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The Epizootic hemorrhagic disease virus (EHDV), an orbivirus, is the etiological factor of a fatal hemorrhagic disease of wild ruminants. A subset of EHDV serotypes, including the Ibaraki strain of EHDV2 (EHDV2-Ibaraki), infect and cause disease in cattle, thus posing a potential threat to livestock. As a member of the Sedoreoviridae family, the EHDV particle is devoid of a membrane envelope and is predicted to employ endocytic pathways for infection. However, the degree of dependence of EHDV2-Ibaraki on specific internalization pathways while infecting bovine cells (its natural host) is unknown. The endosome alkalinizing agent ammonium chloride blocked EHDV2-Ibaraki infection of Madin-Darby Bovine Kidney (MDBK) cells with dependence on its time of addition, suggesting the criticality of endosomal pH for the completion of early stages of infection. Treatment of cells within the alkalinization-sensitive window (i.e., before endosomal processing) with inhibitors of actin polymerization, macropinocytosis (amiloride), or dynamin GTPase activity (dynasore or dynole), or with the cholesterol-depleting agent methyl-β-cyclodextrin, failed to reduce EHDV2-Ibaraki infection. In contrast, in this same treatment time frame, ikarugamycin potently inhibited infection. Moreover, ikarugamycin inhibited interferon induction in infected cells and induced the accumulation of enlarged Rab7- and lamtor4-decorated vacuoles, suggesting its ability to block viral processing and modify late-endosome compartments. Notably, ikarugamycin treatment at initial infection stages, augmented the infection of MDBK cells with the vesicular stomatitis virus while inhibiting infection with bluetongue virus serotype 8. Together, our results point to differential antiviral effects of ikarugamycin on viruses dependent on distinct sets of endosomes for entry/processing.

Charasomes are convoluted plasma membrane domains in characean green algae. They are known to form in response to light via secretion of trans-Golgi network (TGN) vesicles and local inhibition of endocytosis. Charasomes are involved in the acidification of their aqueous environment, thereby facilitating photosynthesis-dependent carbon uptake. Charasome formation is reversible to allow cells to adapt to different light conditions. Here, we show that darkness-induced degradation of charasomes involves the formation of coated pits and coated vesicles. The darkness-induced degradation of charasomes can be inhibited by 1-2 µM ikarugamycin (IKA), which is considered to be a specific inhibitor of clathrin-dependent endocytosis. At a much higher concentration (100 µM), IKA also significantly reduces the internalization of styryl dyes, indicating uptake via clathrin-coated vesicles (CV).We are the first to present evidence, based on fine structure investigation, that IKA does not interfere with the formation of clathrin coat, but inhibits the detachment and/or further processing of coated vesicles. Both charasome degradation and constitutive endocytosis are also significantly inhibited by sterol complexing agents (methyl-β-cyclodextrin and filipin). The absence of an additive effect, when applied together with IKA, suggests that charasome degradation and constitutive endocytosis (measured via styryl dye uptake) is not inhibited due to membrane retrieval via lipid rafts, but due to clathrin coat formation requirement of a specific set of sterols. Analysis of Chara australis clathrin proteins revealed two heavy chains and several light chains with sequence peculiarities, suggesting functional and/or species specific differences. The data obtained indicate that clathrin plays a central role not only in constitutive endocytosis but also in the degradation of charasomes, thereby representing a valuable system for studying targeted exo-and endocytosis. [ABSTRACT FROM AUTHOR]