Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains

Human embryonic kidney HEK cells 293T and 293β5 (25) and Madin Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Thermo Scientific) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1× nonessential amino acids, 10 mM HEPES, 1 mM sodium pyruvate, and 100 U/ml penicillin-streptomycin solution (Thermo Scientific). Delayed brain tumor (DBT) cells were maintained in minimal essential medium (MEM) supplemented with 10% tryptose phosphate broth, 5% FBS, 100 U/ml penicillin-streptomycin, and 2 mM l-glutamine. Cells were maintained in a humidified environment at 37°C and 5% CO₂.

Codon-optimized MERS S-containing sequences for a C-terminal C9 epitope tag were purchased from Genscript and subsequently cloned into pcDNA3.1+ between the EcoRI and NotI restriction sites. pcDNA3.1-229E-S-C9 and pcDNA3.1-hAPN plasmids were provided by Fang Li, University of Minnesota. pcDNA3.1-SARS-S-C9 and pcDNA3.1-ACE2-C9 plasmids were provided by Michael Farzan, Scripps Research Institute. pcDNA3.1-HA5-QH-trypsin site was provided by Lijun Rong, University of Illinois—Chicago and was previously described (26). The pHEF-VSV-G plasmid was obtained from BEI Resources. pcDNA3.1-murine carcinoembryonic antigen-related cell adhesion molecule (mCEACAM) was described previously (27). C-terminal Flag-tagged human DPP4 (hDPP4) plasmid pCMV6-Entry-hDPP4 (catalog no. RC209466) (CMV stands for cytomegalovirus) was purchased from OriGene. pCAGGS-TMPRSS2-FLAG and pCAGGS-TMPRSS2-S441A-FLAG were previously constructed (7). TMPRSS11D (HAT) was obtained from Open Biosystems and cloned into pCAGGS between SacI and XhoI restriction sites. pCMVSport6-human CD9 was purchased from Open Biosystems. CD9 and scramble control short hairpin RNA (shRNA) constructs flanked by the U6 promoter and a RNA polymerase III stop sequence were engineered into the pUC57 vector by Genscript. The pNL4.3-HIVluc (luc stands for luciferase) plasmid was provided by the NIH AIDS Research and Reference library. pΔEGFP-S15-mCherry (EGFP stands for enhanced green fluorescent protein) (28) was provided by Edward Campbell, Loyola University Chicago. pEGFP was provided by Chris Wiethoff, Loyola University Chicago.

Monoclonal mouse antibodies against CD9 (clone M-L13), CD63 (clone H5C6), and CD81 (clone JS-81) were obtained from BD Pharmingen. Monoclonal mouse antibody against transferrin receptor (clone H68.4) was obtained from Zymed Laboratories. Rabbit anti-Flag and horseradish peroxidase (HRP)-conjugated anti-β-actin antibodies were obtained from Sigma-Aldrich. Mouse antirhodopsin (C9) antibodies were obtained from Millipore. Rabbit anti-CD13 (APN) antibodies were obtained from Abcam. Mouse anticalnexin antibodies were obtained from Cell Signaling. A mouse monoclonal antibody to IAV H1 hemagglutinin (HA) (clone PY102) was provided by Balaji Manicassamy, University of Chicago. Secondary antibodies were purchased from Invitrogen and include Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 488-conjugated goat anti-mouse, and Alexa Fluor 568-conjugated goat anti-mouse antibodies. Donkey anti-goat, goat anti-mouse, and HRP-conjugated goat anti-rabbit antibodies were purchased from Thermo Scientific.

Influenza A/Puerto Rico/8/1934 H1N1 (PR8) containing a Gaussia luciferase (Gluc) reporter gene (29) was provided by Peter Palese, Mount Sinai School of Medicine. PR8-Gluc stocks were produced using a standard protocol (30). Briefly, MDCK cells were inoculated with PR8-Gluc and incubated in DMEM supplemented with 0.2% bovine serum albumin (BSA). At 30 h postinfection (hpi), the progeny were collected, treated with tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma), and used to infect fresh MDCK cells at a multiplicity of infection (MOI) of 1. Supernatants were then collected, clarified by centrifugation, aliquoted, and stored at −80°C. Two strains of recombinant mouse hepatitis viruses (MHV), MHV-A59 and MHV-JHM, each containing a firefly luciferase (Fluc) reporter gene, were produced, and the titers of virus on DBT cells were determined as described previously (31).

