Fig 1.
BEX1 limits viral infection in vivo.
A) qPCR for BEX1 mRNA expression (normalized to Rpl7 mRNA) in WT mouse hearts in uninfected conditions and one week following infection with CVB3. B) qPCR for the CVB3 RNA genome (relative to Rpl7) in WT and BEX1 KO hearts that were not infected or infected for 2, 7, and 28 days with CVB3. C) Immunostaining of WT and BEX1 KO cardiac sections in uninfected conditions and one week following infection with CVB3. Replicating CVB3 was detected using a double-stranded RNA-binding antibody and is shown in red, while the cell outlines were stained in green with wheat germ agglutinin stain. Scale bar = 125μm. D) Quantification of the percent of cardiomyocytes that were infected in WT and BEX1 KO cardiac sections shown in panel C. Statistical analyses: t-test for panels A and D; 2-way ANOVA for panel B. (* p<0.05 CVB3-treated WT vs. uninfected WT; # p<0.05 BEX1 KO vs. WT same treatment).
Fig 2.
Immune cell infiltration is impaired in BEX1 KO hearts during CVB3 infection.
A) Hematoxylin and Eosin staining was conducted in WT and BEX1 KO mice following the indicated treatments. Scale bar = 125μm. B) The gating of CD45+ cells in representative WT and BEX1 KO 7 days post-infection hearts is shown for total leukocyte abundance using flow cytometry. D-K) Total Leukocytes (C), T lymphocytes (D), total monocytes (E), natural killers (F), B lymphocytes (G), myeloid-derived cells (H), neutrophils (I), infiltrating monocytes (J), and macrophages (K) were evaluated in WT and BEX1 KO hearts at the indicated post-infection time points using flow cytometry. d = days. Statistical analyses by 2-way ANOVA. (* p<0.05 CVB3-treated WT vs. uninfected WT; & p<0.05 CVB3-treated BEX1 KO vs. uninfected BEX1 KO; # p<0.05 BEX1 KO vs. WT same treatment).
Fig 3.
Loss of BEX1 increases CVB3-induced cardiac damage.
A) Percent change in body weight of WT and BEX1 KO mice over the course of infection is shown. B-C) Heart weight (B) and spleen weight (C) (both normalized to body weight) of each genotype over the course of infection is shown. D) Percent ejection fraction was measured in WT and BEX1 KO mice in uninfected conditions and four weeks following infection. E-F) Cardiac fibrosis was evaluated using Masson’s trichrome stain (scale bar = 200μm) (E), and it was quantified using ImageJ (F). Statistical analyses by 2-way ANOVA. (* p<0.05 CVB3-treated WT vs. uninfected WT; & p<0.05 CVB3-treated BEX1 KO vs. uninfected BEX1 KO; # BEX1 KO vs. WT same treatment).
Fig 4.
BEX1 overexpression protects against CVB3 infection.
A) Western blot for BEX1 and GAPDH (loading control) in isolated neonatal rat cardiomyocytes. B) qPCR showing abundance of CVB3 RNA (normalized to Rpl7) 12 hours after infection (MOI = 1). C) Western blot for BEX1 and GAPDH (loading control) in cardiac protein extracts from WT and BEX1 transgenic mice. D) qPCR showing abundance of CVB3 RNA (normalized to Rpl7) in hearts of uninfected or infected (seven days) WT and BEX1-overexpressing mice. Statistical analyses by 2-way ANOVA. (* p<0.05 CVB3-treated WT vs. uninfected WT; & p<0.05 CVB3-treated BEX1 KO vs. uninfected BEX1 KO; # p<0.05 BEX1 KO vs. WT same treatment).
Fig 5.
Loss of BEX1 increases susceptibility to multiple viruses ex vivo.
A) qPCR of the CVB3 genome (normalized to Rpl7) from WT and BEX1 KO mouse embryonic fibroblasts (MEFs) infected with CVB3 (MOI = 1) for 24 hours. B-C) Percent of MEFs (WT or BEX1 KO) infected with influenza (B) or Sendai virus (C) after 24 hours, as measured by flow cytometry of GFP-tagged viral capsids. D-F) ELISA assessing IFN-β protein present in growth medium of WT or BEX1 KO MEFs 24 hours after infection with CVB3 (D), influenza (E), or Sendai virus (F). G-I) Rescue of antiviral function in BEX1 KO MEFs by treatment with recombinant IFN-β for cells infected with CVB3 (G), influenza (H), or Sendai virus (I). Statistical analyses by t-test (A-F) and two-way ANOVA (G-I). (# p<0.05 BEX1 KO vs. WT same treatment; * p<0.05 between indicated groups).