Basolateral entry and release of New and Old World arenaviruses from human airway epithelia.
ABSTRACT Transmission of arenaviruses from rodent hosts to humans is generally thought to occur through inhalation or ingestion of dust or droplets containing viral particles. Here we demonstrate that two identified arenavirus receptors, alpha-dystroglycan (alpha-DG) and transferrin receptor 1 (TfR1), are expressed in polarized human airway epithelia. Lymphocytic choriomeningitis virus strains with high or low alpha-DG affinity and Junin virus, which binds TfR1, efficiently infected polarized epithelia only when applied to the basolateral surface or when injury compromised tight junction integrity. Viral egress from infected epithelia exhibited basolateral polarity. This study demonstrates that respiratory entry of arenaviruses occurs via basolateral receptors.
- SourceAvailable from: jvi.asm.org[Show abstract] [Hide abstract]
ABSTRACT: Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both, primary and systemic infection phases. NiV entry and spread from polarized epithelia therefore determine virus entry and dissemination within a new host, and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were only detected after infection with high virus doses, at time points when the cell monolayer integrity was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein M. Studies with stably M-expressing, and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal.Journal of Virology 01/2013; · 5.08 Impact Factor
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ABSTRACT: The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membrane and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis .Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a di-leucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of CT 2 and 4 amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression of apical or basolateral surface in polarized Madin-Darby canine kidney cells; whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surface. Interestingly, in rL536A and rL537A mutants the F protein expression was more on apical than basolateral surface and this effect was more pronounced in rL537A mutant. We conclude that these wild type residues in the NDV F CT have effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.Journal of Virology 07/2013; · 5.08 Impact Factor
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ABSTRACT: We empirically tested the long-standing hypothesis of codivergence of New World arenaviruses (NWA) with their hosts. We constructed phylogenies for NWA and all known hosts and used them in reconciliation analyses. We also constructed a phylogenetic tree of all Sigmodontinae and Neotominae rodents and tested whether viral-host associations were phylogenetically clustered. We determined host geographical overlap to determine to what extent opportunity to switch hosts was limited by host relatedness or physical proximity. With the exception of viruses from North America, no phylogenetically codivergent pattern between NWA and their hosts was found. We found that different virus clades were clustered differently and that Clade B with members pathogenic to humans was randomly distributed across the rodent phylogeny. Furthermore, viral relatedness within Clade B was significantly explained by the geographic overlap of their hosts' ranges rather than host relatedness, indicating that they are capable of host switching opportunistically. This has important bearings on their potential to become panzootic. Together, these analyses suggest that NWA have not codiverged with their hosts and instead have evolved predominantly via host switching.Molecular Ecology 06/2012; 21(16):4137-50. · 6.28 Impact Factor
JOURNAL OF VIROLOGY, June 2008, p. 6034–6038
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 12
Basolateral Entry and Release of New and Old World Arenaviruses
from Human Airway Epithelia?
Douglas E. Dylla,1Daniel E. Michele,2,3† Kevin P. Campbell,1,2,3and Paul B. McCray, Jr.1,4*
Genetics Ph.D. Program,1Howard Hughes Medical Institute,2Department of Molecular Physiology and Biophysics,3and
Department of Pediatrics,4The Roy J. and Lucille A. Carver College of Medicine,
University of Iowa, Iowa City, Iowa 52242
Received 15 January 2008/Accepted 6 April 2008
Transmission of arenaviruses from rodent hosts to humans is generally thought to occur through inhalation
or ingestion of dust or droplets containing viral particles. Here we demonstrate that two identified arenavirus
receptors, ?-dystroglycan (?-DG) and transferrin receptor 1 (TfR1), are expressed in polarized human airway
epithelia. Lymphocytic choriomeningitis virus strains with high or low ?-DG affinity and Junin virus, which
binds TfR1, efficiently infected polarized epithelia only when applied to the basolateral surface or when injury
compromised tight junction integrity. Viral egress from infected epithelia exhibited basolateral polarity. This
study demonstrates that respiratory entry of arenaviruses occurs via basolateral receptors.
Lymphocytic choriomeningitis virus (LCMV) is generally
noncytopathic, and the most common human disease associ-
ated with infection is aseptic meningitis. Laboratory arenavirus
strains infect several epithelial tissues, including gastric and
lung epithelium (3, 7, 15, 18). LCMV is endemic in rodents,
which serve as a reservoir. Transmission of arenavirus to hu-
mans is believed to occur by more than one route. Evidence
suggests that inhalation of infected particulates plays an im-
portant role (7, 15), as does direct inoculation from animal
bites or abrasions. Rhesus macaques exposed to the Junin
arenavirus by aerosol developed acute illness and died within a
month (15). Additionally, rhesus and cynomolgus macaques
developed morbidity following aerosol infection with LCMV
(7). While the respiratory tract is a proposed route of entry, the
interactions between LCMV and polarized human respiratory
epithelia have not been studied.
