Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus.

Abstract

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.

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Biochem. J. (2012) 443, 851–856 (Printed in Great Britain)doi:10.1042/BJ20120150
851
ACCELERATED PUBLICATION
Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import
able to inhibit replication of HIV-1 and dengue virus
Kylie M. WAGSTAFF*, Haran SIVAKUMARAN†, Steven M. HEATON*, David HARRICH† and David A. JANS*1
*Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia, and †HIV Molecular Virology, Queensland
Institute of Medical Research, Brisbane, QLD 4006, Australia
The movement of proteins between the cytoplasm and nucleus
mediated by the importin superfamily of proteins is essential
to many cellular processes, including differentiation and
development, and is critical to disease states such as viral disease
andoncogenesis.Werecentlydevelopedahigh-throughputscreen
to identify specific and general inhibitors of protein nuclear
import, from which ivermectin was identified as a potential
inhibitor of importin α/β-mediated transport. In the present
study, we characterized in detail the nuclear transport inhibitory
propertiesofivermectin,demonstratingthatitisabroad-spectrum
inhibitorofimportinα/β nuclearimport,withnoeffectonarange
of other nuclear import pathways, including that mediated by
importin β1 alone. Importantly, we establish for the first time that
ivermectin has potent antiviral activity towards both HIV-1 and
dengue virus, both of which are strongly reliant on importin α/β
nuclearimport,withrespecttotheHIV-1integraseandNS5(non-
structural protein 5) polymerase proteins respectively. Ivermectin
would appear to be an invaluable tool for the study of protein
nuclear import, as well as the basis for future development of
antiviral agents.
Keywords:denguevirus,HIV,importinα/β1,ivermectin,nuclear
import.
INTRODUCTION
The movement of proteins between the nucleus and cytoplasm
is essential to key cellular processes such as differentiation
and development, as well as being critical to disease states
such as viral disease and oncogenesis [1–3]. Proteins with
a molecular mass greater than ∼45 kDa generally require
an NLS (nuclear localization signal) to gain entry into the
nucleus via the nuclear envelope-localized NPCs (nuclear pore
complexes). NLSs are generally recognized by members of
the Imp (importin) superfamily of proteins, NLS recognition
commonly being through the Impα adaptor protein within the
Impα/β1 heterodimer, or Impβ1 or homologues thereof directly
[4–7]. Nuclear protein export is an analogous process, whereby
NESs (nuclear export signals) are recognized by the exportin
family of Impβ homologues.
With seven Impαs and >20 Impβs in humans and a wide
variety of known NLS/NES sequences, the lack of specific
inhibitors hampers analysis of the functional roles of these
various transporters; currently, the exportin/CRM1 (chromosome
region maintenance 1)-specific inhibitor LMB (leptomycin
B) is the only widely accepted commercially available
compound to inhibit nuclear transport. Although other inhibitory
compounds are beginning to be developed [8–16], including
compoundsthatarestructurallyrelatedtoLMBsuchasratjadone,
peptide-based inhibitors and several small-molecule inhibitors
[17–21], these are not widely available and have not been extens-
ively tested. Clearly, there is an urgent need for new and specific
inhibitors of components of the mammalian cell nuclear transport
machinery.
Previouslywedevelopedahigh-throughputscreeningapproach
to identify inhibitors of viral protein nuclear import [22]. As
aproofofconcept,wetargetedtheinteractionoftheIN(integrase)
protein from HIV-1 with its nuclear import receptor Impα/β.
Fromthisscreening/cross-screeningprocess,weidentifiedseveral
specific inhibitors of IN nuclear import, including mifepristone,
but we also identified inhibitors that appeared to act on Impα/β-
mediated nuclear import generally. One of these was ivermectin,
a broad-spectrum anti-parasite medication used in humans most
commonly to treat nematode infections such as onchocerciasis
(river blindness) [23], as well as scabies [24] and lice [25].
In the present study, we investigated the effects of ivermectin
treatment on the subcellular localization of numerous NLS-
bearing cargo proteins, demonstrating that ivermectin is a potent
inhibitor of Impα/β1-dependent transport, with no effect on
proteins containing NLSs recognized by alternative nuclear
import pathways. Importantly, it can be used to inhibit both
HIV and DENV (dengue virus) infection, both of which rely
on Impα/β1-dependent transport of IN and NS5 (non-structural
protein5)respectively[3,26]forefficientviralproduction,raising
theintriguingpossibilitythatdrugsanalogoustoivermectincould
be potent broad-spectrum antiviral agents.