Vesicular stomatitis virus (VSV)-based pseudovirus particles (pp) were produced by the method of Whitt (32). Briefly, 293T cells were transfected with plasmids encoding viral glycoproteins. Two days later, cells were inoculated for 2 h with VSVΔG-luc (32), rinsed extensively, and incubated for 1 day. Supernatants were collected, centrifuged at 800 × g for 10 min to remove cellular debris, and stored in aliquots at −80°C. HIV-based pp were produced as previously described (28). Briefly, 293T cells were cotransfected with pNL4.3-HIV-luc and pcDNA plasmids encoding the appropriate glycoproteins, and where indicated, pΔEGFP-S15-mCherry was also cotransfected. After 2 days, supernatants were collected, centrifuged at 1,000 × g at 4°C for 10 min to remove cell debris, and stored in aliquots at −80°C.

Two shRNA constructs were used, one designed to target CD9 and the other a scrambled control. 293β5 cells were cotransfected with 0.05 μg/10⁶ cells of pCDNA3.1-hDPP4 along with 1 μg/10⁶ cells of the indicated shRNA plasmid or a pUC57 construct lacking the shRNA. Stable transfectants were selected in DMEM supplemented with 10% FBS (DMEM–10% FBS) containing 1.2 mg/ml of G418 (Thermo Scientific) for neomycin resistance on the DPP4 plasmid. Cells underwent selection for at least 7 days before being used in assays.

DBT cells or 293β5 cells were transfected with appropriate plasmids encoding viral receptors or proteases, divided into 96-well cluster plates, and incubated for 30 min at 37°C with the antibodies indicated in the figures at 0.12 μg/μl (∼10⁷ antibodies/cell). The viruses indicated in the figures were then added for 2 h at 37°C, and then the cells were rinsed, incubated at 37°C for 6 h (MHV and PR8), 16 h (VSV), or 48 h (HIV). For PR8, cells were not lysed, and media were analyzed for secreted Gluc. For the other viruses, cells were lysed in passive lysis buffer (Promega). Luciferase levels in media or lysates were measured after the addition of either Fluc substrate (Promega) or Gluc substrate (New England BioLabs) using a Veritas microplate luminometer (Turner BioSystems).

To measure antibody binding, 293β5 cells were lifted with Accutase (Millipore), pelleted, and resuspended to 10⁶ cells/ml in phosphate-buffered saline (PBS) supplemented with 2% FBS (PBS–2% FBS) containing the antibodies indicated in the figures at 0.12 μg/μl. After 30 min at 37°C, cells were rinsed three times by pelleting and resuspension in PBS–2% FBS and then incubated for 30 min at 4°C with Alexa Fluor 488-conjugated donkey anti-mouse IgG. After sequential rinsing, cell fluorescence was detected using a BD C6 Accuri flow cytometer. To measure HIV pp binding, 293β5 cells, transfected with empty pCMV6 or with pCMV6-Entry-hDPP4, were suspended in PBS–2% FBS. The cells were divided, and aliquots were incubated for 30 min at 37°C with tetraspanin antibodies at 0.12 μg/μl. The cells were chilled and then incubated for 1 h on ice with HIV-mCherry-MERS S. The cells were rinsed three times by pelleting and resuspension, and mCherry fluorescence was detected using a BD C6 flow cytometer or a BD LSRFortessa flow cytometer as indicated. All flow cytometric data were analyzed using FlowJo software.

DBT cells were transfected with 0.5 μg of pEGFP, and a total of 4 μg of a pCAGGS empty vector or TMPRSS2 plasmid per 10⁶ cells. Twenty-four hours after transfection, the cells were lifted with trypsin, washed three times with cold PBS supplemented with 2% FBS, and sorted using a BD FACSAria cell sorter. Live, green fluorescent protein (GFP)-positive (GFP⁺) cells were plated and incubated at 37°C overnight before antibody blockade experiments were performed as described above.