Alpha-dystroglycan (?-DG) has been identified as a receptor
for some arenaviruses, including the Old World arenaviruses
Lassa fever virus and certain strains of LCMV, as well as clade
C New World arenaviruses, which include Oliveros and Latino
viruses as its sole members (4, 24). Some LCMV strains show
little dependence on ?-DG (23). Ubiquitously expressed, dys-
troglycan is transcribed as a precursor peptide and posttrans-
lationally cleaved to yield ?-DG and ?-DG, noncovalently
linked peripheral and integral proteins, respectively (13). To-
gether they form an important transmembrane junction con-
necting the intracellular cytoskeleton and extracellular matrix.
The receptor for the clade B New World arenaviruses, repre-
* Corresponding author. Mailing address: 240F EMRB, Depart-
ment of Pediatrics, Carver College of Medicine, University of Iowa,
Iowa City, IA 52242. Phone: (319) 335-6844. Fax: (319) 335-6925.
† Present address: Department of Molecular and Integrative Phys-
iology, The University of Michigan, Ann Arbor, MI 48109.
?Published ahead of print on 16 April 2008.
FIG. 1. ?-DG expression in human airway epithelia. (A) Reverse
transcription-PCR was performed using cDNA derived from primary
epithelia using primers specific for ?-DG or human GAPDH (20 or 30
cycles). The human-GAPDH control confirmed mRNA was isolated
properly. Results are representative of findings for three different human
specimens. (B) Antibodies specific for?-DG or ?-DG were used to detect
protein expression in a positive-control mouse myoblast cell line (C2C12)
and an immortalized human airway epithelial cell line (NuLi).
sented by Machupo, Guanarito, Junin, and Sabia viruses, was
identified as transferrin receptor 1 (TfR1) (11, 17).
We examined the expression and localization of the identi-
fied New World and Old World arenavirus receptors in polar-
ized primary cultures of human airway epithelia. We first asked
whether ?-DG is an available receptor for LCMV in human
airway epithelia. Well-differentiated primary human airway ep-
ithelia were prepared as previously described (14). RNA was
isolated from polarized airway epithelia using TRIzol (15596-
026; Invitrogen). cDNA was generated using SuperScript II
reverse transcriptase (18064-022; Invitrogen). Reverse tran-
scription-PCR was performed with primer sets designed for
?-DG (?-DG-F [5? GGTGAAGATCCCGTCAGACACTTT
3?] and ?-DG-R [5? ACCACAGGGATAAACTGTAGGTGC
3?]) or human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (HGAPDH-F [5? GTCAGTGGTGGACCTG
ACCT 3?] and HGAPDH-R ?5? AGGGGTCTACATGGCA
ACTG 3?]). While ?-DG mRNA levels were undetectable af-
ter 20 PCR cycles, the mRNA was readily detected after 30
cycles (Fig. 1A).
Immunoblotting confirmed that dystroglycan protein was
present in samples of immortalized airway epithelia. An im-
mortalized human respiratory airway cell line (NuLi) (28) and
positive-control C2C12 mouse myoblast (ATCC CRL-1772)
cell lysates were probed using antibodies specific for ?-DG
(AP83) or ?-DG (IIH6) (10). Both cell types produced abun-
dant ?-DG, detected as a band of approximately 43 kDa (Fig.
1B). The airway cell ?-DG protein appeared as a broad smear,
with a more-prominent band detected at approximately 150
kDa. A likely reason for the increased size and variation in
?-DG molecular weights in airway epithelia compared to those
with C2C12 cells is differential glycosylation (8). The less-
abundant signal in airway epithelia may also represent in-
complete recognition of glycosylated isoforms by the anti-
body or shedding of the noncovalently linked peripheral
protein (22, 27).
To localize ?-DG and TfR1 expression in polarized airway
epithelia, immunohistochemistry was performed. Epithelia
were pretreated apically with 100 ?l of 1,000-U/ml collagenase
(Sigma C-9407) diluted in 50:50 Dulbecco’s modified Eagle
medium–Ham’s F-12 medium (11320-033; Gibco) supple-
mented with 2% Ultroser G (15950-017; Biosepra) for 2 h at
37°C to remove the extracellular matrix components exposing
apical sialic acid residues as previously described (26). ?-DG
immunolocalization studies utilized a Cy3-labeled ?-tubulin
antibody (1:100; no. C-4585; Sigma) to label the apical surface
cilia and a previously described ?-DG antibody, IIH6 (1:20)
(10). ZO-1 antibody (1:100; no. 61-7300; Zymed) and an anti-
TfR1 antibody (1:100 CD71; no. 555534; BD Pharmingen)
were used for TfR1 immulocalization. All immunohistochem-
istry was performed under permeablizing conditions by block-
ing in a 0.5% Triton X solution. Control samples were incu-
bated with 1:200 isotype antibody. Following primary antibody
incubation, the epithelia were washed and incubated with ap-
propriate secondary antibodies. Primary and secondary anti-
bodies were applied to both the apical and basolateral cell
surfaces. The epithelia were then mounted on slides and im-
aged using laser scanning confocal microscopy.