MATERIALS AND METHODS
Generation of GFP (green fluorescent protein)-fusion protein
bacterial and mammalian expression plasmids
Bacterial or mammalian cell expression vectors encoding GFP-
tagged IN, SV40 (simian virus 40) T-ag (large tumour antigen),
Abbreviations used: CLSM, confocal laser-scanning microscopy; DENV, dengue virus; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine
serum; GFP, green fluorescent protein; hCMV, human cytomegalovirus; Imp, importin; IN, integrase; LMB, leptomycin B; NES, nuclear export signal; NLS,
nuclear localization signal; NS5, non-structural protein 5; PIC, pre-integration complex; PTHrP, parathyroid hormone-related protein; SRY, sex-determining
region of the Y chromosome; sulfo-NHS-biotin, sulfo-N-hydroxysuccinimide biotin; SUMO, small ubiquitin-related modifier; SV40, simian virus 40; T-ag,
large tumour antigen; Tat, transactivator of transcription; TRF1, telomeric repeat factor-binding protein 1; VSV-G, vesicular stomatitis virus glycoprotein.
1To whom correspondence should be addressed (email david.jans@monash.edu).
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DENV NS5, tumour-suppressor protein p53, hCMV (human
cytomegalovirus) processivity factor UL44 and polymerase
UL54, TRF1 (telomeric repeat factor-binding protein 1),
SRY (sex-determining region of the Y chromosome), PTHrP
(parathyroid hormone-related protein), histone H2B, the SUMO
(small ubiquitin-related modifier)-conjugating E3 ligase UBC9,
Tat (transactivator of transcription) protein from HIV-1 [27,28],
and the chromatin remodelling factor aF10 [29] were generated
using the Gateway cloning technology (Invitrogen) and vector
pGFP-attC, for GFP-fusion protein expression in bacteria, or
pDest53 (Invitrogen), for GFP-fusion protein expression in
mammalian cells as described previously [30].
Cell culture and transfection
HeLa (human cervical adenocarcinoma) cells were cultured in
DMEM (Dulbecco’s modified Eagle’s medium) supplemented
with 10% (v/v) FBS (fetal bovine serum), 1 mM L-glutamate,
1 mM penicillin/streptomycin and 20 mM Hepes at 37◦C in 5%
CO2. At 24 h before transfection, cells were seeded on to glass
coverslips (15 mm×15 mm). LipofectamineTM2000 (Invitrogen)
was used according to the manufacturer’s instructions to transfect
DNA into the HeLa cells. Where appropriate, cells were treated
with ivermectin at a final concentration of 25 μM for 1 h before
imaging. Cells were imaged live 24 h after transfection by
CLSM (confocal laser-scanning microscopy) (Bio-Rad 1024ES
or Olympus FV1000) using a ×60 or ×100 oil-immersion
objective as described previously [30,31]. Digitized images were
analysed using the ImageJ version 1.43g public domain software
(NIH) to determine the ratio of nuclear (Fn) to cytoplasmic
(Fc) fluorescence (Fn/c) according to the formula: Fn/c =
(Fn−Fb)/(Fc−Fb), where Fb is background autofluorescence
[5,32,33]. Statistical analysis was performed using Welch’s test
and the GraphPad Prism 5.0 software.
Protein purification and Impα/β dimerization
GFP-tagged NS5 protein was purified from bacteria as a His6-
tagged protein under denaturing conditions [34], whereas Imp
proteinswerepurifiedfrombacteriaasGST-fusionproteinsunder
native conditions as described in [34–36]. Where appropriate,
Impα and Impβ were pre-dimerized at 13.6 μM for 15 min at
room temperature (22◦C) to generate the Impα/β heterodimer for
binding studies.
