We propose to identify the cellular and molecular mechanisms required for entry of feline calicivirus (FCV) into polarized epithelial cells (PECs), the cell type first encountered during natural infection. FCV is a prevalent pathogen with high morbidity (up to 100% in shelters), and occasionally high mortality (40-60%) associated with spontaneously and unpredictably arising virulent FCV isolates. Despite widespread vaccination, FCV-related diseases continue to afflict cats.

FCV replicates in PECs of the oropharynx and upper respiratory tract. Feline junctional adhesion molecule A (fJAM-A), the cellular receptor for FCV, is concentrated at intercellular tight junctions (TJs) between these cells, and is also present the on basolateral, but not on their apical surfaces. In healthy epithelia, TJs are inaccessible to viruses. Thus, an important question is how does FCV access its receptor in the TJs of epithelial cells. Viral infection of PECs often requires cellular components and activation of specific cellular signaling pathways not required for infection of other cell types. Published studies of FCV entry have not used cells representative of those infected during natural infection. Feline mammary epithelial cells (FMECs) are polarized, form tight junctions, express feline JAM-A (fJAM-A) at junctional complexes, and are susceptible to infection with FCV. Using FMECs, we expect to uncover basic mechanisms of FCV cellular entry pertinent to natural infection.

Hypothesis: FCV infection of FMECs requires activation of cellular signaling and endocytic pathways that differ from those required to infect non-polarized cells.

Aim 1: Determine binding patterns and mechanisms of uptake of FCV in FMECs. We hypothesize that FCV attaches to a binding intermediate on the apical surface of FMECs, which mediates trafficking of FCV to tight junctions, followed by endocytosis and infectious entry. We will use confocal microscopy to follow synchronized FCV infection of polarized FMECs. Virus and a variety of endosomal and tight junction markers will be tracked over time. In addition we will use standard approaches to inhibit or block various endocytic pathways to define the cellular requirements for FCV entry into polarized cells.

Aim 2: Identify cellular signals required for FCV uptake and infection of FMECs. We hypothesize that infectious entry of FCV into PECs requires the activation of specific cellular signaling pathways. We will use a library of inhibitors of kinases and phosphatases to screen for signaling pathways required for FCV entry. In addition, we will use a similar inhibitor approach to test whether actin re-modeling is required for FCV entry into polarized epithelial cells.