?-DG displayed no distinct polarity, localizing to both apical
and basolateral membranes, with some specimen-to-specimen
FIG. 2. Immunolocalization of arenavirus receptors in human airway epithelia. En face (A to G) and corresponding confocal vertical sections
(inset) of primary human airway epithelia following immunohistochemistry are shown. The localization of ?-DG was detected by isotype control
antibody (A) or ?-DG IIH6 monoclonal antibody (B and C). ?-DG expression is represented by a green (FITC) signal, while a red signal indicates
cilium-specific ?-tubulin Cy3 labeling of the apical surface. (D) TO-PRO 3 (blue) was used to label nuclei. The vertical focus in panel D lies
beneath the tight junctions to display basolateral membrane labeling. Scale bar in panel A ? 20 ?m; n ? 12 epithelia from 6 different donors. (E
to G) The expression localization of TfR1 detected by secondary antibody only (E) or CD71 monoclonal antibody (F and G). TfR1 expression is
represented by a green (Alexa Fluor 488) signal, while a red signal (Alexa Fluor 568) indicates tight junction boundaries detected by ZO-1
antibody. The vertical planes of focus in panels F and G differ to demonstrate unique localization; n ? 10 epithelia from 5 different donors.
VOL. 82, 2008NOTES6035
variation noted (Fig. 2B to D). This is consistent with previous
observations made with mouse trachea (9). Diffuse intracellu-
lar expression was also observed. No colocalization with cilia
was detected. In contrast, TfR1 exclusively localized to baso-
lateral membranes (Fig. 3F and 3G) below the tight junction
boundary designated by ZO-1.
Having localized arenavirus receptors in airway epithelia, we
examined the polarity of entry and exit of LCMV strains with
differing affinities for ?-DG as a receptor and the entry polarity
of Junin virus. Three different infectious LCMV strains, Arm-
strong 53b (a gift from John Harty, University of Iowa), Arm-
strong clone 13, and WE54 (a gift from Michael Oldstone,
Scripps Research Institute), were studied. Armstrong 53b has
a low affinity for ?-DG, and the latter two display high ?-DG
affinities (4, 23, 24). Cultures of primary human airway cells
were infected either apically or basolaterally at a multiplicity of
infection (MOI) of 1 (in 100 ?l of culture medium) for 2 h, and
then the surfaces were rinsed and culture medium replaced.
Forty-eight hours postinfection, basolateral culture media
were collected and the apical surface of each culture was also
rinsed with medium and collected for viral exit titer determi-
nation. Cultures were then fixed with 2% paraformaldehyde
for 10 min and subsequently blocked with 5% bovine serum
albumin plus 0.3% Triton X-100 for 1 h at room temperature.
Following two phosphate-buffered-saline washes, a 1:500 dilu-
tion of guinea pig LCMV polyclonal antibody (1, 2) (gift of
Dan Bonthius, University of Iowa) was incubated on both
surfaces of the culture for 1 h at 37°C. After two washes, a
1:1,000 dilution of anti-guinea pig fluorescein isothiocyanate
(FITC)-conjugated secondary antibody (FI-7000; Vector Lab-
oratories) was applied under the same conditions as for the
primary antibody. Epithelia were mounted on slides and im-
aged using fluorescence microscopy.
Attenuated Candid 1 Junin virus (supplied by Michael Buch-
meier, Scripps Research Institute) (12) was similarly applied to
either the apical or basolateral surfaces of primary airway
epithelia cultures at an MOI of ?0.04 (in 100 ?l) for 2 h.
Afterward, cultures were rinsed and culture media replaced.
Three days postinfection, epithelia were processed as de-
scribed above. Junin monoclonal antibody specific for the nu-
cleoprotein, (J3.6.2) (12) at a 1:500 dilution was incubated on
both surfaces of the culture overnight at 4°C. After rinses, a
goat antimouse Alexa Fluor 488 (1:500; no. A-21121; Invitro-
gen) antibody was applied for detection.
