Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium - PubMed (original) (raw)

Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium

Eva Böttcher et al. J Virol. 2006 Oct.

Abstract

Host cell proteases that cleave the hemagglutinin (HA) of influenza viruses in the human respiratory tract are still not identified. Here we cloned two human type II transmembrane serine proteases with known airway localization, TMPRSS2 and HAT, into mammalian expression vector. Cotransfection of mammalian cells with plasmids encoding HA and either protease resulted in HA cleavage in situ. Transient expression of either protease in MDCK cells enabled multicycle replication of influenza viruses in these cells in the absence of exogenous trypsin. These data suggest that TMPRSS2 and HAT are candidates for proteolytic activation of influenza viruses in vivo.

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Figures

FIG. 1.

FIG. 1.

Cleavage of influenza virus HA by coexpression with TMPRSS2 (left panel) and HAT (right panel). A549 cells were cotransfected with pCAGGS-HA (lanes 2 to 5) and empty pCAGGS vector (lanes 2 and 3), pCAGGS-TMPRSS2 or pCAGGS-HAT (lane 4), and pCAGGS-TMPRSS2(S441A) (lane 5, left panel). Mock transfections were done with empty pCAGGS plasmid (lane 1). We analyzed cell lysates prepared 48 h after transfection by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as described in the text. For a positive control of HA cleavage, transfected cells were incubated with 10 μg of TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (Sigma)/ml for 20 min at 37°C prior to lysis (lane 3).

FIG. 2.

FIG. 2.

Multicycle viral replication in MDCK cells transfected with TMPRSS2 (left column), HAT (middle column), or proteolytically inactive protease mutant TMPRSS2(S441A) (right column). Ten hours after transfection with protease-encoding plasmids, the cultures were infected with influenza viruses A/Memphis/14/96 (H1N1) (top row), A/Mallard/Alberta/205/98 (H2N9) (middle row), and A/Texas/6/96 (H3N2) (bottom row) at a multiplicity of infection of 0.01. We incubated infected cultures for 24 h and immunostained them for viral nucleoprotein as described previously (16).

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