Biotinylation of Imp proteins
Impα was biotinylated as described previously [34] using the
sulfo-NHS-biotin (sulfo-N-hydroxysuccinimide biotin) reagent
(Pierce). Briefly, 3.5 mg of Imp was incubated with 250 μl of
sulfo-NHS-biotin (1 mg dissolved in 150 μl of water) on ice
for 2 h. Unbound biotin was removed via a PD-10 column
(GE Healthcare) and the resulting biotinylated protein was
concentrated in an Amicon-30 concentration device.
AlphaScreen-based binding assay
The AlphaScreen assay was performed in triplicate in 384-
well white opaque plates (PerkinElmer) [34]. Briefly, 2 μl of
375 nM (30 nM final concentration) His6-tagged protein was
added to each well, followed by 20 μl of Imp at the appropriate
concentration (generally 0–60 nM), prepared by serial dilution
in PBS, and incubation for 30 min at room temperature. All
subsequent additions and incubations were made under subdued
lighting because of the photosensitivity of the beads. A 1 μl
volume of a 1:10 dilution (in PBS) of the acceptor beads and
1 μl of 2.5% BSA were added simultaneously and incubated for
90 min at room temperature. A 1 μl volume of a 1:10 dilution of
the donor beads was then added to give a final sample volume
of 25 μl and the mixture was incubated at room temperature for
2 h. The assay was quantified on a PerkinElmer FusionAlpha
plate reader, triplicate values were averaged, and titration curves
(three-parameter sigmoidal fit) were plotted using the Sigmaplot
graphingprogram.Valuesinthe‘hookingzone’,wherequenching
of the signal has occurred through the presence of too much of
either binding partner, were excluded from the final plot as
previously [30,34,37]. These were used to determine the optimal
Imp concentrations for the library screen.
HIV infectivity assay
HeLacellsplatedonto24-wellplatesat40–50%confluencewere
infectedwith200 ng(capsidequivalent)/wellofVSV-G(vesicular
stomatitisvirusglycoprotein)-pseudotypedNL4-3.Luc.R-E-HIV
in DMEM containing 50% (v/v) FBS and incubated at 4◦C for
2 h to synchronize infection, followed by incubation at 37◦C.
At 2 h (for mifepristone) or 6 h (for ivermectin) after infection,
viruswasremoved,thecellswerewashedwithPBS,andduplicate
wells were treated with DMEM/10% (v/v) FBS containing
ivermectin at 25 or 50 μM or mifepristone at 100 and 200 μM.
At 8 h after infection, medium was removed, and cells were
washed with PBS and fresh DMEM/10% (v/v) FBS, followed
by incubation at 37◦C. At 48.5 h after infection, medium was
removed,cellswereharvestedandlysed,andlysateswereassayed
for luciferase activity (Steady-Glo reagent; Promega) and protein
concentration (Bradford assay), according to the manufacturer’s
instructions.
DENV infectivity assay
Vero cells cultured in DMEM/10% (v/v) FBS were pre-treated
for 4 h with 25 or 50 μM ivermectin, or 25 μM mifepristone,
and then infected with DENV-2 (New Guinea C) at an MOI
(multiplicity of infection) of 4 as described in [31,38,39], and the
cells were maintained in DMEM/2% (v/v) FBS. At various times
after infection, the culture medium was collected and virus titres,
calculated as plaque-forming units/ml, were determined using
plaque assays in C6/36 cells as described previously [31,38,39].
RESULTS AND DISCUSSION
Ivermectin inhibits the nuclear import of multiple Impα/β1 cargo
proteins
We recently used high-throughput screening to identify several
compounds, including mifepristone, as specific inhibitors of IN
recognition by Impα/β1 [22]. In addition, several compounds,
including ivermectin, were identified that were also able to
inhibit recognition by Impα/β1 of SV40 T-ag [4], raising the
possibility that ivermectin may specifically inhibit Impα/β1-
dependent nuclear import in general, and thereby represent a
valuable tool to study nuclear transport. To build on these
preliminaryobservations,anumberofnuclear-localizingproteins
were expressed as GFP-fusion proteins in HeLa cells and treated
with or without ivermectin or mifepristone as a control (Figure 1
and Table 1). These included IN and T-ag, as well as the
Impα/β-recognized proteins DENV NS5, p53 and hCMV UL44,
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Ivermectin inhibits nuclear import
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Figure 1
import, whereas mifepristone specifically inhibits IN nuclear accumulation
Ivermectin inhibits Impα/β1- but not Impβ1-dependent nuclear
(A) Typical CLSMimages of HeLacells expressing the indicated GFP-fusion proteins 24h after
transfection, treated with or without 25μM ivermectin or 50μM mifepristone as indicated for
1 h before imaging. (B) Results (mean+
suchasthosein(A)todeterminethenucleartocytoplasmicfluorescenceratio(Fn/c);*P <0.01.
