Inhibiting Human Parainfluenza Virus Infection by
Preactivating the Cell Entry Mechanism
S. F. Bottom-Tanzer,a,b K. Rybkina,a,b J. N. Bell,a,b C. A. Alabi,c C. Mathieu,a,b,d M. Lu,e S. Biswas,a,b,c M. Vasquez,f
M. Porotto,a,b,g J. A. Melero,f V. Más,f A. Mosconaa,b,h,i
aDepartment of Pediatrics, Columbia University Medical Center, New York, New York, USA
bCenter for Host-Pathogen Interaction, Columbia University Medical Center, New York, New York, USA
cRobert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA
dCIRI, International Center for Infectiology Research, Immunobiology of Viral Infections Team, INSERM U1111, University Claude Bernard Lyon 1, CNRS, UMR5308, Ecole
Normale Supérieure de Lyon, Lyon, France
eDepartment of Microbiology, Biochemistry and Molecular Genetics, Rutgers Biomedical and Health Sciences, Public Health Research Institute, Newark, New Jersey, USA
fCentro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain
gDepartment of Experimental Medicine, University of Campania Luigi Vanvitelli, Naples, Italy
hDepartment of Microbiology & Immunology, Columbia University Medical Center, New York, New York, USA
iDepartment of Physiology & Cellular Biophysics, Columbia University Medical Center, New York, New York, USA
ABSTRACT Paramyxoviruses, specifically, the childhood pathogen human parainflu-
enza virus type 3, are internalized into host cells following fusion between the viral
and target cell membranes. The receptor binding protein, hemagglutinin (HA)-
neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into
the cell through a coordinated process involving HN activation by receptor binding,
which triggers conformational changes in the F protein to activate it to reach its
fusion-competent state. Interfering with this process through premature activation of
the F protein has been shown to be an effective antiviral strategy in vitro. Conforma-
tional changes in the F protein leading to adoption of the postfusion form of the
protein—prior to receptor engagement of HN at the host cell membrane—render
the virus noninfectious. We previously identified a small compound (CSC11) that im-
plements this antiviral strategy through an interaction with HN, causing HN to acti-
vate F in an untimely process. To assess the functionality of such compounds, it is
necessary to verify that the postfusion state of F has been achieved. As demon-
strated by Melero and colleagues, soluble forms of the recombinant postfusion
pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to
generate postfusion-specific antibodies. We produced novel anti-HPIV3 F
conformation-specific antibodies that can be used to assess the functionality of com-
pounds designed to induce F activation. In this study, using systematic chemical
modifications of CSC11, we synthesized a more potent derivative of this compound,
CM9. Much like CSC11, CM9 causes premature triggering of the F protein through
an interaction with HN prior to receptor engagement, thereby preventing fusion and
subsequent infection. In addition to validating the potency of CM9 using plaque re-
duction, fusion inhibition, and binding avidity assays, we confirmed the transition to
a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3
conformation-specific antibodies. We present both CM9 and these newly character-
ized postfusion antibodies as novel tools to explore and develop antiviral ap-
proaches. In turn, these advances in both our molecular toolset and our understand-
ing of HN-F interaction will support development of more-effective antivirals.
Combining the findings described here with our recently described physiologically
relevant ex vivo system, we have the potential to inform the development of thera-
peutics to block viral infection.
Citation Bottom-Tanzer SF, Rybkina K, Bell JN,
Alabi CA, Mathieu C, Lu M, Biswas S, Vasquez M,
Porotto M, Melero JA, Más V, Moscona A. 2019.
Inhibiting human parainfluenza virus infection
by preactivating the cell entry mechanism.
mBio 10:e02900-18. https://doi.org/10.1128/
mBio.02900-18.
Editor Stacey Schultz-Cherry, St. Jude
Children's Research Hospital
Copyright © 2019 Bottom-Tanzer et al. This is
an open-access article distributed under the
terms of the Creative Commons Attribution 4.0
International license.
Address correspondence to A. Moscona,
am939@cumc.columbia.edu.
This paper is dedicated to J. A. Melero.
This article is a direct contribution from a
Fellow of the American Academy of
Microbiology. Solicited external reviewers: Ruth
Karron, Johns Hopkins School of Public Health;
John Williams, Children's Hospital of Pittsburgh
and University of Pittsburgh Medical Center.
Received 7 January 2019
Accepted 10 January 2019
Published 19 February 2019
RESEARCH ARTICLE
Host-Microbe Biology
crossm
January/February 2019 Volume 10 Issue 1 e02900-18 ® mbio.asm.org 1
IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are in-
ternalized into host cells by fusion between viral and target cell membranes. The re-
ceptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein
(F) facilitate viral fusion and entry into cells through a process involving HN activa-
tion by receptor binding, which triggers conformational changes in F to activate it
to reach its fusion-competent state. Interfering with this process through premature
activation of the F protein may be an effective antiviral strategy in vitro. We identi-
fied and optimized small compounds that implement this antiviral strategy through
an interaction with HN, causing HN to activate F in an untimely fashion. To address
that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies
that can be used to assess the functionality of compounds designed to induce F ac-
tivation. Both the novel antiviral compounds that we present and these newly char-
acterized postfusion antibodies are novel tools for the exploration and development
of antiviral approaches.
KEYWORDS antiviral, conformational antibody, fusion activation, paramyxovirus, viral
fusion, viral glycoprotein antibody
Acute respiratory infection is the leading cause of child mortality worldwide (1, 2).More than 20% of all acute lower respiratory infections are associated with
paramyxovirus infection, and greater than 14% result in death (2). Paramyxoviruses and
pneumoviruses account for the majority of childhood croup, bronchiolitis, and pneu-
monia cases (3), with human parainfluenza virus 3 (HPIV3) infections alone resulting in
11% of childhood respiratory hospitalizations in the United States (3, 4). There are
currently no vaccines or antiviral therapies for parainfluenza viruses.