293β5 cells were transfected with the indicated plasmid DNAs, incubated for 2 days, and then cooled to room temperature (RT). Antibodies and HIV-mCherry pp were added, and the cells were incubated for 30 min at RT and then for 10 min at 37°C and returned to RT. Alexa Fluor-conjugated secondary antibodies were applied for 10 min at RT, along with Hoechst 33258 (Molecular Probes). The cells were rinsed with PBS, fixed with 3.7% paraformaldehyde in 100 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 6.8), mounted using PermaMount, and imaged with a DeltaVision microscope (Applied Precision) equipped with a digital camera (CoolSNAP HQ; Photometrics), using a 1.4-numerical-aperture 60× lens objective. Images were deconvolved with SoftWoRx deconvolution software (Applied Precision). Colocalization was measured and quantified using Imaris version 6.3.1 (Bitplane Scientific Solutions).

Adherent 293β5 cells (∼10⁵/cm²) were rinsed with ice-cold PBS, incubated for 30 min at 4°C with 1 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) in PBS, rinsed, and then incubated for 20 min at 4°C with 100 mM glycine in PBS. The cells were rinsed with PBS, then incubated for 20 min at 4°C in morpholineethanesulfonic acid (MES) buffer (25 mM MES [pH 6.0], 125 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂) containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) detergent (catalog no. 220201; Calbiochem) or 1% Triton X-100 detergent (Sigma). Cell lysates (10⁷/ml) were removed from plates and emulsified by 20 cycles of extrusion through 27-gauge needles. The nuclei were removed by centrifugation, and the lysates were mixed with equal volumes of 80% (wt/vol) sucrose in MES buffer, placed into Beckman SW60 tubes, and overlaid with 3 ml of 30% (wt/vol) sucrose and then with 0.5 ml of 5% (wt/vol) sucrose, both in MES buffer. Samples were centrifuged with a Beckman SW60 rotor at 370,000 × g for 18 h at 4°C. Fractions were collected from air-gradient interfaces. Biotinylated proteins in gradient fractions were bound to streptavidin agarose beads (Pierce). Nonreducing dot blotting and Western blotting procedures were used to identify the distributions of proteins in gradient fractions, as described previously (33).

PR8 or MERS pp were incubated at 37°C for 30 min with equal volumes of low-density (LD) or high-density (HD) sucrose gradient fractions or with 2.5 U trypsin/reaction (in 50 μl total) (Sigma). Treated PR8 and MERS pp were divided, and proteins in one set of aliquots were precipitated with trichloroacetic acid and analyzed by Western blotting. The other set of aliquots was used to transduce 293β5 cells. Cells transduced with MERS pp were pretreated for 1 h with 10 μM leupeptin (Sigma) or without leupeptin, inoculated for 2 h, rinsed, and incubated without leupeptin for 18 h. The cells were then lysed, and luciferase levels were measured. Cells infected with PR8 viruses were infected at an MOI of 1, rinsed after 2 h, and incubated for an additional 6 h. Media were collected, and Gluc levels were measured.

Tetraspanins facilitate the entry of many viruses, including hepatitis C virus (34), human papillomavirus (35), and IAV (23). To determine whether entering CoVs also utilize tetraspanins, we evaluated the effects of tetraspanin antibodies on infection by mouse hepatitis virus (MHV) strains A59 and JHM. To this end, murine DBT cells were incubated for 30 min with mouse monoclonal antibodies against the CD9, CD63, and CD81 tetraspanins or with an equimolar mixture of the three (anti-Tspan). A monoclonal antibody against transferrin receptor (anti-TfR) was used as an isotype-matched control for general cell coating by antibodies. The cells were inoculated with luciferase-expressing recombinant MHV-A59 or MHV-JHM for 2 h, and then unadsorbed viruses and antibodies were rinsed away. As measured by luciferase levels at 8 hpi, the two viruses were significantly inhibited by all three tetraspanin antibodies, with the antibody combination (anti-Tspan) inhibiting the A59 and JHM strains by ∼50% and ∼90%, respectively (Fig. 1A). The TfR antibodies did not block the viruses. None of the antibodies interfered with transduction by VSV pp bearing VSV G proteins (VSV pp), indicating that tetraspanin antibodies do not generally suppress virus entry or reporter gene expression.

