Fig 2: Infection and growth of the group A rotavirus (RVA) SA11 strain in cells expressing exogenous type II transmembrane serine proteases (TTSPs). (A) Generation of MA104 cells transduced with lentiviral vectors expressing TMPRSS2 (T2), TMPRSS11D (T11D), TMPRSS11E (T11E), or TMPRSS13 (T13). Expression of TTSPs was confirmed by Western blotting with antibodies specific to the proteins indicated on the left. The full-length and cleaved forms are indicated by closed and open arrowheads, respectively. Asterisks indicate nonspecific bands that cross-reacted with antibodies. (B to D) The RVA SA11 strain grown in the absence of trypsin was inoculated into MA104 or TTSP-expressing cells (T2, T11D, T11E, and T13 cells) and cultured without trypsin for 48 h. MA104 cells infected with SA11 and cultured with trypsin (shown as trypsin) were used as a positive control. (B) Cells were stained with anti-RVA antibody (green) and Hoechst 33342 nuclear dye (blue) at 48 h postinfection. Bars, 100 μm. (C) Total RNA was extracted from cells and analyzed by quantitative reverse transcription (qRT)-PCR. The levels of SA11 VP6 RNA were normalized to β-actin mRNA levels. (D) Viral titers in the cultures were determined using a focus assay. Statistical difference at 72 hpi was measured. The values in the graphs are expressed as the means ± standard deviations (SDs) from triplicate samples. One-way analysis of variance with Dunnett’s test was employed to determine statistical significance. Significant differences are indicated by asterisks: ***, P < 0.001.

Fig 3: Proteolytic cleavage of VP4 by TMPRSS11D. (A) Immature SA11 virions were incubated with the indicated concentrations of recombinant TMPRSS11D (rT11D) or trypsin for 3 h. Thereafter, samples were subjected to Western blotting using anti-SA11 VP5* antibody or anti-TMPRSS11D antibody. Arrowheads indicate the signals of the indicated proteins. (B) Schematic representation of the internally quenched fluorogenic R247 peptide. The peptide sequence corresponds to the amino acid sequence between 242 and 251 of SA11. Through peptide cleavage, EDANS at the C terminus of the peptide was released from the quencher DABCYL at the N terminus and emitted a fluorescence signal. (C and D) In vitro cleavage assay with fluorogenic R247 and R247H peptides. The fluorescence signal from peptides was monitored at the indicated time in different concentrations of trypsin (C) and rT11D (D). The values in the graphs are expressed as the means ± SDs from triplicate samples.

Fig 4: Effect of type II transmembrane serine protease (TTSP) inhibitors and proteolytic inactive mutation into TMPRSS11D on group A rotavirus (RVA) infectivity. MA104 and T2T11D cells infected with the RVA Wa strain were cultured for 48 h with the TTSP inhibitors AEBSF (A), camostat (B), and leupeptin (C). Total RNA was extracted from cells, and the relative levels of Wa NSP3 RNA were measured via qRT-PCR. The β-actin mRNA levels were used as reference control. (D) Generation of MA104 cells transduced with a lentiviral vector expressing the protease-inactive mutant TMPRSS11D-S368A with codon optimization (T11Dopt-S368A). The expression of TMPRSS11D-S368A was confirmed by Western blotting. (E) Viral titers in the cultures were determined using a focus assay at 48 h postinfection by the RVA Wa strain. The values in the graphs are expressed as the means ± SDs from triplicate samples. *, P < 0.05; ***, P < 0.001 by one-way analysis of variance with Dunnett’s test.

Fig 5: Proteolytic activation of group A rotaviruses (RVAs) on the surface of type II transmembrane serine protease (TTSP)-expressing cells. (A) Cell surface expression levels of TMPRSS2 and TMPRSS11D in MA104 and T2T11D cells. Nonpermeabilized cells were double-stained with anti-TMPRSS2 and anti-TMPRSS11D antibodies and analyzed via flow cytometry. Infectivity of immature virions in TTSP-expressing cells. Immature virions were harvested from MA104 cells infected with the RVA Wa strain (B) or Vero cells infected with the Sendai virus (SeV) (C) in the absence of trypsin. A portion of immature virions was activated by trypsin pretreatment. The infectivity of immature (blue bars) and mature (red bars) virions was determined using a focus assay. The values in the graphs are expressed as the means ± SDs from triplicate samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Dunnett’s test (B) or two-tailed Student's t test (C).