Paramyxovirus entry, including HPIV3 entry, is mediated by fusion of the viral and
target host cell membranes at the cell surface. Virus-cell fusion results from coordinated
action of the two envelope glycoproteins that comprise the viral entry machinery—a
receptor binding protein, hemagglutinin neuraminidase (HN), and a fusion protein (F).
Upon binding to sialic acid-containing target receptors, HN, a molecule with both
receptor binding and cleaving activities, triggers and activates the F protein (5). Once
F is activated, the hydrophobic fusion peptide inserts into the target host membrane
and undergoes a series of structural rearrangements leading to association between
heptad repeats (HR) at the C terminus and N terminus of the molecule (HRC and HRN,
respectively) and subsequent fusion between the viral and cellular membranes (6).
The process of viral fusion and the extent to which it occurs are mediated by the
various functions of HN and F. HN moderates receptor binding and cleavage, as well as
stabilization and activation of the F protein. HN, a type II transmembrane protein, gives
rise to these functions via coordination between its cytoplasmic domain, membrane-
spanning region, stalk region, and a globular head, which contains the primary sialic
acid binding site and neuraminidase active site, as well as a second sialic acid binding
site that modulates activation of F. F influences the extent of fusion through its
prefusion stability, kinetics of activation, and precursor cleavability. Fusion is moderated
through a balance of these functions, with timing also playing an essential role.
Activation and the subsequent conformational change of the F protein must occur
when F is in contact with the target cell membrane. If F assumes its elongated
intermediate state or its postfusion state prematurely, the virus is rendered noninfec-
tious. This sequence of events presents a possible antiviral target (3). We sought to
identify molecules that irreversibly inactivate the viral fusion complex by stimulating
HN to trigger F prior to receptor engagement. In a computational screen for small
molecules that bind to the globular head domain of HN, we identified a compound that
inhibited entry of a laboratory-adapted strain of HPIV3 into target monolayer cells and
prevented infection. This compound interacts with HN prior to receptor engagement,
thereby causing premature F activation and precluding viral entry (7). The prototype
molecule (“CSC11”) interacts with HN without altering receptor binding avidity or
receptor cleaving (neuraminidase) activity. The compound showed virucidal activity
Bottom-Tanzer et al. ®
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and an antiviral effect on laboratory-adapted HPIV3 in both cultured monolayer cells
and human airway epithelium (HAE) cells (7).
The prototype compound provided proof of concept for the strategy of inducing
HPIV to self-inactivate and points to the possibility of developing a new class of
antivirals (7–10). We carried out systematic chemical modifications of each structural
group in the CSC11 compound and assessed the efficacy of the derived compounds on
the infectivity and other key properties of clinical isolate (field strain) viruses in
authentic host tissues. We have also assessed the functions of receptor binding,
receptor cleaving, and fusion as well as viral entry in the presence of these compounds
and identified a modification that improves the antiviral effect (activity) without
modulating receptor binding or cleaving activity. To further assess the functionality of
these compounds in vitro, it is necessary to verify that the postfusion state of F has been
achieved. As demonstrated previously by Melero and colleagues (11, 12), genetically
engineered recombinant postfusion F proteins can be used to generate postfusion
antibodies (Abs). To assess the functionality and mechanism of action of these com-
pounds, we produced novel anti-HPIV3 postfusion antibodies (11, 12). The results
presented here demonstrate the ability to make use of F protein pretriggering as an
antiviral strategy and as a way to engineer a more potent antiviral. Here, we identified
the mechanism of action of this new class of antiviral compound by analyzing the effect
of these molecules on the functions and structures of HPIV3 surface glycoproteins.
RESULTS
Effect of chemical modification of prototype compound on antiviral activity.
The structures of CSC11 and of two selected derivatives that were targeted in a series
of chemical modifications to identify the functional groups responsible for stimulating
HN to activate F are depicted in Fig. 1A. We determined whether modification of each
CSC11 functional group—the thiophene R1, the hydroxyphenol R2, and the triazole R3
(see Fig. S1 in the supplemental material)—alone or in combination could preserve
antiviral function and/or affect other parameters involved in the interaction between
the small molecule and HN. Thirty-three molecules were synthesized and assessed for
efficacy at inhibiting viral entry, as measured by plaque formation, for both laboratory-
adapted strains and clinical isolate HPIV3. All 33 new compounds inhibited HPIV3
infectivity in a plaque reduction assay to various degrees, but among these, only 2
showed improved inhibitory efficacy over our prototype CSC11. The modified mole-
FIG 1 Strategy of synthesis of new inhibitors of viral entry and comparison of newly synthesized
inhibitors of viral entry with CSC11. (A) Chemical structure of CSC11 compound. Each group—thiophene,
hydroxyphenol, or triazole (alone or in combination)—has been modified to increase activity, resulting
in structures of variations of the following CSC11 compounds shown: CM9 and CM28. (B) Comparison of
properties of CSC11-derived compounds. CV1 cells grown in a monolayer culture were infected in the
presence of increasing concentrations of the compounds. Viral entry was assessed by plaque reduction
assay. Values corresponding to 50% inhibitory concentrations (IC50) and IC90 were then calculated for
inhibition of entry by HPIV3 laboratory-adapted reference strain and HPIV3 clinical strain viruses. The
values are representative of data from three experiments.
Inhibiting HPIV by Preactivating Cell Entry Mechanism ®
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cules that preserved antiviral efficacy against clinical isolate viruses were chosen for
further analysis. As shown in Fig. 1B, the concentrations at which 50% and 90%
inhibition of viral entry was achieved for CSC11 and the following two newly synthe-
sized modified compounds: CM9, where the 1,2,4-triazole group on CSC11 was replaced
with a nitrile group, and CM28, where the thiophene, 1,2,4-triazole, and hydroxyl
groups of CSC11 were replaced with a 2,3-dichlorothiophene group, a nitrile group, and
a methoxy group, respectively.
