Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification

by Selleckchem | Oct 20 2023

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV's ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3 h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib-an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyxo-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells.

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Tilapia Lake Virus (TiLV) is a recently identified virus that poses a significant threat to global tilapia aquaculture, which has implications for food security. TiLV belongs to the family Amnoonviridae and is classified in the monotypic genus Tilapinevirus. It shares some features with orthomyxoviruses but has distinct characteristics.

The genome of TiLV consists of ten segments of single-stranded, negative RNA. Interestingly, nine of the ten major proteins encoded by TiLV's genome do not show any similarity to known viral or cellular proteins. This highlights the unique nature of TiLV and its divergence from other viruses.

The mechanism by which TiLV enters tilapia cells was not previously understood. However, a study was conducted to investigate the molecular requirements for TiLV entry. The researchers measured the entry window of TiLV (approximately 3 hours) and applied various inhibitors that target regulators of endocytic functions to determine their effect on TiLV entry.

The study found that inhibiting dynamin activity using compounds like dynasore or dynole, or depleting cholesterol using methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and the production of infectious viral particles. This suggests that TiLV enters tilapia cells through a dynamin-mediated endocytic pathway that is dependent on cholesterol.

In addition to dynamin and cholesterol, the study also revealed the involvement of the cytoskeleton in TiLV entry. Inhibiting actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole partially inhibited TiLV infection within the entry window.

Surprisingly, the study showed that TiLV entry was not affected by inhibitors of endosomal acidification (such as NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), or erlotinib (an inhibitor of the endocytic Cyclin G-associated kinase, GAK). This indicates that TiLV entry is independent of clathrin and pH.

In summary, TiLV enters tilapia cells through a dynamin-mediated endocytic pathway that is dependent on cholesterol and the cytoskeleton, but independent of clathrin and pH. These findings distinguish TiLV from the prototypical orthomyxovirus, influenza A virus, and highlight its unique requirements for cellular entry.