Elsevier

Vaccine

Volume 30, Issue 51, 7 December 2012, Pages 7374-7380
Vaccine

Hemagglutinin activating host cell proteases provide promising drug targets for the treatment of influenza A and B virus infections

https://doi.org/10.1016/j.vaccine.2012.10.001Get rights and content

Abstract

Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for infectivity and spread of the virus. Some years ago, we identified TMPRSS2 and HAT from human airways as activating proteases of influenza A viruses containing a monobasic HA cleavage site. Therefore, these proteases are considered as potential drug targets. In this report, first we show that HA of influenza B virus is activated by TMPRSS2 and HAT, too. We further demonstrate that benzylsulfonyl-d-arginine-proline-4-amidinobenzylamide (BAPA), which is a potent inhibitor of HAT and TMPRSS2, efficiently suppresses virus propagation in TMPRSS2-expressing human airway epithelial cells by inhibition of HA cleavage. BAPA treatment reduced virus titers of different influenza A and B viruses more than 1000-fold and delayed virus propagation by 24–48 h at non-cytotoxic concentrations. A combination of BAPA with the neuraminidase (NA) inhibitor oseltamivir carboxylate efficiently blocked influenza virus replication in airway epithelial cells at remarkable lower concentrations for each compound than treatment with either inhibitor alone. Our studies provide a novel and potent approach for influenza chemotherapy that should be considered for influenza treatment.

Highlights

Protease inhibitors provide novel drugs for influenza A and B virus treatment.► Combination chemotherapy using protease inhibitors and neuraminidase inhibitors.► Host factor-directed drugs for influenza treatment.

Introduction

Influenza (flu) is a highly contagious acute infection of the respiratory tract that affects millions of people during annual epidemics and occasional occurring pandemics. Seasonal outbreaks are caused by both influenza A and B virus strains that co-circulate with varying predominance and may give rise to severe morbidity and mortality equally. Furthermore, the emergence of a new influenza A virus for which there is little or no immunity in the human population may provoke an influenza pandemic as has been the case in 1918, 1957, 1968 and recently in 2009.

Influenza viruses are enveloped viruses and contain two spike glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). The HA mediates binding to N-acetyl-neuraminic acid containing cell surface receptors and fusion of viral and endosomal membranes, in order to release the viral genome into the host cell [8]. The NA cleaves N-acetyl-neuraminic acid from carbohydrate moieties facilitating entrance and egress of the virus [8], [20].

The HA is synthesised as a fusion incompetent precursor protein HA0 that is post-translationally cleaved by a host cell protease into the disulfide-linked subunits HA1 and HA2 [9], [27]. Cleavage of HA0 is a prerequisite to undergo conformational changes at low pH in the endosome that trigger membrane fusion and, therefore, essential for viral infectivity. The amino acid sequence at the HA cleavage site varies between viral strains, and can affect tissue tropism, spread and pathogenicity of the virus [9], [27]. Most avian and mammalian influenza A viruses, including the subtypes H1 and H3 that currently infect humans, and influenza B viruses, contain a single arginine (R) at the HA cleavage site and are activated by trypsin in vitro [17], [18]. Relevant trypsin-like proteases are present in a limited number of tissues such as the respiratory- and the intestinal tract, thereby restricting virus infection to these tissues. We demonstrated that the type II transmembrane proteases (TTSP) TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease) present in the human respiratory epithelium cleave influenza A virus HA at a monobasic cleavage site [2]. Recently, the TMPRSS2-related protease TMPRSS4 was shown to cleave HA with monobasic cleavage site, too [6]. In contrast, the HA of highly pathogenic avian influenza viruses of subtypes H5 and H7 is cleaved after the multibasic motif R-X-R/K-R by the subtilisin-like proteases furin and proprotein convertase 5/6 (PC5/6) [9], [14], [28]. The ubiquity of these enzymes mediates systemic infection with an often fatal outcome.