CSC11 and CM9 do not block HN-receptor interaction for laboratory-adapted
strains or clinical isolate HPIV3. We assessed the effect of the two new compounds
(CM9 and CM28) compared to CSC11 on HPIV3-HN receptor binding in a hemadsorp-
tion (HAD) assay that quantitates the binding of sialic acid receptor-bearing erythro-
cytes (red blood cells; RBC) to HN/F coexpressing cells. For comparison, we used
zanamivir (4-GU-DANA), which inhibits HN-receptor binding (13). CSC11 and CM9 (in
contrast to zanamivir) did not affect receptor binding for either laboratory-adapted or
clinical strain HNs (Fig. 2A and B), confirming that the inhibitory mechanism does not
involve inhibition of receptor binding. In contrast, CM28 partially inhibited binding and
therefore was not assessed further. We next assessed CM9 and CSC11 for their effect on
neuraminidase activity. The compounds had no inhibitory effect on viral neuraminidase
activity (Fig. 2C and D), indicating that CM9 does not interfere with HN’s primary
receptor binding/cleaving site (site I) for either strain of HPIV3.
CSC11 and CM9 exert a virucidal effect on clinical strain viruses. We next asked
whether inhibition of viral entry is attributable to a direct and temperature-dependent
virucidal effect prior to virus-target cell interaction, in line with our hypothesis that the
compounds stimulate HN to trigger F at 37°C. Virions were incubated with the com-
pounds at 37°C or 4°C for 60 min, and, after removal of the compounds, the infectivity
of the treated virions was assessed by plaque reduction assay. Pretreatment of the virus
with CSC11 and CM9 (but not with zanamivir) at 37°C had an irreversible effect on
infectivity in both the laboratory and clinical strains (Fig. 3A). Despite removal of these
compounds prior to the assay, viral entry was reduced by virtually 100% by the
presence of CM9 for both viruses. Pretreatment at 4°C did not significantly inactivate
FIG 2 Small-molecule activity effects on HN-receptor interaction and neuraminidase activity. (A to B)
Sensitivity of HN receptor binding to increasing concentrations of viral entry inhibitors (white bars,
zanamivir; striped bars, CM28; gray bars, CSC11; black bars, CM9) was quantified by hemadsorption at 4°C
on cells expressing HPIV3 HN from a laboratory-adapted reference strain (A) or a clinical isolate strain (B).
The results are shown as absorbance at 405 nm reflecting the binding of red blood cells (RBC) (y axis) as
a function of test compound concentration (x axis). Each point represents the mean of results from 3
experiments ( standard deviations [SD]), each of which was performed in triplicate. (C and D) Relative
neuraminidase activity in the presence or absence of the indicated compounds (x axes) was assayed at
37°C and pH 5 on cell monolayers transiently expressing HN from a clinical strain (C) or a laboratory-
adapted strain (D). Each bar represents results of triplicate experiments  standard deviations; data are
expressed as relative fluorescence units (RFU)/s.
Bottom-Tanzer et al. ®
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either virus (data not shown), consistent with the hypothesis that these compounds
affect F-triggering, which cannot occur at this temperature. To assess particle integrity,
we quantitated viral RNA in infectious and noninfectious preparations as we did
previously for CSC11 (7). The viral RNA levels in samples pretreated with CM9 were
similar to those in samples treated with either dimethyl sulfoxide (DMSO) or zanamivir
(data not shown), indicating that the decreased infectivity was due to viral inactivation
and not to a loss of viral particle integrity.
CM9 inhibits viral infection in human airway epithelium. For clinical utility, an
effective agent must limit the viral life cycle in authentic tissue. We established
previously that a human airway epithelium (HAE) model closely reflects the in vivo
behavior of a panel of HPIV3 variants (9, 14), and we employed this system to test a
sialidase-based inhibitor, showing a direct correlation between the HAE and in vivo
results (15). The HAE model was previously used to characterize the cell specificity of
respiratory syncytial virus (16) and HPIV3 (17), studies that confirmed that the model
replicates the paramyxovirus-HAE interactions occurring in the human lung. We as-
sessed the effect of CM9 compared to that of CSC11 on viral infection in HAE (Fig. 3B).
HAE were infected with the HPIV3 clinical isolate in the presence of CM9, CSC11, or
zanamivir (which, as described above, inhibits by interfering with HN-receptor binding).
After 90 min at 37°C, the inoculum was removed and the HAE cells were incubated at
37°C. The virus released from the apical surface was collected 3 days postinfection. The
percentages of reduction in viral titer compared to the results seen with untreated
infections are shown on the y axis. The viral titer in CM9-treated tissues was reduced by
80%, while the viral titer in those treated with CSC11 was reduced by approximately
55%. Zanamivir, since it blocks HN-receptor binding, is also effective via this alternative
mechanism. These results support the idea of the efficacy of CM9 in the natural host
and will lead to future experiments with more-potent analogs of this compound in an
animal model.
Evaluation of mechanism of action of CM9: development of conformation-
specific anti-F antibodies. To directly determine whether the F protein achieves a
triggered conformation as a result of compound treatment, we developed a set of
antibodies to detect this state of F. Soluble F HRN and HRC regions were linked to form
a protein that adopts a six-helix bundle (6HB) conformation. This structure mimics the
postfusion form of the F protein that arises after the HRN and HRC regions have
become associated (see Fig. 4A). Following a strategy similar to that used for human
pneumovirus F proteins, the immunization with the 6HB peptide allowed the isolation
of three specific monoclonal antibodies against the postfusion F conformation. This
specificity was observed by antibody binding to a purified postfusion soluble HPIV3 F
and to the 6HB peptide but not to either HRC or HRN alone (see Fig. 4B). Stabilization
FIG 3 Assessment of virucidal action and ex vivo efficacy. (A) Aliquots of HPIV3 laboratory-adapted
reference strain (left) or clinical strain (right) viral preparations were incubated with 3 mM solutions of the
indicated compounds for 60 min at 37°C or left untreated. The compounds were removed using filtration
columns, and the resulting viral infectivity was determined by plaque reduction assay. The effect on
infectivity of the pretreatment with the compounds is shown as percent inhibition of the number of
plaques compared to the number without preincubation with compounds. (C) Human airway epithelia
(HAE) were infected with 4,000 PFU of the HIPV3 clinical strain in the presence of 3 mM CM9, CSC11, or
zanamivir. After 90 min of incubation at 37°C, the inoculum was removed and the HAE were incubated
at 37°C. Virus released from the apical surface was collected at 3 days postinfection. The percent
reduction of the viral titer compared to results from untreated samples is shown on the y axis.
