Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
__________________________________________________________________
Microscopy as a useful tool to study the proteolytic activation of influenza
viruses
Pawel Zmora and Stefan Pöhlmann*
Infection Biology Unit, German Primate Center, Kellnerweg 4, 37075 Gӧttingen, Germany
*
Corresponding author: spoehlmann@dpz.eu
The influenza virus surface protein hemagglutinin (HA) mediates the first step in the viral replication cycle, viral entry into
target cells. For this, HA binds to cellular receptors, proteins or lipids modified with α-2,3 or α-2,6 sialic acid, and
facilitates the fusion of the viral and the endosomal membrane – a process essential for infectious entry. However, the HAprotein is synthesized as an inactive precursor, HA0, and must be activated by proteolytic cleavage to acquire the capacity
to fuse membranes. Since influenza virus does not encode proteases and HA does not undergo autocatalytic activation, the
virus critically depends on host cell proteases for acquisition of infectivity and the respective enzymes are potential targets
for therapeutic intervention. The type II transmembrane serine proteases (TTSPs), in particular TMPRSS2, were shown to
activate influenza virus and other respiratory viruses in cell culture. Moreover, a recent study demonstrated that expression
of TMPRSS2 is essential for spread and pathogenesis of H1N1 influenza viruses in mice. In this review, we provide an
overview of the proteolytic activation of influenza virus, with the main focus on type II transmembrane serine proteases,
and we outline how microscopy can be used to analyze the cellular localization of HA activation.
Keywords: influenza virus; hemagglutinin; type II transmembrane serine proteases; cellular localization
1. Influenza, a major source of global morbidity and mortality
Influenza, or shortly flu, is an infectious respiratory disease, characterized by the sudden onset of fever, dry cough and
headache and/or myalgia [1]. The influenza symptoms can be confounded with the common cold, caused by e.g.
coronavirus infection, but are more severe and can entail serious complications, such as bacterial pneumonia, which can
take a fatal course. Persons at high risk for severe influenza are young children and the elderly, pregnant women and
patients with a compromised immune system [1].
A hallmark of influenza viruses is the capability to constantly change their genetic information: The continuous
acquisition of amino acid changes in proteins exposed to major immune pressure, hemagglutinin (HA) and
neuraminidase (NA), is called antigenic drift and is responsible for the annual influenza epidemics (seasonal influenza).
Seasonal influenza is believed to be responsible for 3-5 million cases of severe illness and 250,000-500,000 deaths
worldwide [1]. The economic burden associated with seasonal influenza was estimated to amount to $87.1 billion USD
in the US alone [2]. As a consequence of the antigenic drift, the formulation of vaccines has to be constantly adapted to
the circulating viral strains and vaccination has to be repeated annually in order to protect from influenza.
The exchange of entire genetic segments (reassortment) between different influenza viruses, termed antigenic shift,
can result in the emergence of novel influenza viruses, against which the human population is immunologically naïve.
As a consequence, these viruses might cause an influenza pandemic, which can have dramatic medical, social and
economic consequences. The worst influenza pandemic of the 20th century unfolded in 1918: The so called Spanish
influenza, caused by a virus of the H1N1 subtype, is believed to have killed between 30 to 50 million people [3-5].
Other influenza pandemics of the 20th century were the Asian influenza in 1957 (caused by an H2N2 virus), the Hong
Kong influenza in 1968 (caused by an H3N2 virus) and the Russian influenza in 1977 (caused by an H1N1 virus). The
first influenza pandemic of the 21st century started in spring of 2009 in Mexico [6], and within a few months the new
virus (again of the H1N1 subtype) spread around the world, causing 100,000 - 300,000 deaths [7, 8]. The 2009
pandemic virus, which continues to circulate as a seasonal influenza virus, was a reassortant of a swine, human and
avian influenza viruses, and the disease it caused was thus called ‘swine flu’.
Current influenza therapy targets two viral proteins: M2 and NA [9]. M2 inhibitors (adamantanes) prevent viral
uncoating while NA inhibitors (oseltamivir, zanamivir) block the release of progeny virions from infected cells.
