Infection with Usutu Virus Induces an Autophagic Response in Mammalian Cells

Abstract

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection.

The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis

“…For viral infections, transfected cells were infected (MOI, 1 PFU/cell) as described above for infections in liquid medium, and subsequent experiments were performed at 48 h posttransfection. The extent of siRNA silencing was analyzed by Western blotting (29,35) using specific antibodies against nSMase2.…”

“…Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 min and processed for immunofluorescence as described previously (29,35). Cells were observed using a Leica TCS SPE confocal laser scanning microscope and an HCX PL Apo ϫ63/1.4 oil immersion objective.…”

“…HeLa or Vero cells infected with WNV were fixed in 4% paraformaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 24 h p.i. and processed for electron microscopy as described previously (29,35). Samples were examined using a JEOL JEM-1010 electron microscope (JEOL, Japan) operated at 80 kV, and images were acquired using a digital camera (4K64K TemCam-F416; Tietz Video and Image Processing Systems GmbH, Gauting, Germany).…”

Usutu virus: current knowledge and future perspectives

“…42,43 Moreover, infection with USUV triggered an autophagic response in the host cell ( Figure 4). 44 Autophagy is an important cellular pathway that plays important roles in viral infections and pathogenesis, and the upregulation of the autophagic pathway has been well documented for various flaviviruses, 45 although the involvement of autophagy in WNV infection is still controversial. 46 Infection with USUV induces endoplasmic reticulum stress, which results in activation of a cellular signaling response termed unfolded protein response, 44 consistent with that observed for other members of the Flavivirus genus.…”

“…Treatment with inhibitors of autophagy 3-methyladenine or wortmannin also showed a significant reduction in the virus yield in USUV infection, since this virus takes advantage of autophagy for replication. 44 Even fewer data have been published regarding vaccines against USUV, as vaccine development against some flaviviruses present several drawbacks: relatively low incidence, cost-effectiveness, implementation for wild life vaccination, availability of animal models, and so on. For instance, in the case of USUV, only laboratory suckling mice, but not adult mice, exhibit susceptibility to infection, thus hampering the analysis of the immune responses and complicating the evaluation of vaccine candidates against this pathogen.…”