−S.E.M., n>89) for quantitative analysis of images
and the Impβ1-recognized TRF1. As expected [22], IN nuclear
accumulation was significantly inhibited in the presence of both
ivermectin and mifepristone, whereas T-ag nuclear accumulation
was inhibited by ivermectin but not mifepristone. Importantly,
all of the other cargo proteins were unaffected by mifepristone
treatment, consistent with mifepristone being a highly specific
inhibitor of IN nuclear accumulation. Significantly, nuclear
accumulation of all of the cargo proteins containing Impα/β1-
recognized NLSs was reduced (P<0.01) in the presence of
ivermectin,whereasnosucheffectwasobservedforTRF1,which
is transported to the nucleus in a manner dependent on Impβ1
alone. These results imply that ivermectin is a broad-spectrum
inhibitor of Impα/β1-mediated nuclear import, through an effect
on the Impα/β1 heterodimer.
Ivermectin does not affect nuclear accumulation of cargo proteins
containing NLSs recognized by other Imps
To confirm the specificity of ivermectin action, various GFP-
fusion proteins containing NLSs recognized by a variety of Imps
were expressed in HeLa cells and treated with/without ivermectin
for 1 h before imaging. Results (Figure 2 and Table 1) indicate
that ivermectin only inhibited the nuclear accumulation of hCMV
UL54, which contains classical Impα/β1-recognized NLSs
[40,41]. In contrast, no effect was seen on SRY or PTHrP, which
both contain NLSs recognized by Impβ1 [6,42,43], consistent
with that observed for TRF1. Interestingly, histone H2B, which
Table 1
nuclear import of a range of cargoes imported by different nuclear import
pathways
Summary of data for the effects of ivermectin or mifepristone on
Peptidefragmentsareindicatedbyparentheses,indicatingtheresiduesofthepeptide;otherwise
proteins were full-length. NT, not tested. AF10 is transported into the nucleus independently of
Imps through direct interactions with nucleoporins [29]. Tat has been reported to be recognized
directly by Impβ1 [27], but shown to have an Imp-independent nuclear import mechanism
in vitro [28].
Protein/peptide fragment Import pathwayIvermectinMifepristone
GFP
GFP–AF10-(696–794)
GFP–ppUL44
GFP–p53
GFP–UL54-(1145–1161)
GFP–T-ag-(111–135)
GFP–IN
GFP–NS5
GFP–TRF1-(337–441)
GFP–SRY
GFP–PTHrP-(66–94)
GFP–Tat-(46–64)
GFP–H2B
GFP–UBC9
–
–
Impα/β
Impα/β
Impα/β
Impα/β
Impα/β
Impα/β and Impβ1
Impβ1
Impβ1 and calmodulin
Impβ1
Impβ1 (?)
Multiple Impβs
Imp13
No effect
No effect
Inhibits
Inhibits
Inhibits
Inhibits
Inhibits
Inhibits
No effect
No effect
No effect
No effect
No effect
No effect
No effect
NT
No effect
No effect
NT
No effect
Inhibits
No effect
No effect
NT
NT
NT
NT
NT
Figure 2
affect other nuclear import pathways
Ivermectin is a broad-spectrum Impα/β1 inhibitor that does not
HeLa cells transfected to express the indicated GFP-fusion proteins were treated with or
without 25μM ivermectin for 1h before live-cell imaging 24h after transfection. Results
(mean+
−S.E.M., n>68) were determined as described in Figure 1(B); **P <0.001.
contains at least two NLSs and is thought to be imported into
the nucleus by multiple different Impβ homologues [44–46] was
also not affected by ivermectin, implying that ivermectin does
not affect these various nuclear import pathways. Likewise, the
SUMO-conjugating enzyme UBC9, which is imported into
the nucleus through the action of Imp13 [47], was not affected
by ivermectin. These results (summarized in Table 1) indicate
thativermectinisspecificforImpα/β1-recognizednuclearimport
cargoes, and has no effect on any of the other nuclear
import pathways tested, including that mediated by Impβ1 alone.