Inhibiting HPIV by Preactivating Cell Entry Mechanism ®
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of the F construct used (see Fig. 4A) in its postfusion conformation was confirmed by
electron microscopy (EM) after observation of cone-shaped molecules resembling
previously described HPIV3 F trimers (see Fig. S2). Among the three antibodies isolated,
the PA3/F3 demonstrated the best dose-response for both the soluble F and the 6HB.
Specificity toward HPIV3-triggered F protein was further confirmed via cell-based
enzyme-linked immunosorbent assay (ELISA) using heat-activated F protein (Fig. 5),
adapting a cell-based ELISA that we had used previously (12). HEK 293T cells expressing
either HPIV3 (clinical strain) or measles F proteins (as a control for specificity) were
incubated at 55°C for the indicated times. We and others have previously shown for
measles F protein that at this temperature, the thermal energy induces the F protein to
adopt a posttriggered state (18, 19), making this a good control. After incubating the
three monoclonal antibodies (MAbs) (1:1,000 dilution) with the cells at 4°C for 1 h,
antibody binding was quantified using a -galactosidase (-Gal) reporter system (19).
Maximal recognition of triggered HPIV3 F occurred at 60 min at 55°C; the antibodies, as
expected, did not recognize measles F-expressing cells (these were detected by anti-
measles F conformational antibodies; data not shown). The most sensitive of the three
antibodies, PA3/F3, was used to further explore the mechanism of action of CM9 and
CSC11.
FIG 4 Generation and analysis of monoclonal antibodies specific for the postfusion conformation of
HPIV3 F. (A) Scheme of the constructs of hPIV3 F used in this study for isolation of postfusion-specific
monoclonal antibodies. The F protein scheme shows the last amino acid of its ectodomain (493), the
fusion peptide (gray), the cleavage site (arrow), the C-terminal 6His tag (dark gray), the foldon sequence
(dark red), the signal peptide (blue), and the HRC and HRN domains. The sequence used for the 6HB is
shown at the bottom. (B) Proteins were tested for binding in ELISA to the MAbs indicated in each panel.
Results are shown as abundance at 492 nm on the y axis.
FIG 5 Triggering of HPIV3 F protein at 55°C: identification of the activated state of F. 293T cells
expressing HPIV3 F protein (or measles F protein) were incubated overnight at 37°C. The cells were then
incubated at 55°C for the indicated times and then stained with mouse MAbs recognizing the triggered
conformation of HPIV3 F at 4°C. The values on the y axis represent the amounts of conformational
antibody binding in relative luminescent units (RLU) and indicate the averages  standard errors of the
means (SEM) of results from three independent experiments performed in triplicate.
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CM9 acts by causing untimely structural rearrangement of the F protein. To
determine whether CM9 causes premature activation of F, we used the antibodies
developed as described above as well as a biochemical assay that we previously used
to detect conformational change in F upon fusion activation (7). For the biochemical
assay, we took advantage of the relative resistance of the native (prefusion state) F
protein to protease K degradation. The posttriggered F is sensitive to degradation (7,
20, 21); receptor engagement by HN causes F to become sensitive to protease degra-
dation, and conversion to protease sensitivity serves as a marker for F activation. CM9
treatment caused F to become protease sensitive (Fig. 6A), indicating that it has been
activated. Cells expressing HN and F were preincubated with or without receptor
engagement, and with or without CSC11 or CM9, and were then transferred to 37°C for
an hour to allow fusion to occur. The HN/F complexes were immunoprecipitated and
subjected to proteinase K digestion and Western blot analysis. When receptor engage-
ment was permitted in the absence of any compound (first lane of each quadruplet),
the F protein was susceptible to protease degradation and was increasingly degraded
by the higher concentrations of protease. In the absence of receptor engagement
(blocked by zanamivir; second lane of each quadruplet) the levels of F protein remained
nearly constant, indicating that before receptor engagement, F was protected from
proteinase K degradation. In the presence of CSC11 or CM9 (third and fourth lanes of
each quadruplet), the F protein became susceptible to protease digestion and was
degraded as fully as it had been when it was activated by HN-receptor engagement.
Note that in the samples treated with CSC11 or CM9, zanamivir was present to block
HN-receptor engagement (in the second lanes), indicating that it is solely the interac-
tion with CSC11 or CM9 that accounts for the conversion to protease susceptibility. The
observed conformational change in F induced by CSC11 or CM9 required the presence
of the receptor-binding protein, HN.
