Tick-Borne Flavivirus Inhibits Sphingomyelinase (IsSMase), a Venomous Spider Ortholog to Increase Sphingomyelin Lipid Levels for Its Survival in Ixodes scapularis Ticks

Abstract

Our previous study showed that cells from medically important arthropods, such as ticks, secrete extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Understanding the molecular determinants and mechanism(s) of arthropod-borne flavivirus transmission via exosome biogenesis is very important. In this current study, we showed that in the presence of tick-borne Langat Virus (LGTV; a member of tick-borne encephalitis virus complex), the expression of arthropod IsSMase, a sphingomyelinase D (SMase D) that catalyzes the hydrolytic cleavage of substrates like sphingomyelin (SM) lipids, was significantly reduced in both Ixodes scapularis ticks (in vivo) and in tick cells (in vitro). The IsSMase reduced levels correlated with down-regulation of its activity upon LGTV replication in tick cells. Our data show that LGTV-mediated suppression of IsSMase allowed accumulation of SM lipid levels that supported membrane-associated viral replication and exosome biogenesis. Inhibition of viral loads and SM lipid built up upon GW4869 inhibitor treatment reversed the IsSMase levels and restored its activity. Our results suggest an important role for this spider venomous ortholog IsSMase in regulating viral replication associated with membrane-bound SM lipids in ticks. In summary, our study not only suggests a novel role for arthropod IsSMase in tick-LGTV interactions but also provides new insights into its important function in vector defense mechanism(s) against tick-borne virus infection and in anti-viral pathway(s).

Tick‐virus interactions: Current understanding and future perspectives

“…31 During infection of I. scapularis ticks and I. scapularis ISE6 cultured cells, infection with Langat virus was shown to interfere with the action of sphingomyelinase and greatly increase the concentration of sphingolipids in the cell membrane. 33 This resulted in increased viral assembly, as well as a substantial increase in the production of exosomes; exosomes have been confirmed as a means of transmission of Langat virus between cells. 34 It is possible that the CD36 receptor is responsive to the composition of these viral exosomes as well as the envelope of complete flaviviruses, allowing it to act upon them directly to enable sensing.…”

“…The effects of some viruses can be modelled using less-pathogenic analogues. In many of these studies, tick-borne flaviviruses were modelled by the use of Langat virus, 33,34,78,92,99,105 a naturally attenuated version of TBEV capable of infecting multiple species of ticks in the Ixodes genus. Similarly, CCHFV can be modelled through the use of Hazara virus, 84,109 which is a much less pathogenic but closely related nairovirus that can be utilized under BSL2 and ACL2 conditions.…”

“…This system is usually utilized for the processing of apoptotic cells, although it is also capable of sensing some infection‐derived lipids, including sphingolipids 31 . During infection of I. scapularis ticks and I. scapularis ISE6 cultured cells, infection with Langat virus was shown to interfere with the action of sphingomyelinase and greatly increase the concentration of sphingolipids in the cell membrane 33 . This resulted in increased viral assembly, as well as a substantial increase in the production of exosomes; exosomes have been confirmed as a means of transmission of Langat virus between cells 34 .…”

An Experimental Murine Model to Study Acquisition Dynamics of Tick-Borne Langat Virus in Ixodes Scapularis

“…LGTV-infection of ticks, large volumes (1-2ml) of concentrated virus stocks (such as 10 7 or 10 8 pfu/ml) and longer incubations (17 days) are required for generation of small batch of infected ticks. Also, only 60-70% of ticks were capable of acquiring pathogen loads by body absorbance or bathing in the virus stocks 8,10 .…”

“…IsSMase neutralizes the Th1 cytokine response towards a Th2-induced cytokine response and modulates CD4+ T cells in order to express interleukin 4 (IL-4) 10,30 . Our recent studies focused on understanding the molecular mechanisms during pathogen-vector-host interactions have revealed different survival strategies used by microbes [6][7][8][9][10]16,28,29 . In the current study, utilizing LGTV as a model pathogen, we established a method to infect naïve I. scapularis by feeding these ticks on…”