Abstract
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi’s sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi’s Sarcoma [1], is closely associated with two B-cell malignancies, namely the primary effusion lymphoma [2] and the plasmablastic variant of multicentric Castleman’s disease [3](reviewed in [4])
We identified and mutated conserved residues in the N-terminal domain of the gH/gL glycoprotein complex of KSHV and the related monkey virus rhesus monkey rhadinovirus (RRV) that are critical for the interaction with cellular receptors from the Eph family
An evolutionarily conserved motif in domain I of gH is critical for Eph receptor interaction Based on the known structures of the herpes simplex virus type 2 and EBV gH/gL complex [22,30] and the conserved nature of this glycoprotein complex, it can be assumed that gH of KSHV comprises domain I (D I) to domain IV (D IV), a transmembrane domain (TM) and a short C-terminal intravirion domain (IVD)
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi’s Sarcoma [1], is closely associated with two B-cell malignancies, namely the primary effusion lymphoma [2] and the plasmablastic variant of multicentric Castleman’s disease [3](reviewed in [4]). The interaction of the respective gH/gL glycoprotein complex of KSHV and RRV with members of the Ephrin receptor tyrosine kinase (RTK) family of proteins (Ephs) is a conserved feature in the entry process of both rhadinoviruses. Despite very divergent primary sequences of gH and gL of RRV 26–95 and 17577, both isolates were found to interact with a broad spectrum of A- and B-type Eph receptors and to bind EphB3 with the highest avidity [16]. Both KSHV and RRV require the presence of gH as well as gL in the gH/gL complex for Eph-interaction
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