Using the experimental conditions described for Fig. 6A, we assessed whether
incubation with CSC11 or CM9 induced exposure of the postfusion epitope recognized
by MAb PA3/F3. Cells cotransfected with HN and F, along with CM9 or CSC11, were
incubated with zanamivir to prevent receptor engagement at 4°C and 37°C. After
incubation, transfected cells were stained with a monoclonal postfusion antibody
(PA3/F30; postfusion, F was quantitated using high content image analysis based on
FIG 6 CM9 and CSC11 trigger formation of the postfusion conformation detected by limited proteolysis
(A) or conformational monoclonal antibodies (B). (A) Monolayers of cells coexpressing F and HN were
incubated for 60 min in medium supplemented with 3 mM CM9 or CSC11 and/or 2 mM zanamivir. The
cells were lysed, and the envelope glycoproteins were immunoprecipitated and then incubated in the
presence of increasing concentrations (0, 1, and 2) of proteinase K. Representative Western blots
show F proteolysis detected by polyclonal anti-F HRC antibodies. F0 (uncleaved) and F1 (cleaved)
proteins are indicated with arrows. After receptor engagement by HN (first lane in each quadruplet) or
CSC11 or CM9 treatment (third and fourth lanes in each quadruplet), F becomes susceptible to protease
degradation. When receptor engagement is blocked by zanamivir and no other compound is present, F
is not degraded (second lane in each quadruplet). (B) Cells cotransfected with HN and F or transfected
with F only were incubated in the presence of zanamivir alone, zanamivir and CM9, or zanamivir and
CSC11 and stained using PA3/F3 monoclonal antibodies. The proportions of positively stained cells were
determined using Cell Profiler (20,000 cells were read for each condition from biological triplicates) and
are shown as percentages of cells expressing posttriggered F on the y axis ( SD).
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cell surface fluorescent staining. In the presence of CM9 and CSC11 at 37°C (and of
zanamivir [to prevent HN-mediated F activation]), the F protein was converted to its
posttriggered form. Recognition of the postfusion epitope occurred only in CM9- or
CSC11-treated cells (Fig. 6B), demonstrating the ability of CM9 and CSC11 to induce the
posttriggered conformation of F protein in the presence of HN, without receptor
binding. We quantified the fold increase in the abundance of the posttriggered epitope
compared to level seen with the same treatment at 4°C (see Table 1). Taken together,
the results from these two methods—limited proteolysis and posttriggered epitope
recognition—show that the small molecules promoted the posttriggered state of HPIV3
F in the absence of receptor engagement.
DISCUSSION
Paramyxovirus infection begins when the receptor binding protein engages the
cellular receptor and, through a coordinated series of events, allows activation of the
fusion protein and successful viral entry. Dysregulation of this process—whether by
small-molecule antivirals (7) or by enhanced HN-F interaction—is detrimental to infec-
tivity in the natural host. This is underscored by the differences that have recently
become apparent between clinical isolate and laboratory-adapted strains of HPIV3 (14,
22, 23). Highly fusogenic laboratory-adapted virus is remarkably infectious under
conditions of growth on immortalized cells and yet yields low titers when used to infect
HAE cells (8, 9). However, when passaged several times on this ex vivo model, variants
evolved with decreased efficiency of fusion activation and reduced interaction between
HN and F (24), characteristic of clinical strains. Clinical strains, while they grew efficiently
in both HAE and in vivo models, failed to grow on immortalized cells, demonstrating
the profound specificity of the HN/F machinery for the authentic host (23). Even brief
culture in immortalized cells provides a selection pressure that increases fusion acti-
vation from the lower activity that is required for fitness in the lung (14). Given the
precision required for F activation both temporally and in terms of strength of inter-
action (24–26), this activation process provides an ideal target for our antiviral ap-
proach.
Targeting the fusion process of paramyxoviruses and other enveloped RNA viruses
has been considered in other contexts. Several small molecules that prevent RSV F
refolding have been identified, with one progressing to a clinical trial (27). Mechanistic
studies suggest an association between the small molecules and the F protein, with the
virus remaining infectious upon detachment of the small molecules (28). In addition to
paramyxovirus activity, broadly neutralizing antibodies (29) and small molecules (30–
32) against influenza virus target the conserved HA stem region in an effort to inhibit
pH-induced conformational changes that mediate uncoating and virus-endosome
fusion. The antiviral strategy that we propose here is conceptually different, since we
searched specifically for small molecules that irreversibly inactivated the viral fusion
machinery. One such compound was previously identified for influenza virus (32), and
its use follows a rationale similar to that behind the proposed use of low pH (33) or
aqueous arginine-based nasal sprays (34, 35) as effective influenza antivirals. Further,
cellulose acetate phthalate (CAP) elicits its antiviral activity on HIV glycoprotein gp41 in
a similar manner (36). Fusion inhibition is achieved with CAP by inducing the formation
of the terminal 6-helix bundle conformation of gp41 and has recently been proposed
in a semen-induced, intravaginally delivered anti-HIV drug (37). This suggests that
TABLE 1 Ratios between the positive cells with and without the premature-triggering
compounda
Compound Fold increase at 4oC Fold increase at 37oC
HN F  zn 0 0
HN F  zn  CSC11 0.80  0.42 4.97  1.38
HN F  zn  CM9 0.85  0.81 7.42  0.52
aP value for CSC11 0.05; P value for CM9 0.01.
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premature activation of the fusion protein as an antiviral strategy can be employed not
only for paramyxoviruses similar to HPIV3 but for other RNA viruses as well.
Targeting F activation in the way that we propose has an important added benefit.
By activating an irreversible viral process and rendering all affected virions inert, we
address the growing clinical concerns surrounding antiviral resistance. For RNA viruses
in particular, a high polymerase error rate contributes to nimble evasion of antiviral
activity (38–40), making the use of receptor analogs potentially an ineffective approach
(41, 42). In the case of influenza antivirals, for example, rapid viral evolution has
necessitated repeated development of new antiviral approaches and continued sur-
veillance, especially in children and immunocompromised populations (43, 44).
