Novel Type II Transmembrane Serine Proteases, MSPL and TMPRSS13, Proteolytically Activate Membrane Fusion Activity of the Hemagglutinin of Highly Pathogenic Avian Influenza Viruses and Induce Their Multicycle Replication

Abstract

Host cellular proteases induce influenza virus entry into cells by cleaving the viral surface envelope glycoprotein hemagglutinin (HA). However, details on the cellular proteases involved in this event are not fully available. We report here that ubiquitous type II transmembrane serine proteases, MSPL and its splice variant TMPRSS13, are novel candidates for proteases processing HA proteins of highly pathogenic avian influenza (HPAI) viruses, apart from the previously identified furin and proprotein convertases 5 and 6. HAs from all HPAI virus H5 and H7 strains have one of two cleavage site motifs, the R-X-K/R-R motif with R at position P4 and the K-K/R-K/T-R motif with K at position P4. In studies of synthetic 14-residue HPAI virus HA peptides with these cleavage site motifs, furin preferentially cleaved only HA peptides with the R-K-K-R motif in the presence of calcium and not peptides with the other motif, whereas MSPL and TMPRSS13 cleaved both types of HA peptides (those with the R/K-K-K-R motif) efficiently in the absence of calcium. Full-length recombinant HPAI virus HA with the K-K-K-R cleavage motif exhibited poor susceptibility to cleavage in the absence of MSPL or TMPRSS13 and the presence of furin in infected cells, but it was converted to mature HA subunits in transfected cells expressing MSPL or TMPRSS13, with membrane-fused giant-cell formation. This conversion and membrane fusion were suppressed by inhibitors of MSPL and TMPRSS13. Furthermore, infection with and multiplication of genetically modified live HPAI virus A/Crow/Kyoto/53/2004 (H5N1) with the K-K-K-R cleavage site motif were detected only in MSPL- and TMPRSS13-expressing cells.

Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic

“…Among the few exceptions to these rules are HAs which contain the unusual tetrapeptide K-K-K-R [104]. These HAs are cleaved by the serine protease TMPRSS13 [105].…”

Matriptase, HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

“…In addition, a carbohydrate side chain at Asn-11 of HA1 has been demonstrated to interfere with cleavage of this HA by steric hindrance, and loss of the glycosylation was associated with ubiquitous HA cleavage and virulence (57). Recently, the type II transmembrane protease MSPL/TMPRSS13, which is expressed in several tissues, has been shown to cleave the HA at such a motif and to enable systemic infection independent of furin (58). Therefore, the strict separation into HA with monobasic and multibasic cleavage sites that are activated by trypsin-like proteases and furin, respectively, does not apply to all influenza virus isolates, and attention should be drawn to unusual cleavage sites.…”

Influenza virus activating host proteases: Identification, localization and inhibitors as potential therapeutics

“…These latter types of cleavage sites were found in some H9 virus isolates of LPAIV, which are activated by TMPRSS2, HAT and by matriptase (Garten and Klenk, 2008;Baron et al, 2013;Böttcher-Friebertshäuser et al, 2014). Another unusual cleavage site motif was detected in some highly pathogenic H5 and H7 viruses containing the sequence -K-K-K-R- (Röhm et al, 1996;Senne et al, 1996), which is cleaved by TMPRSS13/MSPL, but not by furin (Okumura et al, 2010). Distribution of the protease in tissues and organs influences the spread of virus progeny through the organism.…”