Analysis of the spread of a wild type MV expressing the green fluorescent protein (GFP) in human airway well-differentiated epithelial sheets revealed that MV infects only columnar cells connected by the apical adhesion complex13
. Thus we thought that MV might target an intercellular junctional protein to enter the airway epithelium. To narrow the search for this receptor, we initially compared genome-wide transcription in permissive (H358 and H441) and non-permissive (H23 and H522) airway epithelium cell lines13
. For these cells high quality genome-wide microarray analyses are available (GEO microarray data GSE8332)14
. We identified 175 transmembrane proteins preferentially expressed in permissive cells. Among these, we expressed cDNAs of 22 that either had top preferential expression ratios, or interesting biological characteristics. None of these proteins, including four claudins from the tight junction, and E-cadherin and nectin-3 from the adherens junction (Supplementary Table 1
, footnote), conferred susceptibility to MV infection.
Next we performed a genome-wide expression analysis based on mRNA extracted from all seven epithelial cell lines from human airways or bladder previously characterized as permissive (3 lines) or not (4 lines)13
. This time, we observed significant enrichment of 222 mRNAs for surface-associated proteins (GEO microarray data GSE32155). We selected 16 genes with high expression ratios in both screens, interesting biological characteristics, or both. In addition, we selected the genes with the top 12 expression ratios not already represented in the first analysis (for details see Supplementary Table 1
). Non-permissive Chinese hamster ovary cells were transfected with expression plasmids and subsequently infected with GFP-expressing MV.
In one instance, we observed GFP expression followed by syncytia formation (
, central panel). The plasmid transfected in these cells coded for adherens junction protein PVRL4/nectin-4. The corresponding mRNA had the 9th
highest preferential expression ratio in the second screen (Supplementary Table 1
, #9). Nectin-4 is a single pass type I transmembrane protein of the immunoglobulin superfamily8,15
. Its long (3.7 kb) mRNA was initially detected only in human trachea among somatic tissues8
, but a recent study documented expression in skin, lung, prostate, and stomach16
Identification of nectin-4 as candidate MV receptor
We assessed the levels of nectin-4 protein expression in the seven epithelial cell lines used for gene expression profiling. FACS analyses with specific antibodies confirmed high levels of expression in the three cell lines permissive for MV infection (
, top row). Three of the non-permissive cell lines did not express nectin-4, while the fourth showed variable expression levels (
, bottom row). We also purchased four nectin-4 specific siRNAs, and assessed whether transfection of H358 cells with these affects MV entry. Indeed, three siRNAs strongly reduced infection, and in particular siRNA 4_1 almost completely abolished it (
, right panel). We then documented that nectin-4 is functionally equivalent to the proposed epithelial receptor EpR13
through cell fusion assays (Supplementary Fig. 1
). We also showed that neither the other three human nectins nor the related poliovirus receptor PVR/CD15517
have MV receptor function (Supplementary Fig. 2
). Remarkably, alpha-herpesviruses use ubiquitous nectin-1 as receptor, and the same is true for nectin-218
. While this paper was in review, another group documented in cancer cells that nectin-4 is an epithelial cell receptor for MV19
All four nectins share the same overall structure defined by three extracellular immunoglobulin-like domains (V and two C2-type domains, VCC), a single transmembrane helix, and an intracellular domain. To map the domain interacting with MV H, we took advantage of two recombinant soluble forms of nectin-4: VCC-Fc and the shorter V-Fc15
, which were used to block MV infection. As shown in
, both forms were similarly effective: 1 μg/ml solutions sufficed for about 50% reduction of syncytia formation.
V domain of nectin-4 supports strong binding to MV H
An independent mapping approach relied on two nectin-4 specific antibodies, N4.40 and N4.61. While N4.40 recognizes one of the two C domains, N4.61 recognizes the V domain15
. Again, different dilutions of either antibody were added before virus inoculation.
shows that while a 0.5 μg/ml N4.61 solution inhibited entry almost completely, 100 times more concentrated N4.40 did not inhibit virus entry. Thus the soluble nectin-4 V domain and anti-V antibodies block infection.
To further characterize the interactions of soluble H and purified virus particles with nectin-4 and SLAM, we separated the same amount of soluble forms of both receptors by non-reducing polyacrylamide gel electrophoresis, and transferred them to membranes. documents that binding of H to partially denatured nectin-4 (2nd and 3rd lanes) is at least as strong as binding to partially denatured SLAM (1st lane). documents stronger binding of virus (left panel) and of soluble H protein (top right panel) to VCC-Fc than to V-Fc nectin-4 (left panel).
We then sought to determine the kinetic parameters of binding native nectin-4 (VCC-Fc) to native H. The soluble complete extracellular domain of SLAM was used as control (
). The measured dissociation constant (Kd
) of SLAM was 93.5 nM, which compares well with 80 nM measured previously20
. The Kd
of H and nectin-4 was 20 nM: while the koff
of both reactions was similar, the kon
of nectin-4 and H was almost 5 times faster than that of SLAM and H (
). Since the Kd
of the CD46 and vaccine H interaction is about 79 nM20
, nectin-4 is the cellular protein bound by H with strongest affinity. However, when CHO cells stably expressing either SLAM or nectin-4 were infected, we documented about 5 times more efficient MV infection in the SLAM-expressing CHO cells (Supplementary Fig. 3)
. Thus parameters other than the Kd
, like accessibility of the receptor-binding region, influence virus spread in this system.