Here, we began with CSC11, a prototype compound that we previously demon-
strated pretriggers F to its postfusion conformation in the absence of receptor engage-
ment (7). Given that it reduced infectivity by approximately 60%, we sought to increase
its effectiveness by systematically modifying functional groups on the compound. After
screening the resulting compounds, we selected the most potent and further tested
them. We present a novel compound that results in a nearly 100% reduction of
infection upon preincubation with both laboratory-adapted and clinical isolate strains
of HPIV3 and an almost 40% increase in efficacy. To determine the mechanism by which
these small compounds inhibit fusion, we first used a previously developed biochem-
ical assay which exploits the relative resistance of the prefusion F state to protease K
degradation and the sensitivity of the postfusion state (7, 20, 21, 45). Indeed, CSC11 and
CM9 promoted protease sensitivity in the presence of HN, indicating the ability of the
compounds to induce the postfusion conformation of F through interaction with the
receptor binding protein. Given the complex series of conformational changes that F
adopts throughout the fusion sequence, we endeavored to determine the exact F
protein structure that CSC11 and CM9 induced and to refine further our understanding
of the mechanism as a whole. The available experimental evidence suggested that the
F protein adopted a 6HB conformation, so we developed monoclonal antibodies to
fusion proteins in this conformation. When we applied these antibodies to HN/F-
transfected cells incubated with CSC11, CM9, or zanamivir alone, we observed recog-
nition of the 6HB conformation only under the compound treatment conditions.
Additionally, we observed a significantly higher percentage of cells expressing postfu-
sion F under conditions of treatment with CM9 than with CSC11, consistent with our
initial observations of efficacy.
Compounds that activate viral fusion machinery in the absence of receptor binding
rendering virus particles uninfectious have potentially significant clinical applications.
By using our understanding of the exquisitely sensitive and specific nature of the
paramyxovirus fusion process, we have developed several compounds that exert
profound virucidal effects in both in vitro and physiologically relevant ex vivo models.
The optimized CM9 will be assessed in a small-animal model that represents HPIV3
disease, as a counterpart to the human airway model. To further elucidate the mech-
anism of action of these compounds, we developed novel monoclonal antibodies
specific for the postfusion conformation of F. Not only did they confirm our hypothesis
but they also will be invaluable for future structural studies of HPIV3 F, a protein against
which a historically limited repertoire of antibodies has been available. Now that we
have validated these antibodies as being specific for the posttriggered HPIV3 F, they
can be used in a wide range of settings to confirm F activation and can be applied in
high-throughput assays to screen for activated F. Together, these advances provide not
only the foundation for a new antiviral strategy toward RNA viruses but also the means
to further refine future studies.
MATERIALS AND METHODS
Cells and viruses. 293T (human kidney epithelial) and CV-1 (African green monkey kidney) cells were
grown in Dulbecco’s modification of Eagle’s medium (DMEM; Thermo Fisher) supplemented with 10%
fetal bovine serum and antibiotics. Stocks of laboratory-adapted HPIV3 were made in CV-1 cells from
virus that was subjected to plaque purification four times. Virus was collected 36 to 48 h postinfection
and stored at 80°C.
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January/February 2019 Volume 10 Issue 1 e02900-18 mbio.asm.org 9
Stocks of clinical strain viruses were prepared in HAE cultures. HAE cultures were infected by applying
200 l of EpiAirway growth medium containing 4,000 PFU of the HPIV3 clinical isolate to the apical
surface for 90 min at 37°C. Following incubation, the medium containing the inoculum was removed, and
cultures were grown at 37°C and fed each day with 1 ml of medium basolaterally. Three to 5 days
postinfection, viruses were harvested by adding 200 l of Opti-MEM (Thermo Fisher) apically for 30 min
at 37°C. The suspension was then collected, and viral titers were determined via plaque assay as
described previously (9).
HAE cultures. The EpiAirway AIR-100 tissue model (MatTek Corporation) consists of normal human-
derived tracheobronchial epithelial cells that have been cultured to form a pseudostratified, highly
differentiated mucociliary epithelium closely resembling that of airway epithelial tissue in vivo. Upon
receipt from the manufacturer, HAE cultures were transferred into six-well plates (containing 1.0 ml
medium per well), with the apical surface remaining exposed to air and incubated at 37°C in 5% CO2
overnight, as previously described (9).
Infection and treatment of HAE cells and measurement of viral titers from infected HAE cells.
HAE cultures were infected by applying 200 l of EpiAirway medium containing 4,000 PFU of laboratory-
adapted or clinical strain HPIV3. Viral suspensions were applied to the apical surface of HAE tissues for
a 90-minute adsorption period at 37°C in the presence or absence of 3 mM CSC11, CM9, or zanamivir.
At 90 min, the medium containing the inoculum was removed, and cultures were placed at 37°C and fed
each day with 1.0 ml medium via the basolateral surface. Viruses were harvested by adding 200 l
Opti-MEM per well to the apical surface of HAE cultures and allowed to equilibrate for 30 min at 37°C.
The suspension was then collected, and viral titers were determined as previously described (9).
Chemicals. Zanamivir (4-GU-DANA) was prepared from Relenza Rotadisks (5 mg zanamivir with
lactose). A 50 mM stock solution was prepared by dissolving each 5-mg blister capsule in 285 l
Optimem (Gibco). Alternatively, zanamivir (Acme Biosciences) was dissolved in Opti-MEM to reach a
concentration of 50 mM and stored at 80°C. CSC compound derivatives were synthesized by Charn-
wood Molecular Ltd. Stock solutions (100 mM) were prepared in DMSO. Stock solutions were stored at
20°C.
Transient expression of HPIV3 HN and F constructs. HPIV3 HN and F genes from laboratory strains
and clinical isolate viruses were cloned into pCAGGS as previously described (22). The constructs used in
the assay for F protein triggering, HN-N-Venus and F-C-CFP, were prepared as previously described (10).
Transfections were performed with Lipofectamine 2000 (Invitrogen), according to the manufacturer’s
instructions.
Plaque reduction assay. The effect of the compounds on plaque number was assessed as described
previously (7, 13, 46). Briefly, CV-1 cell monolayers were infected with 100 PFU of the indicated virus in
the presence of serial dilutions of the indicated compounds. After 90 min, the plates were overlaid with
either agarose or methylcellulose; At 24 h later, the agarose was removed, cells were fixed and
immunostained, and the plaques (or single infected cells for the clinical strain) were counted.
