Human SCARB2-Mediated Entry and Endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus

CPEB4 knockout mice exhibit normal hippocampus-related synaptic plasticity and memory.

Regulated RNA translation is critical to provide proteins needed to maintain persistent modification of synaptic strength, which underlies the molecular basis of long-term memory (LTM). Cytoplasmic polyadenylation element-binding proteins (CPEBs) are sequence-specific RNA-binding proteins and regulate translation in various tissues. All four CPEBs in vertebrates are expressed in the brain, including the hippocampal neurons, suggesting their potential roles in translation-dependent plasticity and memory. Although CPEB1 and CPEB3 have been shown to control specific kinds of hippocampus-related LTM, the role of CPEB2 and CPEB4 in learning and memory remains elusive. Thus, we generated CPEB4 knockout (KO) mice and analyzed them using several behavioral tests. No difference was found in the anxiety level, motor coordination, hippocampus-dependent learning and memory between the KO mice and their wild-type (WT) littermates. Electrophysiological recordings of multiple forms of synaptic plasticity in the Schaffer collateral pathway-CA1 neurons also showed normal responses in the KO hippocampal slices. Morphological analyses revealed that the CPEB4-lacking pyramidal neurons possessed slightly elongated dendritic spines. Unlike its related family members, CPEB1 and CPEB3, CPEB4 seems to be dispensable for hippocampus-dependent plasticity, learning and memory

Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

<div><p>Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90β siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90β resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90β and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. <i>In vivo</i> studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.</p> </div

No CPEB4 protein was detected in the KO tissues.

<p>(<b>A</b>) Western blot analysis. Expression of CPEB4 in the WT and KO tissues was detected using affinity-purified polyclonal CPEB4 antibody. Equal transfer and loading were determined with LRP130 antibody. (<b>B</b>) Distribution of CPEB4 in subregions of the brain was revealed by immunohistochemical staining of coronal sections using the same affinity-purified CPEB4 antibody. (<b>C</b>) High magnification of CPEB4 images were shown in the hippocampus and amygdala areas. Both assays showed no immunodetectable signals in the KO tissues. Scale, 0.2 mm unless specified.</p

CPEB4 KO mice performed normally in the Morris water maze (MWZ) and contextual fear conditioning tests.

<p>(<b>A</b>) The WT and KO mice normally responded in the hidden platform version of MWZ within 4 trials/day for 4 consecutive days. (<b>B</b>) In the probe trial, the platform was removed, and the percentage of time that the mouse spent on navigating in each quadrant was analyzed. Both of the groups of mice searched closer to the target quadrant where the platform had been previously placed. (<b>C</b>) The motor ability and visual acuity of the mice were assessed by the swimming speed in the cued version of MWZ, in which the platform was indicated by a visible flag. (<b>D</b>) Contextual fear conditioning. The freezing response during habituation (hab) and acquisition was analyzed for 2 min per trial. A foot shock (2-sec 0.5 mA) was given at the end of habituation and the first three conditioning trials. During the extinction phase, the mouse was placed in the chamber for 30 min without reinforcing the shock. The freezing level was analyzed every 6 min for 5 segments. A week after the extinction training, the consolidated extinction memory was recalled by monitoring the freezing behavior for 6 min in the original chamber. Numbers in parentheses denote the number of mice in each group used for the study. All of the data were expressed as the mean ± SEM.</p

The dendritic spine length was slightly increased in CPEB4 KO neurons.

<p>(<b>A</b>) The WT and KO neurons were transfected with the GFP plasmid on DIV14 and fixed on DIV18 for GFP immunostaining. Representative images of the whole neurons and dendritic spine area are shown. Scales, 10 µm unless denoted. (<b>B</b>) Quantification analysis of dendritic spine morphology. Approximately 30 pyramidal neurons in each group (WT, green; KO, red) were collected from three independent cultures and analyzed using the MetaMorph software. The cumulative probability curves of density, length and width of dendritic spines in each group (∼3000 spines) were plotted and analyzed for the statistical difference between groups using Student’s <i>t</i>-test.</p

Behavioral characterization of CPEB4 KO mice.

<p>The anxiety level and exploratory behavior of WT and KO mice were assessed using the (<b>A</b>) open field and (<b>B</b>) elevated plus maze (EPM) tasks. (<b>A</b>) The mice were placed in an arena and recorded for 1 h. The entry times, staying duration and traveling distance in the center during the first 10 min movement were analyzed and presented as bar graphs. The total traveling distance in the arena in every 10 min for 1 h was shown. (<b>B</b>) The percentage of entry and staying duration into the open arms vs. the total arms in the 5 min recording of movement in the EPM was analyzed. (<b>C</b>) Motor coordination ability was assessed by the rotarod test. The latency in which the mouse was able to stay on the rotating rod of various speeds was analyzed. Numbers in parentheses denote the number of mice in each group used for the study. All of the data were expressed as the mean ± SEM.</p

Genetic deletion of CPEB4 did not affect LTP in the SC pathway of adult (9-12-week-old) hippocampal slices.

<p>Basal synaptic transmission, (<b>A</b>) input-output (I–O) curve and (<b>B</b>) paired-pulse (PP) facilitation were normal in the KO slices (<i>P = </i>0.63 and 0.91, respectively, two-way ANOVA). No significant difference was observed between the WT and KO slices in LTP evoked by (<b>C</b>) one train (1X) of high frequency stimulation (HFS, <i>P</i> = 0.27 at 50–60 min after stimulation), (<b>D</b>) four trains (4X) of HFS (<i>P</i> = 0.69 at 100–120 min after stimulation), (<b>E</b>) 1X theta burst stimulation (TBS, <i>P</i> = 0.98 at 50–60 min after stimulation) and (<b>F</b>) 4X TBS (<i>P</i> = 0.11 at 100–120 min after stimulation). Statistics from (<b>C</b>) to (<b>F</b>) were analyzed using Student’s <i>t</i>-test. Numbers in parentheses represent the number of recorded slices isolated from 2–4 male mice. Inset: traces represent baseline (black line, 1) and the indicated time after stimulation (gray line, 2). Calibration: 0.5 mV, 20 msec.</p

EV71 infection is impacted by clathrin specific inhibitors.

<p>(A) 3T3-SCARB2 cells were pretreated with different doses of CPZ for one hour prior to the addition of MOI = 0.04 of EV71. (B) 50 or 100 pmoles of siRNA specific to clathrin or 100 pmoles of control siRNA (MOCK) were transfected to 3T3-SCARB2 cells as following the treatment protocol as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030507#pone-0030507-g004" target="_blank">Figure 4</a> described above. After 24 or 48 hours of transfection, cells were following lysed and detected the content of clathrin light chain B (CLTB) by Western blot using anti-clathrin antibody. Relative expression of siRNA-targeting genes shown in the top panel represented the relative CLTB protein levels of the cells transfected with specific siRNA, compared to the expression in 48 hours transfection of the cells with 100 pmoles control siRNA (Mock) as 1.0 was shown. Lysates were also prepared after 24 or 48 hours of EV71 infection and subjected to analyze the expression of synthetic EV71 capsid protein by western blot using MAB979 antibody. The result was shown in the lower panel. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.</p