Treatment is the only defense against pandemic viruses, since the annually applied vaccines are not effective against
these viruses. Unfortunately, however, the success of treatment is compromised by the emergence of resistant influenza
viruses, i.e. viruses that harbor changes in their genetic information, which render them insensitive to inhibition by the
therapeutic agents [10, 11]. Therefore, novel approaches to anti-influenza therapy are urgently required and host cell
factors essential for influenza virus spread but dispensable for cellular survival are attractive targets, since inhibition of
such factors might prevent the emergence of resistant viruses.
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2. Influenza virus – structure and life cycle
The family orthomyxoviridae contains three genera of influenza viruses: Influenza A, B and C viruses. Influenza A and
influenza B viruses are responsible for seasonal influenza but only influenza A viruses (FLUAV) cause pandemics.
FLUAV are divided into subtypes on the basis of the sequence and antigenic properties of their (HA) and (NA)
proteins: 17 HA (H1-H17) and 10 NA (NA1-NA10) are known at present [3, 12]. Apart from HA and NA, the viral
envelope, which is derived from the host cell, contains the M2 protein, an ion channel. Inside the virions, eight genomic
RNA segments are located, which are associated with the viral RNA polymerase proteins. The genomic segments are
coated with nucleoproteins and attached to the envelope via the M1 protein. They encode proteins which facilitate virus
entry, replication and release [13].
2.1 Influenza A virus proteins
The HA binds to the major viral receptor determinant, sialic acid attached to proteins or lipids on the cell membrane
[14], and facilitates viral entry into host cells [13]. The NA has the opposite function: It removes sialic acid from viral
and cellular components and thereby facilitates virus release from infected cells. Moreover, NA promotes FLUAV
penetration of the respiratory mucus and may enhance receptor binding by removing oligosaccharides from HA, which
surround the receptor-binding site [15]. The major function of M2 is the transport of protons from the endosome into
the virion interior, which is required for the release of viral ribonucleoproteins (vRNPs) during disassembly [3]. The
viral RNA-dependent RNA polymerase subunits, PA, PB1 and PB2, are responsible for transcription and replication of
the viral RNAs [13]. The nonstructural proteins NS2 and particularly NS1 are multifunctional and can suppress the
production of host protein (NS1), inhibit the interferon response (NS1) and facilitate nuclear export of viral
ribonucleoprotein complexes (NS2) [13]. The NS proteins are not included in the virions and are mainly localized
intracellularly, as shown in Fig 1A.
A
B
Fig. 1 Localization of the influenza virus proteins NS1 and HA. (A) MDCK cells were infected with a FLUAV A/PR/8/34 mutant
encoding for a NS1 protein fused to red fluorescent protein (RFP) at an MOI 1.0. At 24 h post infection, the cells were fixed with icecold methanol and the NS1-RFP localization was detected by confocal laser scanning microscopy. (B) MDCK cells were infected
with FLUAV A/PR/8/34 wt at an MOI 1.0 and analyzed as described for (A). However, the localization of influenza HA was
examined by immunostaining with rabbit-anti-HA and FITC-conjugated anti-rabbit antibody. Scale bar = 50 µm.
2.2 Influenza A virus replication cycle
In the first step of FLUAV infection, the virus binds via HA to α-2,3- or α-2,6-sialic acids on the host cell surface [16,
17]. After binding to the receptor, virions are internalized via multiple endocytic pathways, including clathrin- and nonclathrin-dependent pathways, which have been visualized at single viral particle level with confocal microscopy [16, 18,
19]. Upon transport of virions into endosomes, the acidic environment triggers conformational changes in HA, which
facilitate the fusion of the viral and the endosomal membrane, as discussed below. Membrane fusion allows the release
of viral RNAs into the cellular lumen and their subsequent transport into the nucleus, where viral genome replication
and transcription proceeds. Thereafter, viral ribonucleoprotein complexes (vRNP) are transported into the cytoplasm in
an NS2-dependent fashion [20]. In the cytoplasm, the viral genetic segments seem to associate in a microtubuleindependent manner via Rab11-positive organelles [21]. M1 and NS2 finally promote transport of aggregated vRNPs to
the plasma membrane, the site of viral budding [22].