Hemadsorption assays. Hemadsorption (HAD) was performed and quantified as previously de-
scribed (47). Briefly, growth medium from HN-transfected 293T cell monolayers in 48-well Biocoat plates
(Becton Dickinson Labware) was aspirated and replaced with 150 l of CO2-independent medium (pH
7.3; Gibco) containing different concentrations of compounds and 1% RBC in serum-free CO2-
independent medium and placed at 4°C for 30 min. The wells were then washed three times with 150
l cold CO2-independent medium. The bound RBCs were lysed with 200 l RBC lysis solution (0.145 M
NH4Cl, 17 mM Tris HCl), and absorbance was read at 405 nm using a Spectramax M5 (Molecular Devices)
microplate reader.
Measurement of neuraminidase activity. Assays were performed in transiently transfected 293T
cell monolayers, as described previously (7, 47, 48). Briefly, 293T cells expressing either laboratory-
adapted or clinical strain HN were added to 96-well plates in CO2-independent medium at pH 5.0. After
addition of reaction mixtures containing 20 mM 2’-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic
acid (Toronto Research Chemicals Inc.) substrate and different concentrations of CSC compounds, the
plates were incubated at 37°C for 1 h. Throughout this period, fluorescence resulting from hydrolysis of
the substrate was read at a 365-nm excitation wavelength and a 450-nm emission wavelength using a
Spectramax M5 microplate reader.
Virucidal assay and real-time (RT)-quantitative PCR analysis. Aliquots of HPIV3 viral preparations
were incubated for 1 h at 37°C in Opti-MEM supplemented with the indicated compounds or DMSO. After
incubation, the samples were diluted with Opti-MEM and cleared of compounds using Ultrafree MC filters
(Millipore). The viral particles were then collected from the filters in Opti-MEM and their infectivity was
determined by plaque reduction assay. Using a one-step real-time quantitative PCR HPIV3 detection kit
(Primer Design Ltd.) and a Realplex2 Mastercycler (Eppendorf), the total amount of viral RNA in each
sample was determined (7).
Biochemical detection of fusion protein triggering. 293T cell monolayers, transiently transfected
with HN-N-Venus and F-C-CFP, or with F-C-CFP alone, were incubated overnight in DMEM supplemented
with 3 mM zanamivir to prevent fusion. The transfected cells were washed with Opti-MEM and incubated
for 1 h at 37°C in Opti-MEM supplemented with 100 g/ml cycloheximide (to prevent de novo protein
synthesis) only or additionally supplemented with 2 mM zanamivir or 2 mM zanamivir and 3 mM
compound. The cells were then lysed in DH buffer (50 mM HEPES, 100 mM NaCl, 0.005 g/ml dodecyl
maltoside, complete protease inhibitor cocktail [Roche]). HN and F were immunoprecipitated from
postnuclear lysates with anti-green fluorescent protein (anti-GFP) antibody-conjugated agarose beads
(Santa Cruz) for 2 h at 4°C, washed, and resuspended in phosphate-buffered saline (PBS) and then
incubated in the absence or presence of proteinase K (Sigma) (0.64 105mg/ml [1] or
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January/February 2019 Volume 10 Issue 1 e02900-18 mbio.asm.org 10
1.28 105mg/ml [2]) for up to 90 min. Complete protease inhibitor cocktail (Roche) was added to
stop proteinase K digestion after 90 min. The protein-bead complexes were supplemented with Laemmli
sample buffer, heated to 99°C for 5 min, and then subjected to SDS-PAGE and Western blot analysis with
polyclonal antibodies to the HRC region of F. Relative protein levels were quantified using a Kodak
ImageStation and Kodak Molecular Imaging software (7, 20, 21).
Protein expression and purification. The HPIV3 heptad repeat (HR) domains at the N and C termini
(HRN and HRC) and the 6 helix bundle complex (6HB) were produced as previously reported (49). Briefly,
the N42-SGG (AKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYSGG) and C37-SGG (SGGVANDPIDISI
ELNKAKSDLEESKEWIRRSNQKLDSIG) fragments were subcloned into the pTMHa expression vector encod-
ing a N-terminal His9-TrpLE chimeric protein (50). The chimeric proteins were expressed in Escherichia coli
strain BL21(DE3)/pLysS (Novagen) and purified from insoluble bacterial inclusion bodies by nickel-
nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography following published procedures (50). The
His9-TrpLE leader sequence was subsequently cleaved off by using cyanogen bromide (CNBr). The
N42(L6)C37 construct was expressed in E. coli by using a modified pET3a vector (Novagen). Cells were
lysed by glacial acetic acid and centrifuged to separate the soluble fraction from inclusion bodies. The
soluble fraction containing protein was subsequently dialyzed into 5% acetic acid overnight at 4°C. All
proteins were purified to homogeneity by reverse-phase high-performance liquid chromatography
(HPLC) (Waters, Inc.) on a Vydac C18 preparative column (Hesperia, CA) using a water-acetonitrile gradient
in the presence of 0.1% trifluoroacetic acid and lyophilized. Protein identities were confirmed by
electrospray mass spectrometry (PerSeptive Biosystems Voyager Elite, Cambridge, MA). For expression of
soluble HPIV3 postfusion F protein, a pCAGGs plasmid encoding the ectodomain (amino acids 1 to 493)
from strain CI-1 was prepared by Epoch Biolabs. To avoid protein aggregation in the case of efficient F
cleavage, this construct also contained a deletion of the first nine residues of the fusion peptide (amino
acids 110 to 118). The foldon trimerization domain was also added at the C terminus of the F ectodomain
flanked upstream by a tobacco etch virus (TEV) protease site and downstream by a Xa protease site,
followed by a 6His tag. Hence, the complete amino acid sequence of the C-terminal was as follows:
SGRENLYFQGGGGGSGYIPEAPRDQAYVRKDGEWVLLSTFLGGTEGRHHHHHH (TEV and Xa sequences are in
italics and underlined, and foldon sequences are in boldface). Other sequences correspond to linkers and
histidines. Plasmid was used to transfect FreeStyle 293-F cells (Invitrogen), and protein F was purified
from media after immobilized metal affinity chromatography (Ni2 column) followed by two rounds of
size exclusion chromatography as previously described (51). The purity and integrity of the purified
protein were analyzed by SDS-PAGE and Coomassie blue staining under reducing conditions.
