Title: Enzymatic regulation of viral entry
Description: Class I membrane fusion involves a series of transitions that can be regulated or mediated by the activities of viral and cellular enzymes. These enzymes ensure that the conformational changes associated with fusion occur at an appropriate time and in an appropriate cell. Here, we describe how the viral neuraminidase (NA) and cellular cathepsin L regulate cellular entry of influenza A viruses and SARS coronavirus (SARS-CoV), respectively. We first show that cellular expression of influenza A virus NA, but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of α-2,3- and α-2,6-linked sialic acid (SA) observed in cells expressing NA or infected with influenza A viruses. Collectively these data show that NA prevents the reinfection of the virus-producing, and limits the frequency of superinfection and perhaps reassortment of influenza A viruses.

We also demonstrate, in a different context, a necessary role for the cysteine protease cathepsin L in the fusion of SARS-CoV with cells expressing the SARS-CoV receptor ACE2. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins, but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Using the exogenous proteases trypsin and Arg-C, which restore entry mediated by SARS-CoV S protein in the presence of the cathepsin L inhibitor, we show that cathepsin L and trypsin cleave ACE2-bound SARS-CoV S protein in nearly identical regions, and define one tryptic site whose cleavage is necessary for S-protein-mediated entry. Alteration of S-protein residue 667 to an alanine blocked the ability of trypsin or Arg-C to rescue entry in the presence of a cathepsin-L inhibitor or NH 4 Cl. Collectively these data indicate that digestion of the SARS-CoV S-protein in the vicinity of residue 667 is necessary for entry of the virus into an ACE2-expressing cell.