Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis

Rhesus rhadinovirus (RRV) is a gammaherpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus linked to the development of Kaposi's sarcoma and several other lymphoproliferative diseases, including primary effusion lymphoma and multicentric Castleman's disease. RRV naturally infects rhesus macaques and induces lymphoproliferative diseases under experimental conditions, making it an excellent model for the study of KSHV. Unlike KSHV, which grows poorly in cell culture, RRV replicates efficiently in rhesus fibroblasts (RFs). In this study, we have characterized the entry pathway of RRV in RFs. Using a luciferase-expressing recombinant RRV (RRV-luciferase), we show that the infectivity of RRV is reduced by inhibitors of endosomal acidification. RRV infectivity is also reduced by inhibitors of clathrin-mediated but not caveola-mediated endocytosis, indicating that RRV enters into RFs via clathrin-mediated endocytosis. Using a red fluorescent protein (RFP)-expressing recombinant RRV (RRV-RFP), we show that RRV particles are colocalized with markers of endocytosis (early endosome antigen 1) and clathrin-mediated endocytosis (clathrin heavy chain) during entry into RFs. RRV particles are also colocalized with transferrin, which enters cells by clathrin-mediated endocytosis, but not with cholera toxin B, which enters cells by caveola-mediated endocytosis. Inhibition of clathrin-mediated endocytosis with a dominant-negative construct of EPS15, an essential component of clathrin-coated pits, blocked the entry of RRV into RFs. Together, these results indicate that RRV entry into RFs is mediated by clathrin-mediated endocytosis.