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K. M. Wagstaff and others
Figure 3 Ivermectin can inhibit HIV-1 and DENV infection
(A) HeLa cells were infected with 200ng (capsid protein-equivalent) of VSV-G-pseudotyped NL4-3.Luc.R-E- HIV, treated with or without the indicated agents (concentration in parentheses, μM) 2 h
after infection for 6h, then medium was removed, and cells were harvested for measurement of luciferase reporter activity. LD50values for ivermectin and mifepristone in 50% confluent HeLa cells
incubatedfor24hwitheachcompoundwere150 μMand33 mMrespectively.Resultsaremeans+
curves for DENV NS5 and the indicated Imps. Assays were performed as described in the Materials and methods section using 30 μM His6-tagged NS5 protein and 30μM biotinylated Impα/β1, in
thepresenceoftheindicatedconcentrationsofivermectin,mifepristoneorDMSO(vehicle)control.(C)Verocellsweretreatedwithorwithout25μM(left)or50μM(right)ivermectinormifepristone
(as indicated) for 3 h before infection with DENV-2. Cell culture medium was collected and viral titres were analysed at various times after infection by plaque assay. PFU, plaque-forming units.
−S.E.M.foranaverageoffourrepeats;*P <0.05,**P <0.01.(B)AlphaScreenbindinginhibition
Ivermectin inhibits infection by HIV-1 and DENV which rely on
Impα/β1-mediated nuclear transport
Nuclearimportofviralproteinsiscriticaltothelifecycleofmany
viruses, including many RNA viruses that replicate exclusively
in the cytoplasm such as DENV, respiratory syncytial virus and
rabies[2,3,31,48,49].InthecaseofHIV,thevirusgeneratesaPIC
(pre-integration complex), consisting of the newly transcribed
viral cDNA and several HIV (e.g. IN) and host proteins. The
PIC is then transported into the nucleus most likely through
the action of IN [26], subsequent to which IN integrates the viral
cDNA into the host cell genome, which is essential for productive
infection[50].OwingtothesecriticalnuclearfunctionsofIN,itis
likely that inhibition of IN nuclear import will impede productive
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Ivermectin inhibits nuclear import
855
HIVinfection.Totestthisformally,HeLacellswereinfectedwith
200 ng/well VSV-G-pseudotyped NL4-3.Luc.R-E- HIV and the
infection was synchronized at 4◦C for 2 h. Duplicate wells were
then treated with ivermectin for 2 h or mifepristone for 6 h and
viral infectivity was measured by relative luciferase activity 48 h
after infection (Figure 3A). Strikingly, compared with DMSO
control wells, treatment with ivermectin at concentrations as low
as 25 μM treatment for as little as 2 h was able to significantly re-
duce virus production; under these conditions, there is essentially
no observable toxicity induced by the various treatments (LD50
values for ivermectin and mifepristone in 50% confluent HeLa
cells incubated for 24 h with each compound were 150 μM and
33 mMrespectively;theassaywasperformedusingtheInvitrogen
Multitox Fluor Multiplex Cytotoxicity Assay). This is consistent
withivermectinbeingabletogenerallyinhibitImpα/β1-mediated
nuclear import, which is essential for HIV infection and the first
demonstration that inhibitors of nuclear import can have potent
antiviral activity. Mifepristone also significantly inhibited HIV
infectivity (Figure 3A), as expected, consistent with its ability to
specifically inhibit IN nuclear import activity.
