Table 1.
Zmora et al.
Fig 1.
TMPRSS2, TMPRSS4 and HAT are conserved between humans and non-human primates.
(A) Amino acid sequence alignment of human (NP_005647.3), rhesus macaque (XP_014988331.1), cynomolgus macaque (XP_015302312.1) and common marmoset (XP_008984973.1) TMPRSS2. Protein alignment was performed by using Vector NTI AlignX. Colors indicate amino acid identity (yellow), conservation (blue) and similarity (green). The catalytic triad is boxed. (B) For analysis of protease expression, 293T cells were transfected with plasmids encoding TMPRSS2, TMPRSS4 or HAT of the indicated species and equipped with an N-terminal myc antigenic tag. Empty plasmid (pCAGGS) served as a negative control. Protease expression in cell lysates was detected via Western blotting with anti-myc antibody. Due to more prominent expression of TMPRSS2 relative to TMPRSS4 and HAT proteins, 10 μl of lysates from TMPRSS2 expressing cells and 20 μl of lysates from TMPRSS4 and HAT expressing cells were loaded for separation by SDS gel-electrophoresis. The expression of β-actin was determined as a loading control. Filled triangles indicate zymogen forms, while empty triangles highlight cleavage products resulting from autocatalytic activation. The results were confirmed in at least two separate experiments.
Fig 2.
TMPRSS2, TMPRSS4 and HAT of non-human primate origin cleave and activate influenza virus hemagglutinin.
(A) 293T cells were transiently cotransfected with plasmids encoding FLUAV HA of the 1918 H1N1 FLUAV and the indicated proteases or empty plasmid (pCAGGS). At 48 h post transfection the cells were treated with PBS or trypsin, and HA cleavage was determined by Western blotting. The HA precursor HA0 (filled triangle) and the surface unit HA1 (empty triangle) are indicated. The expression of β-actin was determined as a loading control. Similar results were obtained in three independent experiments. (B) The indicated proteases were expressed in 293T cells and the cells infected with FLUAV A/PR/8/34 (H1N1) at an MOI 0.01 and treated with either trypsin or PBS. At 48 h post infection, the virus titers were determined by focus formation assay. The average of three to five independent experiments is shown; error bars indicate standard error of the mean. Virus titers measured upon trypsin treatment were set as 100%.
Fig 3.
Serine protease activity is required for influenza A virus spread in respiratory epithelium of human and non-human primate origin.
(A) Expression of TMPRSS2 in precision-cut lung slices (PCLS) of rhesus macaque origin was analyzed employing immunohistochemistry with an antibody raised against human TMPRSS2 which cross-reacts with the rhesus macaque orthologue. Hematoxylin was used for counterstaining. Omission of the primary antibody served as negative control. (B) Precision cut lung slices (PCLS) prepared from human, rhesus macaque, cynomolgus macaque or common marmoset lung were infected with 3 x 104 ffu of FLUAV A/Hamburg/04/2009 (H1N1, human PCLS) or A/PR/8/34 (H1N1, rhesus, cynomolgus, marmoset PCLS) and treated with the indicated amounts of camostat mesylate. At 48 h post infection, the viral titers in the supernatants were tested using focus formation assay. The results of representative experiments performed with triplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in three to five independent experiments. ffu, focus forming units.