Regardless of the ?-DG affinity of the applied LCMV virus,
only epithelia with virus applied to the basolateral surface
yielded infected cells detected by abundant LCMV immuno-
reactivity (Fig. 3B, E, and H). A high percentage of cells
expressed LCMV immunoreactivity, with obvious clusters of
positive cells visualized. These clusters likely represent a local
spread of infection following virus replication rather than cell
division, since well-differentiated epithelia have a low mitotic
index. Very rarely, a positive cell was observed following apical
infection (Fig. 3G). These results imply that only basolaterally
localized ?-DG is efficiently used or that a different receptor is
used. It is possible that correct ?-DG glycosylation for arena-
FIG. 3. Polarity of infection by infectious arenaviruses. Well-differentiated cultures of primary human airway epithelia were analyzed to
determine the polarity of virus entry following either apical or basolateral virus application. Top to bottom, the panels represent apical infection
(A, D, G, J, and M), basolateral infection (B, E, H, K, and N), or apical infection following surface injury by scratching the epithelia with a pipette
tip (C, F, I, L, and O). LCMV Armstrong 53b (A to C), LCMV Armstrong clone 13 (D to F), and LCMV WE54 (G to I) proteins were detected
with a polyclonal antibody followed by FITC-labeled secondary antibody; n ? 12 epithelia from 4 different donors. Junin protein (J to L) was
detected with monoclonal antibody J3.6.2, followed by Alexa Fluor 488 secondary antibody; n ? 6 epithelia from 3 different donors. As a control,
Ad-LacZ (M to O) was applied and cells were stained with 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside. Scale bar (C) ? 100 ?m.
virus recognition is limited to the basolateral surfaces. How-
ever, this is unlikely, because the IIH6 antibody recognizes
glycosylated ?-DG necessary for virus binding (16, 21). Alter-
natively, differences in viral trafficking at the apical and baso-
lateral surfaces due to unique endocytic pathways (20) may
account for the observed differences. Adenovirus serotype 5
expressing ?-galactosidase was used as a control and demon-
strated basolateral entry polarity detected by 5-bromo-4-
chloro-3-indolyl-?-D-galactopyranoside staining, as expected
The polarity of entry for Junin virus was also basolateral
(Fig. 3K), consistent with TfR1 immunolocalization. Despite a
titer-limited MOI ?2 logs lower than that of LCMV strains,
basolateral infection with Junin yielded an average of 212
(?86) nucleoprotein-positive colonies/epithelial sheet studied
(n ? 6 from 3 donors). Apical Junin infection yielded no
positive colonies (n ? 6). Our observed basolateral polarity of
Junin entry contradicts a previous report of apical entry in a
polarized respiratory epithelial cell line (6). We stress that
polarized primary epithelia more accurately model the in vivo
To further confirm that only basolaterally expressed recep-
tors are used by arenaviruses, we physically disrupted tight
junction integrity. Epithelial sheets were gently scratched once
on the apical side with a pipette tip, followed immediately by
application of each LCMV strain apically for 1 h. Two days
postinfection, immunohistochemistry was performed to label
infected cells. LCMV applied to the apical surface of injured
epithelia displayed distinct tracts of labeled cells along scratch
sites (Fig. 3C, F, and I). Similar results were observed with
serotype 5 adenovirus, known to use a basolateral receptor
(CAR) (29). Junin virus did not exhibit similar results, likely
due to a greatly reduced effective MOI. In summary, these
findings further suggest that only basolateral ?-DG serves as a
functional receptor for Old World arenavirus strains.
The polarity of viral egress was examined by collecting apical
washes and basolateral media following infections. For all
three LCMV strains, we observed LCMV release from in-
fected human airway epithelia exclusively from the basolateral
surface (data not shown), consistent with observations that the
entry and exit of many viruses occur from the same surface in
polarized cell types (5, 6, 25, 29). Low starting titers of the
Junin vaccine strain did not allow conclusive evaluation of viral
This study is the first demonstration of polarized entry and
exit of arenaviruses in well-differentiated human airway epi-
thelia. Previously, aerosol delivery of LCMV (7) or Junin virus
(15) to macaques resulted in virus spread to several visceral
organs. Despite the common notion that the respiratory epi-
thelium is a primary site of arenavirus infection in humans, we
were surprised to discover that entry through the apical surface
of polarized airway epithelia does not occur. We speculate that
arenavirus entry in the respiratory tract likely occurs when
epithelial tight junction permeability and integrity are compro-
mised by injury or disease. The virus might also spread lym-
phatically or via primarily infected mononuclear or dendritic
cells. Basolateral release of virus produced in respiratory
epithelia may also facilitate secondary infection of the
spleen and liver, sites associated with arenavirus intragastric
We acknowledge the support of the University of Iowa Cell and
Tissue Core and the Cell Morphology Core, partially supported by the
Cystic Fibrosis Foundation, NHLBI (PPG HL-51670), and the Center
for Gene Therapy for Cystic Fibrosis (NIH P30 DK-54759). This work
was supported by NIH Research Service Award Institutional Training
Grant 2 T32 GM008629, NIH grant RO1 HL-61460 (to P.B.M.), and
grant PPG HL-51670 (to P.B.M.).
Ariadna Arias and Jennifer Springsteen provided valuable technical
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