The Coxsackie Virus and Adenovirus Receptor (Car) is a Required Host Factor for Recovirus Infection : a Putative Enteric Calicivirus Receptor: CAR is a putative entry receptor for rhesus enteric calicivirus infection

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCVs strains can be propagated to high titers in standard non-human primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells which identified the coxsackie virus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that siRNA, anti-hCAR Mab RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9 targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays we identified and validated the coxsackie virus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs are both necessary to convert a non-susceptible cell line susceptible to ReCV infection. Follow up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.