Interspecies Differences in Virus Uptake versus Cardiac Function of the Coxsackievirus and Adenovirus Receptor

Abstract

The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail links CAR to the cytoskeleton and intracellular signaling cascades. In the heart, CAR is crucial for embryonic development, electrophysiology, and coxsackievirus B infection. Noncardiac functions are less well understood, in part due to the lack of suitable animal models. Here, we generated a transgenic mouse that rescued the otherwise embryonic-lethal CAR knockout (KO) phenotype by expressing chicken CAR exclusively in the heart. Using this rescue model, we addressed interspecies differences in coxsackievirus uptake and noncardiac functions of CAR. Survival of the noncardiac CAR KO (ncKO) mouse indicates an essential role for CAR in the developing heart but not in other tissues. In adult animals, cardiac activity was normal, suggesting that chicken CAR can replace the physiological functions of mouse CAR in the cardiomyocyte. However, chicken CAR did not mediate virus entry in vivo, so that hearts expressing chicken instead of mouse CAR were protected from infection and myocarditis. Comparison of sequence homology and modeling of the D1 domain indicate differences between mammalian and chicken CAR that relate to the sites important for virus binding but not those involved in homodimerization. Thus, CAR-directed anticoxsackievirus therapy with only minor adverse effects in noncardiac tissue could be further improved by selectively targeting the virus-host interaction while maintaining cardiac function. IMPORTANCECoxsackievirus B3 (CVB3) is one of the most common human pathogens causing myocarditis. Its receptor, the coxsackievirus and adenovirus receptor (CAR), not only mediates virus uptake but also relates to cytoskeletal organization and intracellular signaling. Animals without CAR die prenatally with major cardiac malformations. In the adult heart, CAR is important for virus entry and electrical conduction, but its nonmuscle functions are largely unknown. Here, we show that chicken CAR expression exclusively in the heart can rescue the otherwise embryonic-lethal CAR knockout phenotype but does not support CVB3 infection of adult cardiomyocytes. Our findings have implications for the evolution of virus-host versus physiological interactions involving CAR and could help to improve future coxsackievirus-directed therapies inhibiting virus replication while maintaining CAR's cellular functions.

The Coxsackievirus and Adenovirus Receptor: Glycosylation and the Extracellular D2 Domain Are Not Required for Coxsackievirus B3 Infection

“…5D and E). This confirms the previously described inability of chCAR to mediate CVB3 infection (36) and is consistent with strong evidence that D1 is directly involved in virus binding (7).…”

“…5A). Among these domains, chCAR-D2 shows the greatest sequence homology to hCAR-D2 (45%), although chCAR does not function as a CVB3 receptor (36). IgSF11-D2 has 31% homology, CD155-D2 has 21% homology, and CD80-D2 has no sequence similarities to hCAR-D2.…”

The PDZ3 domain of the cellular scaffolding protein MAGI-1 interacts with the Coxsackievirus and adenovirus receptor (CAR)

“…CAR is also a critical cell adhesion protein that plays a role in the junctional localization of important scaffolding proteins and cognate signaling proteins, such as PSD-95 and ASIC3 (Excoffon, Kolawole et al 2012). Moreover, the essential nature of CAR may be due to its ability to traffic or modulate the junctional stability of important proteins, such as MAGI-1, connexin 45, β-catenin, and ZO-1, particularly at cell junctions and the cardiac intercalated disc (Lim, Xiong et al 2008, Pazirandeh, Sultana et al 2011, Freiberg, Sauter et al 2014). MAGI-1 is an important scaffolding protein that can create a signalosome with over 20 distinct proteins that are involved in diverse cellular pathways, from adhesion to the regulation of signaling.…”

“…Studies on its endogenous cellular function have shown that CAR is a developmentally essential protein with important cardiac functions (Asher, Cerny et al 2005, Dorner, Wegmann et al 2005, Freiberg, Sauter et al 2014). In polarized epithelia, CAR is primarily a basolateral adhesion protein involved in the maintenance of cell-cell contacts (Walters, Freimuth et al 2002, Excoffon, Hruska-Hageman et al 2004).…”

Cardiac Fibroblasts Aggravate Viral Myocarditis: Cell Specific Coxsackievirus B3 Replication

“…The coxsackievirus and adenovirus receptor (CAR) was identified as the cellular receptor for CVB3 [ 33 – 35 ]. Murine cardiac fibroblasts, cardiomyocytes, and macrophages were tested for the gene expression of CAR.…”