To assess the relevance of nectin-4 expression for MV infection in humans, we relied on primary human airway epithelial cells cultured at an air-liquid interface21
. These cellular sheets closely resemble the human airway: cells develop apical adhesion complexes with tight and adherens junctions, and a well-differentiated morphology consisting of a pseudostratified, ciliated columnar epithelium with goblet and basal cells. In these epithelia, we confirmed nectin-4 mRNA expression (
) at levels slightly higher than those of the Calu3 cell line, which supports efficient MV infection22
. We next transfected the epithelia with specific siRNAs, achieving 90% decrease in nectin-4 mRNA (
). We then infected the cultures and counted on average 4 infectious centers in the negative control siRNA-treated cells while no infectious center, or infected cell, was detected in nectin-4 siRNA treated cells (
). Thus MV infection depends on the presence of nectin-4.
Nectin-4 is necessary for MV infection of well-differentiated human airway epithelia
A second assay of nectin-4 function in well-differentiated human airway epithelia relied on MV-nectin4blind
(originally named MV-EpRblind
), a virus with two amino acid mutations in its H protein disallowing cell entry through the epithelial receptor13
. Supplementary Fig. 4
documents that while MV infectious centers included more than 100 cells, the rare MV-nectin4blind
infections were limited to 1-2 cells. Thus MV must recognize nectin-4 to enter human airway epithelial sheets, and to efficiently spread laterally.
The fact that nectin-4 is transcribed at the highest level in the trachea8
prompted us to consider a mechanism targeting virus emergence to the tracheobronchial airways. To analyze whether MV replicates in nectin-4 expressing cells in an infected host, we inoculated cynomolgus monkeys (Macaca fascicularis
) that can develop the clinical signs of measles23
. To facilitate detection of infectious centers, a GFP-expressing virus was used. Tissues were collected near the peak of acute disease 12 days post-inoculation, and analyses of tracheal sections revealed the expected pathological pattern (Supplementary Fig. 5,
is a correlative analysis of nectin-4 expression and MV replication in epithelia: strongly nectin-4 positive cells were located directly adjacent to infectious centers. These centers consistently included many DAPI (blue) counterstained nuclei, and always lined the tracheal lumen (
, two overlay panels at right; see also paraffin sections in Supplementary Fig. 5,
panels e-g). Remarkably, within infected cells nectin-4 was sometimes expressed at low levels, suggesting virus-induced downregulation. Indeed Supplementary Fig. 6
documented that median nectin-4 cell surface expression in infected lung and bladder epithelial cell lines is about 5 times lower than in uninfected cells.
Nectin-4 expression and infection of monkey tracheal epithelium
Having considered that in cells where nectin-4 is not expressed MV replication cannot occur, we assessed the levels of viral nucleocapsid and nectin-4 mRNA in the trachea and lung tissues of the five infected animals by real-time PCR. Supplementary Fig. 7
documents a high correlation coefficient (r=0.77) between viral and nectin-4 mRNA levels. Moreover, it indicates high nectin-4 expression levels in the trachea and lungs, suggesting that nectin-4 distribution in the airways of cynomolgus macaques is similar to humans.
MV begins its circuit through selected organs of the human body within SLAM-expressing alveolar macrophages and dendritic cells, which ferry it through the epithelial barrier3,4
(Supplementary Fig. 8
). Analyses in primate models indicate that vigorous MV replication occur in primary and secondary lymphatic organs, including tracheobronchial lymph nodes, already 3-5 days after infection4
. A few days’ later, most infected cells in the trachea are of lymphoid or myeloid origin, and located in the sub-epithelial cell layer24
. We collected here tissues at the peak of acute disease, and documented large infectious centers in nectin-4 expressing epithelial cells adjacent to the tracheal lumen. We also observed good correlation between MV and nectin-4 mRNA levels in different parts of the lungs. These data and the experimental demonstration that a virus unable to recognize nectin-4 cannot cross the airway epithelium and is not shed13
, are consistent with targeting of a protein expressed in the trachea for site-directed host exit. Emergence into the tracheobronchial airways appears ideal for aerosol droplet release through coughing and sneezing, filling the air with virus particles ready to infect the next host, and accounting for the extraordinary high reproductive rate of MV in naïve populations25
Nectin-4 is highly expressed in lung, breast and ovarian cancer, for which it is used as a marker9-11
. MV replicates preferentially in cancer cells26
, and spontaneous regressions of different forms of lymphoma were repeatedly observed after natural MV infections. These oncolytic effects are attributed to SLAM-overexpression in transformed lymphocytes6
. On the other hand, a vaccine-lineage MV, which recognizes ubiquitous CD46 in addition to SLAM as receptor, is currently used in ovarian cancer clinical trials12
. Since most ovarian cancers are of epithelial origin, nectin-4 expression is worth testing as a retrospective correlate of MV oncolytic activity. In addition, MV-based clinical trials of lung and breast cancer should be considered. Interestingly, most viruses in oncolysis clinical trials26
exploit junction proteins as receptors27
. It is conceivable that general accessibility of junction proteins in disordered cancer tissue facilitates viral entry, contributing to efficient oncolysis.