Rotaviruses are the major cause of severe acute dehydrating diarrhea in humans and animals worldwide [
2]. In vitro, African green monkey kidney cell (MA104) is the most commonly used cell in rotavirus propagation. Rotavirus entry is an organized multistep process, it has been demonstrated that rotaviruses enter the cell through direct penetration or by receptor-mediated endocytosis [
27,
28]. N-acetylneuraminic (sialic) acid residues, integrins α2β1, α4β1, αvβ3, αxβ2, heat shock cognate protein (hsc) 70 and certain gangliosides were identified as cellular molecules which were responsible for rotavirus entry [
29,
30]. Previous research indicated that cellular cholesterol is required for infection of some rotavirus species [
22‐
25]. Meanwhile, cholesterol depletion impairs the entry of BRV UK strain [
26]. However, BRV strains have various VP4 (P-types) and VP7 (G-types) genotypes and they were clustered into different lineages [
31]. In addition, whether cholesterol affects BRV attachment or after-entry stage have not been reported. In this study, the critical role of cellular cholesterol on specific phase of BRV CH-12 strain was investigated, we found that the cellular cholesterol is sensitive to MβCD treatment. With the treatment of increasing concentrations of MβCD the cholesterol content of plasma membrane was decreased in a dose-dependent manner, and replenishing cellular membrane with exogenous cholesterol partially restored the cholesterol level. In order to determine which stage of BRV life cycle is affected by cholesterol depletion, we first examined the effect of cellular cholesterol depletion on virus binding, the results showed that BRV attachment was independent to cholesterol. However, when cells were treated with MβCD prior to virus entry, BRV infection was decreased with the reduction of cellular cholesterol level in a dose-dependent fashion. The replenishing of exogenous cholesterol enhanced the infectivity of BRV. To investigate whether cholesterol depletion affects virus entry or replication, cells were treated with MβCD after BRV entry, the result revealed that there were no significant differences of viral mRNA and protein levels between treated cells and mock cells. These results indicated that cholesterol depletion from cellular membrane did not affect the replication of BRV but virus entry. As reported, lipid raft is involved in several viruses entry into cells, such as PRRSV [
16], BoHV-1 [
18], human enterovirus [
32], poliovirus [
33] and canine distemper virus [
34], cellular receptors of several viruses are localized in the lipid rafts. Although diverse rotaviruse species interact with different cell-surface molecules, some of these molecules are localized in the lipid rafts [
30]. Hence, cholesterol depletion from cellular membrane influences BRV entry by disturbing lipid-raft-dependent endocytosis. Our results showed that cholesterol depletion had no effect on BRV replication, suggesting that virus replication is independent of lipid rafts, and it is in accordance with the former reports for other viruses [
16,
18].
Previous study demonstrated that Lovastatin-treated cells increased the levels of simian rotavirus proteins [
35]. In the present work, we found that the synthesis of viral mRNA and protein were not affected when cells were treated with MβCD after virus entry, but the virus titers were obviously decreased, it appears that cholesterol-rich microdomains may contribute to assembly or budding of BRV. Lipids membrane microdomains play an important role in numerous cellular functions such as signal transduction, membrane transport and trafficking [
36] and provide platforms for assembly or budding of many enveloped viruses such as influenza virus [
37], HIV [
38] and morbillivirus measles virus [
39]. The mechanism of noneveloped viruses assembly and release is still poorly understood. The release of some noneveloped viruses from cells does not involve cell lysis. Virions transported to specific plasma membrane domains through vesicular transport, inhibition of the accumulation of virus on cell surfaces did not affect viral RNA and protein synthesis but block virus release [
40,
41]. Recently, compounds that could inhibit rotavirus viroplasm formation and decrease the amount of infectious progeny by disrupting lipid droplets (LDs) or fatty acid synthesis were reported [
42,
43]. In Caco-2 Cells, rotavirus VP4 protein has been demonstrated to directly interact with lipid microdomains. At the final stage of assembly, VP4 incorporates into preformed viral particles, lipid rafts provide a platform for the formation of infectious virions [
28]. These data indicated that lipid rafts disrupted by MβCD impaired the mature of BRV particles and decreased the virus titers.
In conclusion, our results suggested that lipid rafts play a vital role in BRV infection and plasma membrane cholesterol was essential to BRV entry and assembly.