While previous studies have found that CD81 knockdown inhibits IAV entry (23), it is not known whether antibodies to CD81 or other tetraspanins also inhibit IAV infection. Thus, we determined whether the tetraspanin antibodies inhibit influenza A/Puerto Rico/8/1934 (H1N1), also known as PR8 IAV. For ease of analysis, we used PR8 containing a Gaussia luciferase (Gluc) reporter gene (29). Many host cells are resistant to PR8 infection, because they do not express proteases that prime viral HA proteins (13). Therefore, we transfected 293β5 cells with plasmids encoding HAT or TMPRSS2 and then infected the cells with PR8 1 day later. HAT will make target cells susceptible to LP IAV, while TMPRSS2 will not (15). By measuring Gluc accumulation in culture media, we determined that transfecting cells with 0.001 μg/well of HAT was sufficient to render cells susceptible to PR8 infection and that increasing HAT transfection generally led to increased infection (Fig. 1B). Knowing this, we examined whether tetraspanin antibodies might block PR8 infection of the HAT-expressing cells. Indeed PR8 infection was significantly inhibited by all three tetraspanin antibodies, with the antibody combination (anti-Tspan) effecting ∼50% blockade. The tetraspanin antibodies did not inhibit VSV pp transductions (Fig. 1C).

To limit our analyses of tetraspanin antibodies to single-cycle infections, we produced several VSV-based pp, each encoding Fluc reporter genes and each containing the S proteins of a relevant human CoV. The virus preparations were designated according to their S proteins (MERS pp, SARS pp, and 229E pp). Their transduction into cells was taken to reflect features of the authentic MERS-CoV, SARS-CoV, and 229E CoV entry processes (7, 33). Transduction-susceptible target cells were established by transfecting 293β5 cells with genes encoding virus receptors: human APN (hAPN) for human coronavirus 229E (HCoV-229E) (36), hDPP4 for MERS-CoV (37), and human angiotensin-converting enzyme 2 (hACE2) for SARS-CoV (38). These target cells were then inoculated with the pp preparations in the absence or presence of tetraspanin antibodies, as was done with authentic viruses. After 1-h inoculation periods, unadsorbed pp and antibodies were removed, and transduction levels were measured the next day by quantifying Fluc gene expression.

The tetraspanin antibodies impaired transduction by all three CoV pp preparations, with MERS pp and SARS pp most notably inhibited (Fig. 2A). TfR antibodies did not affect transductions. Using MERS pp, we further determined that tetraspanin antibodies were inhibitory only at the pp entry stage, with no effects on transduction when added 30 min following virus inoculation (Fig. 2B). Furthermore, inhibition by the tetraspanin antibodies was not related to the viral particle core, as HIV-based MERS pp were blocked equally to VSV-based MERS pp (data not shown). All of these findings were consistent with inhibition of virus entry at TEMs.

To determine whether the antibodies used for virus blockades bound similarly to target cells, we subjected antibody-coated cells to flow cytometry. The inert TfR antibodies and inhibitory tetraspanin antibodies bound similarly (Fig. 3A), indicating that virus blockades do not arise simply from high levels of antibodies on cells. Next, to determine whether antibodies on cells interfere with virus-cell binding, we used fluorescently labeled, HIV-based MERS pp, which we manufactured according to prescribed methods (28). mCherry MERS pp were adsorbed at 4°C to 293β5 cells overexpressing hDPP4 receptors, either in the absence or presence of tetraspanin antibodies. Subsequent flow cytometric analyses revealed that the fluorescent HIV-based MERS pp bound abundantly to the ∼35% of cells that were overexpressing hDPP4 (Fig. 3B, left). Tetraspanin antibodies did not reduce this percentage of cells bound by MERS pp (Fig. 3B, right). Tetraspanin antibodies did slightly reduce the fluorescence intensities of the MERS pp-bound cells (Fig. 3B, compare left red and right gold lines). We conclude that the tetraspanin antibodies only modestly interfered with virus binding to cells.

