Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway.
For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-beta-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake.
Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Andreas
A
Gerondopoulos
Terry
T
Jackson
Paul
P
Monaghan
Nicole
N
Doyle
Lisa O
LO
Roberts
D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006835:Hydrazones / Q000494:pharmacology; D008264:Macrophages / Q000821:virology; D051379:Mice; D029322:Norovirus / Q000502:physiology*; D053586:Virus Internalization; D047392:beta-Cyclodextrins / Q000494:pharmacology
D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D034281:Dynamins