden sind Fehlprodukte der Proteinbiosynthese, sogenannte defekte ribosomale Produkte (DRiPs), die noch während oder kurz nach ihrer Synthese durch das Ubiquitin-Proteasom-System (UPS) abgebaut werden.

Um die Bedeutung der fehlerhaften Proteinsynthese für die MHC-I Antigenpräsentation weiter zu untersuchen, wurde das Gag Polyprotein des humanen Immundefizienzvirus-1 (HIV-1), ein beschriebenes Substrat des DRiP-Pathways, als Modellantigen gewählt. Um Gag in den DRiP-Pathway zu lenken, wurden verschiedene Abbausignale eingeführt und deren Wirkung auf die Synthese, metabolische Halbwertszeit, DRiP-Rate sowie subzelluläre Lokalisation von Gag und die Freisetzung von Virus-ähnlichen Partikeln untersucht. Als Indikator für die Antigenprozessierung wurde das SIINFEKL (SL) Epitop aus Ovalbumin in Gag eingebracht, welches von einem synthetischen, Kodon-optimierten gag Gen (syngag) exprimiert wurde. Es wurde gezeigt, dass der Austausch des Nterminalen Methionins durch Arginin (RGag), welches entsprechend der „N-end Regel“ ein Abbausignal darstellt, zu verstärkter Bildung von Gag-DRiPs in murinen EL4 Zellen führt. Dies korrelierte mit erhöhter MHC-I Antigenpräsentation sowie einer effizienteren T-Zellaktivierung in vitro und in vivo. Die bessere Antigenprozessierung von RGag in murinen Zellen konnte in einer humanen Zellinie bestätigt werden. Darüber hinaus leitete eine stabile N-terminale Fusion von Ubiquitin das Protein noch weitaus effizienter in den MHC-I Pathway.

Das PTAP late (L)-Domänen Motiv in der p6 Domäne von HIV-1 Gag spielt eine essentielle Rolle in späten Stadien der Virusfreisetzung und reguliert die Ubiquitinylierung von Gag. Mutationen von PTAP im Kontext von syngag- oder HIV-1 kodiertem Gag erhöhen die Ubiquitinylierung und DRiP-Rate und steigern die MHC-I Präsentation des SL-Epitops. Diese neu beschriebene Funktion des PTAP Motivs als ein inhärentes Sequenzmotiv, welches den Eintritt von Gag in den MHC-I Pathway reguliert, könnte durch die sequenzspezifische Interaktion mit zellulären Faktoren, insbesondere Bestandteile des UPS, vermittelt werden. Zusammengefasst unterstreichen diese Befunde die Rolle des DRiP-Pathways in der adaptiven Immunität and zeigen mögliche Strategien auf, mit Hilfe derer die MHC-I Präsentation von HIV-1 Gag oder anderen Antigenen gesteigert werden könnte. Dennoch bedarf es weiterer Untersuchungen in vivo, um zu klären, ob auf diese Weise Vakzinierungsstrategien verbessert werden könnten.

List of abbreviations 7

–  –  –

PLC peptide loading complex PM plasma membrane pMHC peptide-MHC complex Pol Polymerase POSH plenty of SH3 PR Protease PSI protein biosynthesis inhibitor PVDF polyvinylidenefluoride RGS regulator of G-protein signaling RING Really interesting new gene RIPA Radioimmunoprecipitation assay RNA ribonucleic acid RNAi RNA interference RP regulatory particle RSV Rous sarcoma virus RT Reverse transcriptase rVV recombinant vaccinia virus SD standard deviation SDS sodium dodecyl sulfate SEA staphylococcal enterotoxin A siRNA small interfering RNA SIV simian immunodeficiency virus