These data strongly suggested that tetraspanin antibodies interfere with virus entry after virus-receptor binding. To explain how the antibodies block viruses, we posited that viruses might associate with TEMs after binding to cells and that the tetraspanin antibodies disrupt some TEM-associated process(es) facilitating virus entry. To determine whether the mCherry MERS pp do indeed appear at TEMs during their cell entry, we incubated chilled 293β5 cells with the fluorescent pp along with tetraspanin CD81 antibodies and then shifted the cells to 37°C for 10 min to permit “patching,” i.e., antibody-mediated tetraspanin cross-linking into larger TEM structures (39). Quantitative confocal microscopy revealed that CD81 colocalized with ∼20% of MERS pp but with only ∼10% of VSV pp (Fig. 3C, right). Absence of “bald,” i.e., viral glycoprotein-free fluorescent pp binding (Fig. 3C, left) confirmed viral glycoprotein-dependent interactions with cells. Similar, but less compelling copatching of IAV pp and CD81 were also observed in these experiments (Fig. 3C). These data indicate that, shortly after binding to cells, some MERS and IAV pp will be present at TEM locations.

TEMs are known to contain a variety of cell surface proteases (22), among which may be one or several CoV- and IAV-priming proteases (7, 8, 13, 14). This led us to hypothesize that tetraspanin antibodies interfere with TEM-associated proteolytic priming, possibly by preventing TEM proteases from accessing receptor-bound viruses. By this hypothesis, overexpression of proteases, or addition of proteases to media, should provide viral access to priming and thereby reduce or eliminate the antiviral activities of the tetraspanin antibodies. Thus, we overexpressed priming proteases in target cells and performed antibody blockade experiments. Using the frequently cited CoV-priming protease TMPRSS2 (8, 40), we found that MHV infections of DBT cells were augmented ∼20-fold by TMPRSS2 overexpression, indicating that this protease is utilized by MHV and is limiting in the DBT cell context. We supplied DBT cells with graded doses of TMPRSS2-encoding plasmids, along with small constant amounts of a GFP reporter plasmid. Following expression, GFP⁺ cells were isolated by FACS and then used in antibody blockade experiments as described above. The results indicated that tetraspanin antibodies blocked MHV infection of normal DBT cells (Fig. 1A and 4A), but not of DBT cells expressing TMPRSS2 (Fig. 4A). Of note, 10⁶ cells transfected with 0.004 μg of TMPRSS2 plasmid contained TMPRSS2 protein levels that were far below our Western blot and immunofluorescence assay detection limits, making it clear that even small amounts of priming proteases nullify the antiviral effects of tetraspanin antibodies (Fig. 4A).

In similar experiments, PR8 and its priming protease HAT were evaluated in entry assays. Here, 293β5 cells were transfected with graded doses of HAT-encoding plasmids and then infected with PR8, either in the absence or presence of tetraspanin antibodies. The results indicated that HAT bypassed the antibody blockades in a dose-dependent manner (Fig. 4B). Furthermore, trypsin-pretreated viruses also bypassed the antibody blockades (Fig. 4B, rightmost columns). Therefore, the hypothesis that proteases mitigate the antiviral activities of tetraspanin antibodies applies to TMPRSS2, HAT, and trypsin proteases and to MHV and PR8 viruses.

Antibodies binding to CD9, CD81, and CD63 inhibited CoV (MHV) and IAV (PR8) entry (Fig. 1). Of these three, CD9 stood out as a candidate for further evaluation, in part because of reports that sperm-egg fusion requires CD9 (41) and that HIV-induced fusion requires endogenous CD9 levels (42). Therefore, we evaluated how different CD9 levels might affect susceptibility to the VSV-based MERS pp transductions. We produced 293β5 cells stably expressing hDPP4 and a shRNA against CD9. Western blotting confirmed that these cells expressed hDPP4 but expressed only 9% of the CD9 in control cells (Fig. 5A). Notably, the reduced CD9 did not affect any change in the levels of DPP4, unlike that observed by Okamato et al. (43). In comparison with the vector control and scrambled shRNA control cells, the CD9 knockdown (KD) cells were 77% less susceptible to MERS pp transduction. Overexpression of CD9 restored transduction susceptibility to the CD9 KD cells (Fig. 5A). The under- and overexpression of CD9 had no effects on VSV G-mediated transductions (data not shown), indicating a MERS S-specific effect of the CD9 knockdown. All of these results demonstrated that CD9 supports MERS at the level of S-protein-mediated virus entry.

