Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV)

Abstract

10The rhesus monkey rhadinovirus (RRV), a γ2-herpesvirus of rhesus macaques, shares many biological 11 features with the human pathogenic Kaposi's sarcoma-associated herpesvirus (KSHV). Both viruses, 12 as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family 13 of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry 14 varies between cell types suggesting the existence of Eph-independent entry pathways. We 15 therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass 16 spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 17 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. In vitro, blocking assays with soluble 18Plxdc2 decoy receptor reduced RRV infection by approx. 60%, while overexpression of Plxdc1 and 2 19 dramatically enhanced RRV susceptibility in otherwise marginally permissive Raji cells. While the 20 Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically 21 interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of 22 RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. 23Mutation of this motif abrogated the interaction with Plxdc1/2 in in vitro assays and reduced RRV 24 infection in a cell-type specific manner. Taken together, our findings characterize Plxdc1/2 as novel 25 interaction partners and entry receptors for RRV and support the concept of the N-terminal domain 26 of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. 27 vivo studies. Two major RRV sequence groups have been identified (4), which are represented by two 33 cloned isolates, RRV 26-95 (5) and RRV 17577 (6). Analogous to KSHV infection, primary RRV infection 34 is asymptomatic in healthy hosts and leads to life-long persistence, most likely in the B cell 35 compartment (7). KSHV is associated with a solid tumor of endothelial origin, Kaposi's sarcoma (KS), 36 and two B cell malignancies, primary effusion lymphoma (PEL) and the plasmablastic variant of 37 multicentric Castleman's disease (MCD), most prominently in the context of human 38 immunodeficiency virus (HIV) infection and in immunocompromised individuals. Similarly, simian 39 immunodeficiency virus (SIV)-positive rhesus macaques developed B cell lymphomas upon 40 experimental infection with RRV strain 17577 (8, 9) and several studies correlated RRV infection with 41 lymphomagenesis in SIV/SHIV-infected animals (10, 11). While RRV is not consistently associated 42 with solid malignancies, RRV has been identified in retroperitoneal fibromatosis tissue (9, 12), similar 43 to retroperitoneal fibromatosis herpesvirus (RFHV)(11). Another shared characteristic of KSHV and 44 RRV is the receptor usage on a range of cell types. Both viruses engage members of the Eph family of 45 receptor tyrosine kinases (Ephs) through their glycoprotein (g)H...

SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

“…SARS1-S was only weakly recognized by the COVID-19 convalescent serum. RRV gHΔ21-27-Fc, an Fc fusion protein of RRV gH that lacks any detectable receptor interactions (28), served as control.…”

“…The cells were then incubated in either convalescent serum at 1:1000 dilution or soluble ACE2-Fc fusion protein at 2 ng/μl, both described elsewhere (58), for 1h in 10% FCS in PBS, followed by one wash in a large volume of PBS and then incubation with Alexa647-coupled anti-human secondary antibody (Thermo Fisher Scientific) at 1:200 in 10% FCS in PBS. The RRV gHΔ21-27-Fc fusion protein, which was used as a control protein, was generated from RRV 26-95 gH-Fc (59) by deletion of the codons for amino acid 21-27, which are important for receptor binding (28), and was produced analogous to the gH-Fc protein in Hahn et al 2013 (59). The cells were then washed once in a large volume of PBS and post-fixated in 2% PFA in PBS before analysis on an LSRII flow cytometer (BD Biosciences).…”

Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposi’s Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell Type-Specific Manner

“…Of these gH, gL, and gB are the most extensively studied (reviewed in (34)). KSHV and RRV enter many cell types through the interaction of the gH/gL complex with members of the Ephrin receptor tyrosine kinase family (Ephs) (35)(36)(37) and, in case of RRV also with members of the Plexin domaincontaining protein family (38). KSHV also interacts with heparan sulfate and integrins (39)(40)(41).…”

“…Following internalization, the viral membrane fuses with the host membrane. Several reports implicate the gH/gL complex together with gB as the minimal set of glycoproteins required for membrane fusion (45,46,38).…”

“…One study reported an enhancing role of IFITMs in the infection of the BJAB B cell line and human dermal microvascular endothelial cells (HMVEC-D) cells by KSHV, EBV, and herpes simplex virus 2 (HSV-2) (21). However, given the considerable differences between KSHV and RRV entry into B cells and different adherent cells (33,36,37), in particular, as KSHV infection of B cell lines is, with a few exceptions, only efficient through cell-to-cell transfer (47)(48)(49), we hypothesized that IFITM-mediated restriction may be dependent on the type of target cell.…”