Adherens junction protein nectin-4 is the epithelial receptor for measles virus.

Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

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 complex[13]. 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 lines[13]. 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 (, 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 ). 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 (, #9). Nectin-4 is a single pass type I transmembrane protein of the immunoglobulin superfamily[8,15]. Its long (3.7 kb) mRNA was initially detected only in human trachea among somatic tissues[8], but a recent study documented expression in skin, lung, prostate, and stomach[16].

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 EpR[13] through cell fusion assays (). We also showed that neither the other three human nectins nor the related poliovirus receptor PVR/CD155[17] have MV receptor function (). Remarkably, alpha-herpesviruses use ubiquitous nectin-1 as receptor, and the same is true for nectin-2[18]. While this paper was in review, another group documented in cancer cells that nectin-4 is an epithelial cell receptor for MV[19].

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-Fc[15], 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.

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 domain[15]. 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 (K) of SLAM was 93.5 nM, which compares well with 80 nM measured previously[20]. The K 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 K of the CD46 and vaccine H interaction is about 79 nM[20], 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 (. Thus parameters other than the K, 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 interface[21]. 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 infection[22]. 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.

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 receptor[13]. 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 trachea[8] 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 measles[23]. 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 ( panels a-d).

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 panels e-g). Remarkably, within infected cells nectin-4 was sometimes expressed at low levels, suggesting virus-induced downregulation. Indeed 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.

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. 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 barrier[3,4] (). 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 infection[4]. A few days’ later, most infected cells in the trachea are of lymphoid or myeloid origin, and located in the sub-epithelial cell layer[24]. 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 shed[13], 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 populations[25].

Nectin-4 is highly expressed in lung, breast and ovarian cancer, for which it is used as a marker[9-11]. MV replicates preferentially in cancer cells[26], 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 lymphocytes[6]. 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 trials[12]. 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 trials[26] exploit junction proteins as receptors[27]. It is conceivable that general accessibility of junction proteins in disordered cancer tissue facilitates viral entry, contributing to efficient oncolysis.

Methods Summary

Viruses

All three GFP-expressing recombinant viruses used here were derived from the wild-type IC-B strain infectious cDNA[28]. They were rescued[29], amplified and titered as described previously[13].

Cells

The human lung cell lines H358 (catalog no. CRL-5807; ATCC), H441 (HTB-174), H23 (CRL-5800), H522 (CRL-5810), and Calu-3 (HTB-55), and the human bladder cell lines SCaBER (HTB-3), T24 (HTB-4), and HT-1376 (CRL-1472) were maintained as instructed by ATCC. The rescue helper cell line 293-3-46[29] was grown in DMEM with 10% FCS and 1.2 mg/ml of G418. Vero/hSLAM cells were kindly provided by Y. Yanagi (Kyushu University, Fukuoka, Japan). Transgenic CHO-nectin1, CHO-nectin2, CHO-nectin4, and CHO-PVR cells were maintained as instructed[10,30]. Expression of the nectin family proteins on these cells was confirmed with specific antibodies.

Online methods include RNA profiling; gene expression knock-down; FACS analysis; fusion assays; inhibition of syncytia formation; overlay binding assays; BIAcore; establishment and infection of airway epithelial sheets; primate infection and histology.