Currently available measures to control influenza in humans are vaccination and antiviral medications that target the viral ion channel protein M2 or NA. Resistance to M2 blockers (adamantanes; amantadine and rimantadine), however, develops rapidly and has limited the usefulness of these compounds. Notably, all influenza A viruses currently circulating in the human population are resistant to M2 blockers [22], [29]. Furthermore, the adamantanes lack activity against influenza B viruses. The NA inhibitors oseltamivir (oral), zanamivir (inhaled) and peramivir (intravenous) are active against both influenza A and B viruses and oseltamivir is the agent favoured for influenza treatment. Emergence of resistance to oseltamivir was reported sporadically, but in 2007 oseltamivir-resistant H1N1 viruses emerged independently of drug use and almost all seasonal H1N1 viruses that circulated since 2008–2009 were resistant against oseltamivir [12]. So far, there is little resistance in the H3N2 viruses and pandemic H1N1 2009 isolates [22], [29] however resistant viruses are likely to spread due to the ongoing use of oseltamivir. Considering the impact of influenza viruses on public health, more extensive profiling of new drug targets is of great interest. We suggest virus-activating host cell proteases as promising drug targets and the development of specific protease inhibitors seems to be a practicable approach for the treatment of viral diseases [11], [28].

We recently designed a series of substrate-analogue peptide mimetic inhibitors of HAT containing a 4-amidinobenzylamide moiety as the P1 residue and demonstrated that influenza A virus propagation in stable MDCK-HAT cells was efficiently suppressed by treatment with these inhibitors [3], [4], [24]. In the present study we investigated the potential of the peptide mimetic inhibitor benzylsulfonyl-d-arginine-proline-4-amidinobenzylamide (BAPA) to suppress influenza A and B virus infection in Calu-3 human airway epithelial cells with endogenous TMPRSS2-expression [5], [13], [25]. We show that BAPA treatment reduced viral titers 1000–10,000-fold by preventing cleavage of HA of progeny virions in Calu-3 cells. Furthermore, BAPA combined with oseltamivir carboxylate displayed highly synergistic effects and efficiently blocked influenza A virus propagation at remarkably lower concentrations than treatment with either inhibitor alone, representing a promising novel combination chemotherapy for influenza treatment.

Section snippets

Cells and viruses

Calu-3 human bronchial epithelial cells were cultured in DMEM/F-12 Ham (1:1) (Gibco) supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin and glutamine, with fresh culture medium replenished every 2–3 days. Madin–Darby canine kidney II (MDCK-II) cells and 293T human embryonic kidney cells were cultured in DMEM supplemented with 10% FCS, antibiotics and glutamine. All cell growth and incubations occurred under humified air conditions at 37 °C and 5% CO2.

The influenza viruses

Proteolytic activation of influenza B virus HA by HAT and TMPRSS2

The influenza B virus HA contains a single arginine at the cleavage site and is cleaved by trypsin in vitro [18]. Therefore, it was reasonable to assume that HAT and TMPRSS2 activate the influenza B virus HA. To test this hypothesis, 293T cells were co-transfected with pCAGGS expression plasmids encoding the HA of B/Lee/40 and either HAT, TMPRSS2 or empty pCAGGS as a control (mock). At 24 h post transfection cell lysates were subjected to SDS-PAGE and cleavage of HA was analysed by

Discussion

Current measures for the control and prevention of influenza are vaccination and antiviral therapies, which target the viral M2 protein or the NA. However, treatment options become more limited due to the development of drug resistant viruses for both M2 and NA inhibitors. Furthermore, the production of influenza vaccines against a newly emerged virus so far requires four-to-six months, highlighting the urgent need for novel and potent therapeutic approaches.

In the present study, we examined

Conflict of interest

All authors declare that no conflict of interests exists.

Acknowledgement

We are grateful to Mikhail Matrosovich for providing antibodies and viruses and Georg Herrler for providing the VSV antiserum. This work was supported by grants from the Deutsche Forschungsgemeinschaft SFB-593-TPB2 and the FAZIT-Stiftung (Y.L.).

References (32)

  • E. Böttcher-Friebertshäuser et al.

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

    J Virol

    (2011)
  • C. Chaipan et al.

    Proteolytic activation of the 1918 influenza virus hemagglutinin

    J Virol

    (2009)
  • X. Duval et al.

    Efficacy of oseltamivir–zanamivir combination compared to each monotherapy for seasonal influenza: a randomized placebo-controlled trial

    PLoS Med

    (2010)
  • W. Garten et al.

    Cleavage activation of the influenza virus hemagglutinin and its role in pathogenesis

    Avian Influenza: Monographs in Virology

    (2008)
  • E.A. Govorkova et al.

    Combination chemotherapy for influenza

    Viruses

    (2010)
  • S. Hallenberger et al.

    Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160

    Nature

    (1992)
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