Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

Abstract

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.

Multiple Classes of Antiviral Agents Exhibit In Vitro Activity against Human Rhinovirus Type C

“…In addition, HRVc15 grown in the presence of either pleconaril or Win 56291 retained infectivity in subsequent reinfection experiments. Together, these results suggest that HRV-C may be less susceptible to the current class of capsidbinding compounds and are consistent with the hypothesis that HRV-C utilizes a receptor pathway for viral entry that is distinct from that used by other HRV groups (7,39). This observation underscores the challenge of developing broadly active HRV capsid inhibitors, since the P1 capsid region exhibits a much higher degree of sequence diversity among HRV groups than the more conserved viral proteins that support intracellular RNA replication (56).…”

“…While this system supports HRV-C replication, the explant model has significant limitations due to low assay throughput and tissue availability. Recently, human airway epithelial (HAE) cultures were shown to support HRV-C replication in vitro and were used by two independent groups to grow clinical HRV-C isolates and HRV-C produced in vitro from cells transfected with genomic HRV-C RNA (38,39). HAE cultures are composed of polarized, fully differentiated airway epithelial cells with active cilia on the apical surface.…”

“…To this end, we independently developed and characterized a full-length infectious HRV-C assay using air-liquid interface-differentiated bronchialtracheal epithelial (HAE) cultures. Recently, two other groups published reports describing similar assay systems for HRV-C replication (38,39). Viral stocks of HRVc15, HRVc11, HRVc24, and HRV16 generated by the transfection of H1-HeLa cells with in vitro-transcribed RNA were tested in a selected set of primary cells and cell lines to determine their permissivity to HRV-C infection (see Fig.…”

“…In addition, the development of anti-HRV agents has been hampered by the lack of screening assays for group C viruses. Unlike HRV-A and -B serotypes, which can be easily propagated in tissue culture, HRV-C has only been shown to replicate in ex-planted sinus mucosal tissue or air-liquid interface (ALI)-differentiated sinus or bronchial epithelial cells (7,38,39).…”

Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication

“…Subsequently, we and others documented RV-C growth in cultures of epithelial cells isolated from airway tissue specimens that had then become redifferentiated under air-liquid interface (ALI) conditions (12,13). Notably, only fully differentiated ALI cultures supported RV-C replication, whereas undifferentiated airway epithelial cell monolayers did not.…”