In the case of DENV, it has been shown previously that, despite
its critical role in viral replication, which occurs in the cytoplasm,
a majority of DENV NS5 protein is found in the nucleus during
certain parts of the virus infectious life cycle and that mutation of
criticalresiduesintheImpα/β1-recognizedNLSseverelyinhibits
virusproduction[3].Asafirststeptowardsinvestigatingtheuseof
ivermectin as an anti-DENV treatment, inhibition of NS5 binding
toImpα/β1wasfirstexaminedusingourestablishedAlphaScreen
protein–protein-binding assay. Ivermectin was found to strongly
inhibitthebindingofImpα/β1toNS5(IC50=17 μM,Figure3B),
but not of Impβ1 alone to NS5 (IC50>22 mM; it should be
noted that NS5 contains a secondary NLS recognized by Impβ1
alone that, in contrast with the Impα/β1-recognized NLS, is
not essential for NS5 nuclear accumulation) [3]. This indicates
that ivermectin is able specifically to disrupt the interaction
between NS5 and Impα/β1. Mifepristone, in contrast, showed no
significant effect on the binding of NS5 to either Imp as expected.
To test whether ivermectin can inhibit DENV infection, Vero
cells were treated with or without ivermectin for 3 h before
infection with DENV-2 (Figure 3C). Ivermectin, in contrast with
mifepristone,almostcompletelyabolishedvirusproductionwhen
utilized at 50 μM, and significantly reduced virus production at
25 μM, consistent with the critical role nuclear import plays
in the DENV life cycle [3]. It should be noted that the present
study was intended to prove the principle that an inhibitor of viral
protein nuclear import could have antiviral properties, and is not
in any way proposing that ivermectin should be used at 25 μM or
anything near that to treat viral disease. In this context, it should
also be remembered that optimization experiments or rigorous
dose-response analysis have not been performed; however,
it does show that inhibitors of nuclear transport can be potent
antiviral agents and provides a platform for further development
of antivirals in the future.
In summary, the results of the present study show that
ivermectin is a novel inhibitor of nuclear protein import
specifically mediated by Impα/β1; the nuclear accumulation of
all Impα/β1-recognized cargoes tested to date can be inhibited
by short treatments with ivermectin under conditions that do not
lead to cytotoxicity, with no effect on nuclear import mediated
by other Imps such as Impβ1 alone or Imp13. Our recent work
demonstrating that ivermectin inhibits binding of Impα2 to IN
and NS5 even in the absence of Impβ1 in the AlphaScreen assay
(S. M. Heaton, K. M. Wagstaff and D. A. Jans, unpublished
work) strongly implies that ivermectin’s mode of action is likely
to be through binding to the NLS-binding pocket of Impα,
thereby preventing it from recognizing NLS-containing cargo
proteins, rather than alternative mechanisms such as interfering
with Impα/β heterodimerization. This is in stark contrast with
small-molecule nuclear import inhibitors directed at Impβ1 that
prevent binding to RanGTP/cargo release [10,14], which are
mostly unsuitable for live-cell work because of uptake and
precipitation issues and not highly efficient in inhibiting nuclear
import in all cells. Most importantly, in the present study, we
have demonstrated for the first time that inhibitors of nuclear
import such as ivermectin can be potent antiviral agents, able
to significantly inhibit the production of HIV-1 and DENV in
infected cell systems.
Apart from the importance of the observations of the present
study in terms of the potential use of ivermectin in the future for
researchpurposes,theresultsimplythatnuclearimportofspecific
viral proteins is clearly a viable target for the development of
urgently needed antivirals to tackle a number of the world’s major
diseases. Compounds that are specific in inhibiting viral protein
nuclearimport,suchasmifepristoneasshowninthepresentstudy,
loom as exciting possibilities in this context, and are the focus of
future work in this laboratory.
AUTHOR CONTRIBUTION
Kylie Wagstaff designed and executed the majority of experiments (except as noted) and
wrote, drafted and edited the paper before submission. Haran Sivakumaran performed the
HIV infectivity assay (Figure 3A). Steven Heaton performed some of the DENV infectivity
assays(Figure3C,left-handpanel).DavidHarrichsupervised/providedtechnicalexpertise
on the HIV infectivity assays. David Jans supervised/provided technical expertise on all
aspects of the experimental procedures and critically evaluated/edited the paper before
submission.
ACKNOWLEDGEMENTS
WethankCassandraDavidforroutinetissuecultureperformedforthepresentstudy.Some
of the confocal imaging used in the present study was performed at the Monash Micro
Imaging facility, Monash University, Clayton.
FUNDING
This work is supported by the National Health and Medical Research Council [project
grant number 606409 and fellowship number APP1002486] and the Australian Research
Council [fellowship number DP110104437].
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