MNV-1.CW164 was kindly provided by Herbert W. Virgin (Washington University School of Medicine, St. Louis, MO, USA) and propagated in RAW264.7 cells to generate a viral stock. Briefly, RAW264.7 cells were infected with MNV at a multiplicity of infection (MOI) of 0.5 in a serum-free medium and incubated for 1 h. Following washing, infected cells were further cultivated in fresh complete media for 2 days. Viral stock was prepared by centrifuging harvested media at 450 × g for 10 min. Intracellular virus particles recovered by three freeze/thaw cycles were harvested by centrifugation at 3,000 × g for 10 min. Virus samples were then concentrated using an Amicon Ultra centrifugal filter unit with a molecular weight cutoff of 50 kDa (Merck Millipore, Billerica, MA, USA) and stored at −80 °C until further use as crude MNV preparation. Purification of MNV was performed as previously described with modifications65. Briefly, crude MNV was pelleted by ultracentrifugation at 150,000 × g for 1 h at 4 °C. The pellet was gently suspended in a suspension buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) at 4 °C and pelleted again by ultracentrifugation. The resultant pellet was dissolved in suspension buffer and was subjected to sucrose density gradient (10–60%) centrifugation at 100,000 × g for 150 min at 4 °C to collect the MNV-containing fractions that were identified by SDS- polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting for the VP1 capsid protein. The fractions were subjected to ultracentrifugation as described above to obtain purified virus particles. These purified virus particles were suspended in PBS and used for virus entry experiments.