SL SIINFEKL

SUMO small Ub-related modifier TAP transporter associated with antigen processing TCR T cell receptor TGN trans-Golgi network TH tyrosine hydroxylase TM transmembrane TOP Thimet oligopeptidase TPPII tripeptidyl peptidase II TRiC tailless complex polypeptide-1 (TCP-1) ring complex TRIM Tripartite interaction motif TRP-2 tyrosinase-related protein-2 Tsg101 tumor susceptibility gene 101 Ub ubiquitin UBL ubiquitin-like UCH ubiquitin-C-terminal hydrolase UEV ubiquitin enzyme 2 variant UFD ubiquitin fusion degradation UPS ubiquitin proteasome system USP ubiquitin-specific proteases VLPs virus like particles zLLL carbobenzoxyl-leucine-leucine-leucinal

Introduction 10

4 Introduction

4.1 The human immunodeficiency virus type 1 The human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS), first described in 1981 (1). The number of people living with an HIV-1 infection world-wide is still increasing and has reached 33.3 million in 2009, with 2.6 million people that were newly infected with HIV-1 and 1.8 million people dying from AIDS, as estimated by the World Health Organization of the United Nations. Although the introduction of highly active antiretroviral therapy (HAART) in the mid nineties has significantly reduced morbidity and mortality among AIDS patients, eradication of the virus from infected individuals has not been achieved and the disease remains incurable. With still very limited access to HIV-1 prevention and treatment in developing countries, the HIV-1 pandemic remains one of the most critical of infectious disease challenges to public health.

Fig. 4.1: Replication cycle of HIV-1 (artwork by Nadine Jänisch). Schematic representation of the major steps in HIV-1 replication. The replication cycle of HIV-1 begins with the attachment of the virus particle to CD4 and one of the coreceptors CXCR4 or CCR5, followed by membrane fusion, virus entry and uncoating. Following reverse transcription, the proviral DNA is integrated into the host cell genome. The late steps of replication start with the transcription of viral genes and the de novo synthesis of viral structural proteins which undergo assembly and budding at the plasma membrane.

Following auto-catalytic activation, the viral protease processes the structural proteins resulting in the formation of a conical core that is typical for a mature, infectious virus particle.

Introduction 11

HIV-1 belongs to the lentivirus subfamily of the Retroviridae, a family of enveloped RNA viruses, that, instead of using their RNA genome directly for virus replication, reversely transcribe it into proviral DNA that is integrated into the host cell genome. The retroviral life cycle, depicted in Fig. 4.1. begins with the binding of infectious viral particles to cellular receptors, in case of HIV-1 CD4 and one of the coreceptors CCR5 (CC chemokine receptor 5) or CXCR4 (C-X-C chemokine receptor type 4), followed by membrane fusion and virus entry. After reverse transcription of the RNA genome and integration of the proviral DNA into the host chromosomes, virus proteins are synthesized and assemble at the plasma membrane (PM). During budding, the Gag polyprotein precursor is processed by the viral protease (PR) after its auto-catalytic activation, resulting in the formation of a conical core which is typical for the mature and infectious HIV-1 particle.

Fig. 4.2: Genomic organization of HIV-1. This figure depicts the complex organization of the HIV-1

genome comprising the canonical retroviral ORFs flanked by long terminal repeat (LTR) sequences:

gag (blue) coding for the polyprotein precursor Pr55 that is processed into the individual structural Gag proteins, pol (green) encoding the enzymes Protease, Reverse Transcriptase, RNaseH and Integrase and env (orange), coding for the glycoprotein precursor gp160 that is processed into gp120 and gp41. Six additional genes code for regulatory proteins that are classified as essential (Tat and Rev) or accessory (Vpr, Vif, Vpu, Nef). A schematic view of a mature HIV-1 virus particle with the incorporated proteins and two copies of RNA is shown below.

The HIV-1 genome is approximately 9 kbp in length and contains the three open reading frames (ORFs): gag encoding the structural Gag (group specific antigen) proteins, pol coding for the viral enzymes PR, Reverse transcriptase (RT), RNase H and Integrase (Int) and env, which encodes the surface glycoproteins gp 41 and gp 120. In addition to those canonical retroviral coding regions, HIV-1, as a complex retrovirus, encodes six

Introduction 12



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