Viral messenger RNAs are translated at free ribosomes in the cytoplasm, with exception of those coding for the
surface glycoproteins, i.e. HA, NA and M2. These proteins are synthesized in the endoplasmic reticulum, processed in
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the Golgi apparatus and transported to the cell membrane, where progeny virions bud [22]. Within the membrane, the
clustering of HA and NA in lipid rafts may commence the budding process [22]. Therefore, the influenza virus HA and
NA can be detected in the cellular membrane by microscopic inspection (Fig 1B). Subsequently, M1 binds to the
cytoplasmic tails of the viral surface proteins and thereby connects them to the vRNPs [23]. Finally, M2 facilitates
membrane scission and release of the budding virions from the infected cells [24].
3. Hemagglutinin facilitates viral entry into target cells
The HA protein facilitates viral entry into target cells. HA is a type I transmembrane protein with an N-terminal
globular head domain, HA1, and a C-terminal stem domain, HA2 [25]. The two main functions of HA, receptor binding
and membrane fusion, are divided between theses domains: HA1 binds to cellular receptors and HA2 drives fusion of
the viral and the endosomal membrane. The receptor binding site in HA1 is located in the membrane distal tip and is
formed by the 190 helix, the 130 loop and the 220 loop, which constitute the three sides of a pocket [26, 27]. The
interaction between HA1 and sialic acid is mediated by extensive hydrogen bounding and van der Waals interaction.
The HA proteins of human FLUAV preferentially bind to α-(2,6)-linked sialic acid while the HA proteins of avian
FLUAV recognize α-(2,3)-linked sialic acid [26, 27]. Abundant expression of α-(2,6)-linked sialic acid in the human
lung and upper respiratory tracts has been documented, while mainly α-(2,3)-linked sialic acid is expressed in the avian
aerodigestive tract. Expression of α-(2,3)-linked sialic acid in human respiratory tissue is confined to the lower
respiratory tract, forcing avian viruses to change their receptor specificity for efficient spread between humans [28].
Finally, both types of sialic acids are present in the upper part of the porcine respiratory system, allowing simultaneous
spread of avian and human influenza viruses in these tissues, which may promote the emergence of new, potentially
pandemic, subtypes of FLUAV.
Upon successful receptor engagement and endosomal uptake, the membrane fusion machinery is activated by the
endosomal acidic pH. The membrane fusion reaction commences with the insertion of a fusion peptide, located at the
N-terminus of HA2, into the target cell membrane [29]. At this stage, HA2 is connected with the target cell membrane
via its fusion peptide and with the viral membrane via its transmembrane domain. Subsequently, the HA2 subunit
collapses in an umbrella-like fashion, thereby bringing the fusion peptide and the membrane anchor into close
proximity. As a consequence, the cellular and viral membranes are pulled together and ultimately fuse [29].
4. Hemagglutinin activation by host cell proteases is essential for viral infectivity
The HA-protein is synthesized as a precursor termed HA0. HA0, despite proper folding, glycosylation and trimerization
is unable to mediate membrane fusion. To acquire its fusogenic potential, HA0 needs to be cleaved by a host cell
protease. The cleavage occurs in a linker sequence between HA1 and HA2, which is localized on a partially surface
exposed loop [27, 30]. The cleavage generates the mature HA1 and HA2 subunits, which remain covalently linked via a
disulfide bond. The N-terminus of mature HA2 contains the fusion peptide, a functional element that plays a key role in
the membrane fusion reaction [25, 29], as discussed above. Although the processing of HA causes minor structural
alterations – the fusion peptide inserts into a negatively charged pocket – it leaves the HA molecule in a metastable,
membrane fusion-competent state. Importantly, proteolytic processing of HA0 by host cell protease is indispensable for
viral infectivity and the responsible enzymes are potential targets for antiviral intervention.
The HA cleavage site is a determinant of the virulence of avian influenza viruses: The HA-proteins of highly
pathogenic avian influenza viruses (HPAIV) contain a multibasic cleavage site, composed of multiple arginine and
lysine residues, which are processed by ubiquitously expressed subtilisin-like endoproteases [31]. As a consequence, the
viruses are efficiently activated in diverse target cells and can replicate systemically, thereby causing fatal disease. In
contrast, low pathogenic avian influenza viruses (LPAIV) possess HA-proteins with a single arginine or, rarely, a single
lysine residue at the cleavage site, which is termed monobasic. It has been posited that monobasic cleavage sites are
recognized exclusively by proteases expressed in the respiratory and gastrointestinal tracts of birds, which limits viral
replication to these organs and prevents induction of severe disease [32, 33].
