Cell and Receptor Tropism of γ2-Herpesviruses
by Anna Katharina Großkopf
Date of Examination:2020-03-23
Date of issue:2021-02-25
Advisor:Dr. Alexander Hahn
Referee:Dr. Alexander Hahn
Referee:Prof. Dr. Uwe Groß
Referee:Prof. Dr. Lutz Walter
Referee:Prof. Dr. Friedemann Weber
Referee:Prof. Dr. Stefan Pöhlmann
Referee:PD Dr. Christian Roos
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Abstract
English
Kaposi’s sarcoma-associated herpesvirus (KSHV), the only rhadino- or γ2-herpesvirus of humans, is associated with Kaposi’s sarcoma (KS) and two B cell proliferative malignancies, primary effusion lymphoma (PEL) and a variant of multicentric Castleman’s disease (MCD). As routes of primary infection, dissemination through the host, and development of virus-associated pathologies are at least partially shaped by viral cell and tissue tropism, it is crucial to understand the contribution of distinct viral glycoproteins and cellular receptor interactions to cell type-specific infection. In this context, the present thesis focuses on members of the Eph family of receptor tyrosine kinases, which were shown to play a role in KSHV infection of various adherent cell lines. While the KSHV gH/gL glycoprotein complex exhibits the highest affinity for EphA2, additional A-type Ephs have been described as interaction partners of KSHV. Even though the gH/gL-Eph interaction was subject of various studies, key questions regarding the role of Ephs in KSHV tropism and pathology remained unanswered. We therefore aimed to identify amino acid residues on the KSHV gH/gL complex that critically mediate the Eph interaction, create Eph detargeted virus recombinants mutated in the identified amino acid residues, and characterize the Eph usage on BJAB cells, as model for cell-to-cell transmission of KSHV into B cells. Similar to KSHV, the related rhesus monkey rhadinovirus (RRV) interacts with Eph receptors while exhibiting differing affinities for individual Eph family members. Comparison of the two viruses allowed us to identify conserved amino acid residues in the N-terminal domain of gH which are critical for the gH/gL-Eph interaction. Mutation of these amino acids in KSHV and RRV recombinants abrogated the viral interaction with Eph receptors and allowed us to analyze the cell type-specific contribution of the Eph family to KSHV and RRV infection. This system was also employed in our second study which identified two additional A-type Ephs as functional KSHV and RRV receptors on BJAB cells. The role of EphA5 and EphA7 in KSHV cell-to-cell transmission and RRV cell-free infection was demonstrated using CRISPR/Cas9-mediated knockout. We furthermore addressed the question whether additional cellular, Eph-independent interaction partners of the gH/gL complex shape the rhadinoviral infection of different cell types. We identified the Plexin domain containing proteins 1 and 2 (Plxdc1/2) as specific interactors for RRV, but not KSHV, and characterized a crucial Plxdc-interaction motif in close proximity to the identified Eph-interacting residues on RRV gH. Receptor function of Plxdcs was demonstrated by lentiviral overexpression of Plxdc1 and 2 in target cells and a Plxdc-detargeted RRV deletion mutant. Collectively, the present studies identify additional A-type Eph members as functional receptors for KSHV and RRV, characterize the role of a novel family of gH/gL-interacting proteins for RRV infection, and underline the importance of the N-terminal domain of the rhadinoviral gH as conserved receptor-binding domain, which mediates the interaction of KSHV and RRV with Eph receptors and the independent interaction of RRV with Plxdc family members.
Keywords: Virus entry; gamma-herpesvirus; Eph receptors; Plexin domain containing protein; KSHV; RRV; gH/gL complex