Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize alphaV
integrins, particularly alphaVbeta6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung
carcinoma cells. The
small interfering RNA (
siRNA) silencing of
integrin beta6 subunit inhibited virus proliferation, confirming that alphaVbeta6 mediates the CAV9
infection. However, siRNAs against
integrin-linked signaling molecules, such as Src, Fyn, RhoA,
phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve
integrin-linked signaling events. CAV9 endocytosis was independent of
clathrin or
caveolin-1 but was restrained by
dynasore, an inhibitor of
dynamin. The RNA interference silencing of beta2-microglobulin efficiently inhibited
virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9
infection was found to depend on Arf6 as both silencing of this molecule by
siRNA and the expression of a dominant negative construct resulted in decreased
virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on
integrin alphaVbeta6, beta2-microglobulin,
dynamin, and Arf6 but independent of
clathrin and
caveolin-1.