Human FLUAV like LPAIV contain a monobasic cleavage sites but the proteases responsible for the activation of
these viruses remained unclear for years. Multiple studies employing mainly recombinant enzymes suggested that
human FLUAV are activated by secreted proteases in the lung and several candidates, e.g. mini-plasmin and clara cell
tryptase, have been described [34-36]. However, pioneering work by Zhirnov and colleagues demonstrated that cellassociated serine proteases activate human FLUAV in cultured respiratory epithelium [37]. These results raised
questions regarding the nature of the responsible enzymes. A landmark study by Böttcher and colleagues suggested that
the type II transmembrane serine proteases (TTSPs), TMPRSS2 and HAT, are attractive candidates, which can activate
all influenza virus subtypes previously pandemic in humans [38].
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5. Type II transmembrane serine proteases: Domain organization and function
Type II transmembrane serine proteases (TTSPs), which comprise four subfamilies, HAT/DESC, hepsin/TMPRSS,
matriptase and corin, exhibit a complex domain organization: The N-terminal cytoplasmic domain is followed by a
transmembrane domain, an extracellular stem region and a C-terminal catalytic domain [39, 40]. The transmembrane
domain anchors the proteins in the plasma membrane and the stem region may promote targeting of TTSPs to specific
membrane microdomains [41]. TTSPs are differentially distributed to apical or basolateral surfaces of polarized cells in
patterns unique for each protease and mislocalization of TTSPs is frequently observed in tumors [42]. The stem region
exhibits a modular organization and can contain combinations of six types of protein domains, including LDLA (lowdensity lipoprotein receptor domain class A), CUB (Cls/Clr urchin embryonic growth factor and bone morphogenic
protein-1 domain) and SR (scavenger receptor cysteine-rich domain). It serves as a regulatory and/or interaction domain
and contributes to the cellular localization, activation, inhibition and/or substrate specificity of TTSPs [43]. The
catalytic domain is characterized by a highly conserved catalytic triad consisting of a serine, aspartate and histidine
residue [39, 40]. The structure of this domain accounts for the catalytic properties and substrate specificities of TTSPs.
TTSPs, due to their localization at the plasma membrane (Fig. 2), seem to be an important component of the cellular
machinery in charge of activation of precursor molecules in the pericellular microenvironment. Substrates of TTSPs
include hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules as well as viral
glycoproteins [39, 40]. Moreover, TTSPs play a role in development and homeostasis [39-41]. For instance,
enteropeptidase, a TTSP expressed on the brush-border membrane of the duodenum, contributes to the digestion of
dietary proteins [44]. The enzyme converts pancreatic trypsinogen into trypsin, which processes food proteins and
activates digestive enzymes in the small intestine. Corin, another TTSP, regulates blood pressure by converting proANP into active ANP and modulates cardiac function by promoting naturiesis, dieresis and vasodilation [45, 46].
Finally, TMPRSS3 and hepsin are required for normal hearing [47, 48].
A
B
Fig. 2 Localization of the type II transmembrane serine proteases TMPRSS2 (A) and HAT (B). MDCK cells were transfected with
plasmids encoding TMPRSS2 and HAT, equipped with an N-terminal myc antigenic tag. At 48 h post transfection, the cells were
fixed with ice-cold methanol and proteases were detected with mouse-anti-myc primary antibody and a RedX-conjugated anti-mouse
secondary antibody. Scale bar = 50 µm.
