Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol

Although noroviruses cause the vast majority of nonbacterial gastroenteritis in humans, little is known about their life cycle, including viral entry. Murine norovirus (MNV) is the only norovirus to date that efficiently infects cells in culture. To elucidate the productive route of infection for MNV-1 into murine macrophages, we used a neutral red (NR) infectious center assay and pharmacological inhibitors in combination with dominant-negative (DN) and small interfering RNA (siRNA) constructs to show that clathrin- and caveolin-mediated endocytosis did not play a role in entry. In addition, we showed that phagocytosis or macropinocytosis, flotillin-1, and GRAF1 are not required for the major route of MNV-1 uptake. However, MNV-1 genome release occurred within 1 h, and endocytosis was significantly inhibited by the cholesterol-sequestering drugs nystatin and methyl-beta-cyclodextrin, the dynamin-specific inhibitor dynasore, and the dominant-negative dynamin II mutant K44A. Therefore, we conclude that the productive route of MNV-1 entry into murine macrophages is rapid and requires host cholesterol and dynamin II.