TY - JOUR
T1 - A conserved Eph family receptor-binding motif on the gH/gL complex of Kaposi’s sarcoma-associated herpesvirus and rhesus monkey rhadinovirus
AU - Großkopf, Anna K.
AU - Ensser, Armin
AU - Neipel, Frank
AU - Jungnickl, Doris
AU - Schlagowski, Sarah
AU - Desrosiers, Ronald C.
AU - Hahn, Alexander S.
N1 - Funding Information:
This work was supported by the German Research foundation, grants CRC 796, TP B1 (to AE), NE 740/2-1 (to FN), HA 6013/1-1 and HA 6013/2-1 (to ASH), by the Wilhelm Sander foundation 2013.105.1 (to AE), by the Interdisciplinary Center for Clinical Research Erlangen (IZKF), grants J44 (to ASH) and A66 (to AE), by the NIH grant 5R37AI063928 (to RCD) and by intramural funding of the German Primate Center - Leibniz Institute for Primate Research (to ASH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Jan Willem Robering and Dr. Anja Boos for providing Lymphatic Endothelial Cells (LEC), Prof. Rüdiger Beer for providing rhesus monkey fibroblasts (RF), Prof. Scott Wong for providing anti RRV gB antibody 3H8.1 and Dr. Effi Wies, Robert Becker and Candice D’Costa for critical reading of the manuscript.
PY - 2018/2
Y1 - 2018/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85042718500&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85042718500&partnerID=8YFLogxK
U2 - 10.1371/journal.ppat.1006912
DO - 10.1371/journal.ppat.1006912
M3 - Article
C2 - 29432452
AN - SCOPUS:85042718500
VL - 14
JO - PLoS Pathogens
JF - PLoS Pathogens
SN - 1553-7366
IS - 2
M1 - e1006912
ER -