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

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.     

Interaction of the C-terminal domains of sendai virus N and P proteins: comparison of polymerase-nucleocapsid interactions within the paramyxovirus family

Interaction of the C-terminal domains of Sendai virus (SeV) P and N proteins is crucial for RNA synthesis by correctly positioning the polymerase complex (L+P) onto the nucleocapsid (N/RNA). To better understand this mechanism within the paramyxovirus family, we have studied the complex formed by the SeV C-terminal domains of P (PX) and N (N(TAIL)) proteins by solution nuclear magnetic resonance spectroscopy. We have characterized SeV N(TAIL), which belongs to the class of intrinsically disordered proteins, and precisely defined the binding regions within this latter domain and within PX. SeV N(TAIL) binds with residues 472 to 493, which have a helical propensity (residues 477 to 491) to the surface created by helices alpha2 and alpha3 of PX with a 1:1 stoichiometry, as was also found for measles virus (MV). The binding interface is dominated by charged residues, and the dissociation constant was determined to be 57 +/- 18 microM under conditions of the experiment (i.e., in 0.5 M NaCl). We have also shown that the extreme C terminus of SeV N(TAIL) does not interact with PX, which is in contrast to MV, where a second binding site was identified. In addition, the interaction surfaces of the MV proteins are hydrophobic and a stronger binding constant was found. This gives a good illustration of how selection pressure allowed the C-terminal domains of N and P proteins to evolve concomitantly within this family of viruses in order to lead to protein complexes having the same three-dimensional fold, and thus the same function, but with completely different binding interfaces.     

Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.     

TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host.     

Virus multiplication and induction of apoptosis by Sendai virus: role of the C proteins

Sendai virus (SeV) P gene encodes a nested set of carboxyl-coterminal proteins (C', C, Y1 and Y2), which are referred to collectively as the C proteins. Characterization of the virus multiplication and cellular responses in HEp-2 cells infected with the recombinant SeV which lacks two (C' and C), three (C', C and Y1) or all the four C proteins revealed that all the recombinant viruses can grow in the cells to various extents, depending, apparently, on the number of species expressing C protein. In reverse proportion to the viral growth ability, these viruses induced apoptosis in the infected cells. These results indicate that Y2 protein has an antiapoptotic activity, and suggest that this activity works in an additive manner with the longer C protein(s) (C' and/or C) of SeV in order to suppress virus-induced apoptosis in the SeV-infected cells. Apparently, the antiapoptotic activity of the C proteins supports virus multiplication in the infected cells.     

TMPRSS2 and ADAM17 Cleave ACE2 Differentially and Only Proteolysis by TMPRSS2 Augments Entry Driven by the Severe Acute Respiratory Syndrome Coronavirus Spike Protein

The type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors.     

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.     

Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2

Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log(10) units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.     

Involvement of the zinc-binding capacity of Sendai virus V protein in viral pathogenesis

The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C(341)S and V-C(365)R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV V(DeltaC), which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.     

Activation of Influenza A Viruses by Host Proteases from Swine Airway Epithelium

Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways.     

Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the Matrix Protein

The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C(83)S, SeV M-C(106)S, and SeV M-C(295)S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C(83)S and SeV M-C(106)S had smaller virus particles than did the wild-type SeV, whereas SeV M-C(295)S had larger and heterogeneously sized particles. Furthermore, SeV M-C(106)S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle.     

Adjuvants and Immunization Strategies to Induce Influenza Virus Hemagglutinin Stalk Antibodies

The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C), and an RNA hairpin derived from Sendai virus (SeV) Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C) also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.     

Sendai virus targets inflammatory responses,as well as the interferon-induced antiviral state,in a multifaceted manner

We have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-beta), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-beta was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-beta mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.     

The Sendai paramyxovirus accessory C proteins inhibit viral genome amplification in a promoter-specific fashion.

Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i) it had lost most of its inhibitory activity on internally deleted DI genome amplification and (ii) its coexpression no longer prevented the recovery of SeV from DNA. However, consistent with the insensitivity of copy-back DI genomes to C protein inhibition, C coexpression did not prevent the recovery of copy-back nondefective viruses from DNA. The inhibitory effects of C coexpression thus appear to be promoter specific.     

Knockout of the Sendai virus C gene eliminates the viral ability to prevent the interferon-alpha/beta-mediated responses.

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.     

Paramyxovirus accessory proteins as interferon antagonists

A new role of the Paramyxovirus accessory proteins has been uncovered. The P gene of the subfamily Paramyxovirinae encodes accessory proteins including the V and/or C protein by means of pseudotemplated nucleotide addition (RNA editing) or by overlapping open reading frame. The Respirovirus (Sendai virus and human parainfluenza virus (hPIV)3) and Rubulavirus (simian virus (SV)5, SV41, mumps virus and hPIV2) circumvent the interferon (IFN) response by inhibiting IFN signaling. The responsible genes were mapped to the C gene for SeV and the V gene for rubulaviruses. On the other hand, wild type measles viruses isolated from clinical specimens suppress production of IFN, although responsible viral factors remain to be identified. Both human and bovine respiratory syncytial viruses (RSVs) counteract the antiviral effect of IFN with inhibiting neither IFN signaling nor IFN production. Bovine RSV NS1 and NS2 proteins cooperatively antagonize the antiviral effect of IFN. Studies on the molecular mechanism by which viruses circumvent the host IFN response will not only illustrate co-evolution of virus strategies of immune evasion but also provide basic information useful for engineering novel antiviral drugs as well as recombinant live vaccine.