6. TMPRSS2: A novel target for therapeutic intervention
After the identification of TMPRSS2 and HAT as FLUAV activating proteases [38], the TTSPs TMPRSS4, matriptase
and MSPL were also shown to cleave and activate HA in cell culture [49-53]. This finding, jointly with the observation
that several secreted proteases can activate HA [34-36], raised the question which of these enzymes contributes to
FLUAV activation in the infected host. Recent research indicates a key role of TMPRSS2: Endogenous TMPRSS2 was
shown to facilitate FLUAV spread in Caco-2 [54] and Calu-3 cells [55] irrespective of the addition of exogenous
trypsin. Moreover, TMPRSS2 and 2,6-linked sialic acid were found to be coexpressed in large parts of the human
aerodigestive tract [56]. Finally, a landmark study by Hatesuer and colleagues showed that TMPRSS2 is essential for
spread and pathogenesis of H1N1 in mice [57]. Evidence for absence of HA processing in TMPRSS2 knockout mice
was obtained, indicating that lack of viral spread in these animals was due to lack of HA activation. In contrast, a H3N2
virus was less dependent on TMPRSS2 for viral amplification [57], suggesting that viruses of this subtype might be able
to employ other enzymes, potentially members of the TTSP family, to ensure their activation. Two subsequent studies
confirmed these results [58, 59] although one reported that also the spread of an H3N2 virus was dependent on
TMPRSS2 [58] and the protease requirements of viruses of theses subtypes warrant further investigation. Finally,
TMPRSS2 was shown to activate the spike proteins of several coronaviruses, including the highly pathogenic SARS[60-62] and MERS-coronavirus [63-65] as well as surface glycoproteins of parainfluenza virus [66] and human
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metapneumovirus [67]. These observations, jointly with the finding that TMPRSS2 knockout is not associated with an
obvious phenotype in the absence of infection [68] suggest that TMPRSS2 is a prime target for antiviral intervention.
7. Determining the cellular localization of hemagglutinin activation
The identification of the cellular location were HA is activated by TMPRSS2 and related enzymes will provide
important insights into the biology of influenza virus infection and will instruct efforts to generate effective inhibitors.
One study suggested that TMPRSS2 and related enzymes may not be able to activate HA during viral entry into target
cells [54], indicating that HA cleavage and activation occurs during HA transport through the constitutive secretory
pathway of infected cells or upon insertion of HA into the plasma membrane. However, Zhirnov and colleagues
obtained evidence for FLUAV activation during binding and uptake into cultured respiratory epithelium [37] and the
contribution of TTSPs to this process requires further analyses. In addition, studies are needed to determine precisely
where in the infected cell TTSPs activate HA. One report indicates that different TTSPs might activate HA at different
locations: Evidence was provided that HAT may cleave newly synthesized HA before or during the release of progeny
virions at the cell surface, while TMPRSS2 processes HA inside the cell [69]. Finally, our unpublished work suggests
that HA-activating TTSPs but not inactivate enzymes colocalize with HA (Fig. 3), suggesting that the appropriate
cellular localization of TTSPs might even be a determinant of HA activation.
Fig. 3 Colocalization of the type II transmembrane serine protease TMPRSS4 and influenza hemagglutinin in infected cells.
MDCK cells were transfected with a plasmid encoding TMPRSS4 with an N-terminal myc antigenic tag. Subsequently, the cells
were infected with influenza A virus A/PR/8/34 (H1N1) at an MOI of 1.0. At 24 h post infection, the cells were fixed with ice-cold
methanol and expression of influenza A virus HA (A, depicted in green) and TMPRSS4 (B, depicted in red) were detected by
immunostaining with antibodies described in Fig. 1B and 2. The merged picture (C) was obtained with ImageJ software. White
squares indicate examples of colocalization of HA and TMPRSS4 (yellow), which were 1.5 x magnified. Scale bar = 50 µm.
8. Conclusions
The proteolytic cleavage of HA by TMPRSS2 is essential for spread and pathogenesis of H1N1 viruses in mice and
TMPRSS2 and potentially also related proteases constitute targets for antiviral intervention. Current research efforts
seek to determine the cellular localization of HA cleavage. Their results should provide important insights into FLUAV
interactions with host cells and should advance inhibitor development. Immunostaining of infected cells and their
analyses by state-of-the art microscopy will be an integral component of these research endeavours.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (PO 716/6-1), the Leibniz Graduate
School Emerging Infectious Diseases and the Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences
(DFG Grant GSC 226/1 and DFG Grant GSC 226/2).
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