Human SCARB2 Acts as a Cellular Associator for Helping Coxsackieviruses A10 Infection

Figures

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  Figure 1. Replication of CVA10 in hSCARB2-expressing cells. Mouse NIH3T3 and 3T3-SCARB2 and human cell line, RD, were infected with 0.1 MOI of CVA10 and then cultured for up to 72 h. The cultured supernatants (sup) and cell lysates (cell) were collected at 8, 24, 48, and 72 h, respectively, and subjected to the plaque-forming assay in RD cells described in the Materials and Methods. (A) Numbers 0, 1, 2, and up to 6 marked on each well were represented as 100, 101, 102, and up to 106, respectively, of serial dilution fold of the collected samples that were subjected to the assay. The photography of forming plaques was taken at 72 h after incubation. (B) The time course of produced CVA10 from the supernatants and lysate parts was calculated and shown. (C) The time course of CVA10 production in the supernatants and lysate parts of RD cell was also calculated. (D) Immunoblots of CVA10 VP1 and GAPDH in the lysates were collected at 8, 24, 48, and 72 h.p.i. by anti-CVA6/CVA10 and GAPDH antibodies, respectively. The symbol **** was used to indicate p < 0.0001, respectively.

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  Figure 2. Association of hSCARB2 with KRM1 in CVA10-infected RD cells. RD cells were inoculated with MOI = 0.1 of CVA10 and cultured for 8 and 24 h before lysate preparation. Uninfected RD cells were cultured in parallel. Next, 50 μg cell lysates were prepared and mixed with the magnetic bead-conjugated antibody against (a) hSCARB2, (b) KRM1, and (c) isotypic antibody specific to GAPDH, individually, for immunoprecipitation (CoIP). The co-precipitates were subjected to Western blot against associated hSCARB2, CVA10 VP1, and KRM1 proteins. Lysates (20 μg) from infected and uninfected RD cells were directly subjected to immunoblot to detect the investigated proteins.

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  Figure 3. The reduction of endogenous hSCARB2 and KRM1 expression impacted CVA10 infection. RD Cells were transfected individually with 50 pmoles of siRNA specific to hSCARB2 (s2651(S1) and s2652(S2)), KRM1 (s38393(K1) and s38394(K2)), or mixed specific siRNA with equal amounts (50 + 50 pmoles) of S1 + K1, S1 + K2, S2 + K1, S2 + K2, or negative siRNA, followed by 48 h of incubation. Infection of siRNA-treated cells with CVA10 (MOI = 0.05) and then cultured for another 24 h then proceeded. (a) Western blotting with the respective antibody examined the expression level of hSCARB2 or KRN1 in the cells. (b) The supernatant and (c) lysate were collected and subjected to a plaque-forming assay to detect the amounts of produced CVA10, and the results were shown. (d) Total amounts of CVA10 production using the sum of the viral amounts from (b,c) are shown. The symbols **, *** and **** were used to indicate p < 0.01, p < 0.001, and p < 0.0001, respectively.

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  Figure 4. Pathogenesis and lethality of hSCARB2-Tg mice infected with CVA10. Scoring of (a) disease score, (b) loss of body weight, and (c) survival rate in 12-day-old hSCARB2-Tg and WT mice injected s.c. with 1.72 × 106 and 1.72 × 107 pfu of CVA10, individually, were monitored daily (day post-infection, D.P.I.) for 15 days and assessed following the criteria described in the Materials and Methods section. The same age of Tg mice injected s.c. with PBS as control was included. The number (n) of mice per group was shown.

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  Figure 5. Viral distribution in the tissues and organs of CVA10-infected mice. Twelve-day-old WT and hSCARB2-Tg mice were injected with 1.72 × 107 pfu of CVA10 s.c. On day 3, post-viral infection, RNA extracted from the (a) muscle, (b) brain, and (c) spinal cord of the mice were subjected to quantitative RT-PCR analysis using primers specific to the VP1 region. β-actin gene expression in each tissue was used as the internal control. A schematic representation of the VP1 gene expression and the statistical average from six mice per group, as described in the Materials and Methods section, is shown. The symbols * and ns were used to indicate p < 0.05 and no significant difference, respectively.

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  Figure 6. Protection of hSCARB2-Tg mice from CVA10 challenge by FI-CVA10 vaccine. (a) The flowchart of vaccine immunization following the challenge of CVA10 in Tg mice was shown. One-day-old Tg mice were s.c. immunized with two doses of FI-CVA10 vaccine or PBS on day 2 and day 8, following s.c. injection of 1.72 × 107 pfu of CVA10 on day 11 was performed. Daily recording of (b) percentage of mice survival, (c) loss of body weight, and (d) disease score for 15 days of hSCARB2-Tg mice receiving PBS or FI-CVA10 was performed. Data represent the mean scores obtained from the individual number of (n) mice per group.

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  Figure 7. Reduction in viruses in the tissues of CVA10-infected mice pre-receiving FI-CVA10 vaccine. On day 3 post-viral infection, RNA was extracted from the (a) muscle, (b) brain, and (c) spinal cord of Tg mice, which had been pre-immunized with PBS or two-dose of FI-CVA10 (described in the legend of Figure 6), and were subjected to quantitative RT-PCR analysis using primers specific to the VP1 region. RNA from the same age of Tg mice without any treatment (Mock) as background control was included. β-actin gene expression in each tissue was used as the internal control to calculate the relative expression of VP1 mRNA. A schematic representation of the VP1 gene expression and the statistical average from five mice per group from the FI-CVA10-treated group and three mice per group from the PBS-treated group (minimizing the number of animals used in the test) is shown. (d) Tg mice (6–8 weeks old) pre-immunized with PBS or two-dose of FI-CVA10 at a 2-week interval. Serum samples collected from individual Tg mice on day 28 post the second shot of FI-CVA10 were assayed for the titer of anti-CVA10 neutralizing antibodies. The results were expressed as titers for each test sample. Bars…