Monoclonal antibodies that recognize the postfusion F. Female BALB/c mice were inoculated
intramuscularly (i.m.) and boosted 4 weeks later with 6HB peptide (20 g for each inoculation). Four days
after the last inoculation, mice were sacrificed by isoflurane (ISOVA) inhalation and their splenocytes
fused to Sp2-0 myeloma cells with polyethylene glycol 4000 (PEG 4000). Hybridomas were selected in
ClonaCell-HY Liquid HAT Selection Medium E (StemCell Technologies). Postfusion-specific antibody
production in hybridoma supernatants was tested by ELISA using the soluble HPIV3 F protein as
described below. Positive cultures were recloned at least twice by limiting dilution using ClonaCell-HY
Medium E (StemCell Technologies). The postfusion specificity of supernatant antibodies after cloning was
confirmed by ELISA using the soluble HPIV3 F protein and the HRC HRN and 6HB peptides.
Hybridoma cells were maintained in ClonaCell-HY Medium E (StemCell Technologies). For antibody
production, flasks were seeded at a confluence of 1 106 cells/ml in hybridoma serum-free medium
(Gibco). To harvest antibodies, cells and medium were centrifuged at 4°C for 10 min at 1,000 relative
centrifugal force (rcf). Supernatant fluid containing antibodies was collected with a 0.22-m-pore-size
filter (Millipore) and stored at 20°C or at 4°C for a maximum of 2 weeks.
Enzyme-linked immunosorbent assay (ELISA). Wells of 96-well microtiter plates were coated with
40 ng/well of purified protein or peptides using PBS buffer for 16 h at 4°C. Nonspecific binding was
blocked with 2% porcine serum–PBS–0.05% Tween. Serial volumes of antibody supernatants were then
added to the wells for 1 h at room temperature, followed sequentially by an excess of anti-mouse IgG
horseradish peroxidase-linked antibody (GE-Healthcare) and substrate (OPD [o-phenylenediamine dihy-
drochloride]; Sigma). Extensive washing with water followed each step. Optical density was read at
492 nm.
Ethics statement. Animal studies were performed under the regulations of Spanish and European
legislation concerning vivisection and the use of genetically modified organisms. Protocols were ap-
proved by the Comité de Ética de la Investigación y del Bienestar Animal of Instituto de Salud Carlos III
(CBA PA 19_2012).
Electron microscopy. Soluble HPIV3 postfusion F was applied to glow-discharged carbon-coated
grids and negatively stained with 1% uranyl formate. Images were recorded on a Gatan ES1000W
charge-coupled-device (CCD) camera in a JEOL JEM-1011 microscope operated at 100 kV.
-Gal assay for detection of fusion protein triggering with F-conformation-specific MAbs. 293T
cells transiently transfected with viral glycoprotein constructs were incubated overnight at 37°C in
complete medium (DMEM, 10% fetal bovine serum [FBS]). At 20 h posttransfection, cells were transferred
to conditions corresponding to the temperatures and times indicated in the figure. Thereafter, cells were
incubated with mouse monoclonal antibodies (MAbs) that specifically detect HPIV3 F in its posttriggered
conformation (1:1,000) for 1 h on ice. Cells were washed with PBS and then incubated for 1 h on ice with
biotin-conjugated anti-mouse secondary antibody (Life Technologies) (1:500). Cells were washed with
PBS and then fixed for 10 min on ice with 4% paraformaldehyde (PFA). Cells were then washed twice
with PBS, blocked for 20 min on ice with 3% bovine serum albumin (BSA)–PBS, washed, and then
incubated for 1 h on ice with streptavidin conjugated with -Gal (Life Technologies) (1:1,000). Cells were
Inhibiting HPIV by Preactivating Cell Entry Mechanism ®
January/February 2019 Volume 10 Issue 1 e02900-18 mbio.asm.org 11
washed with PBS, the -galactosidase substrate (Applied Biosystems) (1:50) was added, and lumines-
cence was measured by the use of a Infinite M1000PRO (Tecan) microplate reader.
Detection and quantitation of triggered F. Ninety-six-well plates were seeded with 293T cells. The
following day, cells were transfected with combinations of HPIV3 laboratory-adapted F and HN and
Lipofectamine 2000. After 3 h, complete medium with 2 mM zanamivir was added to the cells and they
were incubated overnight at 37°C. The next day, cells were brought to 4°C, washed, and overlaid with
cycloheximide and 2 mM zanamivir or with cycloheximide, 2 mM zanamivir, and 3 mM CM9 or CSC11.
The plates were then either left at 4°C or maintained at 37°C for 60 min. Following treatment, plates were
put on ice and incubated with purified PA3/F3 antibody (10 g/ml) for 60 min, washed with Dulbecco’s
phosphate-buffered saline (DPBS), fixed with 4% PFA for 15 min, blocked with 3% BSA, incubated in the
dark for 60 min with 1:500 anti-mouse Alexa Fluor 555 (Thermo Fisher)–3% BSA, and then incubated in
a 1:1,000 dilution of DAPI (4=,6-diamidino-2-phenylindole; Thermo Fisher) for 60 min. Plates were washed,
0.01% sodium azide was added, and plates were imaged via the use of an IN Cell Analyzer. Percentages
of positively recognized cells were determined using Cell Profiler.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/mBio
.02900-18.
FIG S1, PDF file, 0.3 MB.
FIG S2, PDF file, 4.5 MB.
ACKNOWLEDGMENTS
Thanks to Sho Iketani, Ilaria DeVito, and Marion Ferren for contributions to the work.
This work was supported by NIH/National Institute of Allergy and Infectious Diseases
(NIAID) grants R01AI031971 and R01AI114736 to A.M.
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