Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium

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.The ability of the hemagglutinin protein (HA) of influenza viruses to mediate fusion between viral and endosomal membranes during virus entry into the cell depends on cleavage of fusion-incompetent precursor HA0 into disulfide-linked subunits HA1 and HA2 by a host endoprotease. Cleavage of HA is essential for infection and determines viral pathogenicity and tissue tropism (reviewed in references 8, 10, 11, and 22). Thus, the highly pathogenic avian influenza viruses of subtypes H5 and H7 are cleaved at the multibasic motif R-X-R/K-R by ubiquitous subtilisin-like cellular proteases (11, 23) and cause lethal systemic infection in birds. All other influenza A viruses, including human epidemic and pandemic strains, have a single arginine at the HA cleavage site; these viruses can only be cleaved in a limited number of tissues, such as the intestinal tract in birds and the respiratory tract in birds and mammals (11,22).Early studies demonstrated that influenza viruses with monobasic cleavage site can be proteolytically activated in cell culture by the addition of trypsin (12, 13). Less is known about proteases that cleave influenza viruses under conditions of natural infection. Several trypsin-like proteases isolated from rat and swine lung were shown to support replication of influenza viruses in vitro (3,9,18,25). However, it remains unclear whether these proteases play a role in in vivo infection. In the case of human influenza, specific proteases responsible for HA cleavage in the human respiratory tract have not been identified thus far.In search of such proteases, we use a new approach. Instead of isolating and characterizing influenza virus-activating enzymes from respiratory tissues, we clone and express genes of trypsin-like proteases known to be present in the human airway epithelium and test them for cleavage of HA. We have analyzed here two such proteases, TMPRSS2 (5, 14, 21) and HAT (human airway trypsin-like protease) (4, 24, 27, 28), given their previous detection in the human airways and the availability of their full-length coding sequences. As the source of human genetic material for cloning, we used differentiated cultures of human airway epithelial cells grown at the air-liquid interface in serum-free, hormone-and growth factor-supplemented medium as previously described (6,15). These cultures support multicycle replication of human influenza virus...

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH

“…In this study, the culture medium used for the fusion assays included trypsin as the only protease to mediate HA protein cleavage, suggesting that HA cleavage in Dk/Hk (H9N2) requires another protease (e.g. human airway trypsin-like protease or TMPRSS2) (26).…”

Replication Efficiency of Influenza A Virus H9N2: A Comparative Analysis Between Different Origin Cell Types

“…The identification of virus-host interaction is essential to investigate potential cell culture systems for explanation of virus replication and adaptation. The interaction depends on the ability of the virus to enter host cells followed by specific binding to SA receptors and activation of HA protease processing (12).…”

Technical Considerations for the Generation of Novel Pseudotyped Viruses

“…Furthermore, protease usage can be a critical determinant of viral tropism [76]. For seasonal human influenza viruses, the requirement for proteasemediated cleavage of the hemagglutinin in order for it to become fusion competent is a well-documented trait [65,[77][78][79]. However, protease activation is also a well-studied component in the Paramyxoviridae (a family of negative sense single-strand RNA viruses), where the F protein precursor must be cleaved in order to facilitate maturation of the fusion protein [80], and in the Coronaviridae (a family of positive sense, singlestrand RNA viruses) where proteases can be involved in both cleavage of the spike protein and facilitating release from the host cell [81][82][83].…”

“…Therefore, it may be necessary to supplement PV generation protocols with specific proteases. In many studies it has become common practice to co-transfect a protease-encoding plasmid alongside the other plasmids required for PV production [65,77,79,84]. For influenza, the most commonly used proteases when supplied as a plasmid are HAT and TMPRSS2 [77].…”