Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses
Figure 2
Effects on chloroquine on membrane fusion of JE-VLPs and YFV in Vero cells.
(A) 0.1 g/l chloroquine (194 µM) had no notable toxic effect on the cells as indicated by differential interference contrast (DIC) microscopy of untreated cells (left) and chloroquine-treated cells (right). Cells were treated with 0.1 g/l chloroquine and infected with R18-labeled JE-VLPs (200 µl at 17 pM), (B), or with YFV (MOI = 1), (C). Chloroquine blocked VLP membrane fusion, as judged by the lack of R18 fluorescence dequenching in a field of treated cells (red curve) relative to the normal dequenching of R18 in untreated cells (blue curve). (D) Relative qRT-PCR of viral RNA in untreated (mock) and chloroquine-treated Vero cells 1 h post-infection with YFV (MOI = 1). Endosomal and cytosolic fractions were separated and total RNA was extracted from the cytosolic fraction as described in the Materials and Methods. RT-PCR was used to quantify the 3′-UTR of YFV genomic RNA. Error bars represent the standard error of the mean (SEM) of three experiments. Treatment with chloroquine reduced RNA release into the cytoplasm by >95%. (E) Plaque assay with BHK cells infect with YFV (MOI = 0.1) in presence and in absence of chloroquine. BHK cells were infected with MOI 0.1 of YFV in presence and in absence of 0.1 g/l chloroquine. Chloroquine treatment completely inhibited YFV replication. See also Figure S3.