To determine whether CD9 knockdown inhibits MERS pp entry at the proteolytic priming stage of entry, we forced protease overexpression by introducing graded doses of TMPRSS2-encoding plasmids, then transduced cells with MERS pp, and quantified transduction levels with Fluc measurements. The results of these experiments indicated that excess TMPRSS2 restored transduction sensitivity to CD9 knockdown cells, up to the levels of the CD9-replete cells (Fig. 5B). Specifically, 0.3 μg TMPRSS2 plasmid/10⁶ cells increased MERS pp transductions by 4 (±1)-fold in CD9-positive cells and 11 (±3)-fold in CD9 KD cells (Fig. 5B). These CD9-specific differences were statistically significant (P < 0.0001). Thus, the impediments to virus entry brought about by CD9 knockdown were overcome by excess proteases.

Our findings fit with the hypothesis that infecting viruses encounter cell receptors and priming proteases at TEM locations. Antibody binding to TEMs, or omission of particular tetraspanins, interferes with these encounters, reducing infection. We used a biochemical approach to determine whether cell receptors and priming proteases are indeed within TEMs. Surface-biotinylated 293β5 cells were lysed in buffers containing CHAPS, a zwitterionic detergent that emulsifies cell membranes without disrupting primary or secondary TEM interactions (44). After sucrose density gradient fractionation, CHAPS-soluble proteins remained near the bottom of sucrose gradients, designated the high-density (HD) regions, while CHAPS-insoluble protein-lipid complexes floated to the top, low-density (LD) regions. Dot blotting revealed that ∼20% of biotinylated (plasma membrane) proteins were in the LD region (Fig. 6A, top left). Streptavidin pulldown of the isolated HD and LD fractions revealed that all of the detectable cell surface CD9, CD63, and CD81 were present in the LD material (Fig. 6A, bottom left), indicating that the LD fraction includes the TEMs. Notably, cells lysed by Triton X-100 (TX-100), a detergent known to solubilize TEMs, also generated an LD fraction that comprised ∼20% of plasma membrane proteins (Fig. 6A, top right) but was devoid of any cell surface tetraspanins (Fig. 6A, bottom right). Thus, we designated the CHAPS LD subcellular fractions as “TEMs” and the TX-100 LD fractions as “lipid rafts.”

To determine whether CoV receptors and priming proteases partition into TEMs, it was necessary that 293β5 cells were first transfected to overexpress ACE2, APN, CEACAM (the receptor for MHV [45]), or DPP4. The transfected cells were then lysed in CHAPS, and TEMs were isolated by two sequential cycles of flotation on sucrose gradients. Western immunoblotting revealed that ∼90% of ACE2, APN, and CEACAM and ∼50% of the DPP4 were partitioned into TEM fractions (Fig. 6B). Cells overexpressing the virus-priming proteases TMPRSS2 and HAT were similarly fractionated, and roughly 50% of these proteases were found in the TEM fractions (Fig. 6B). Mature TMPRSS2 and HAT exist as linked cleavage products, and some of the TMPRSS2 and HAT that was not found in TEM (LD) fractions may come from shedding of the Flag-tagged TMPR ectodomain fragments into soluble (HD) material. An uncleavable TMPRSS2_S441A mutant more prominently partitioned into the TEM (LD) fractions. This uncleaved integral-membrane TMPRSS2_S441A zymogen may more accurately represent the predominant TEM localization of TMPRs. Some β-actin (<10%) partitioned with TEMs, consistent with known TEM-cytoskeleton interactions (46). Calnexin, a transmembrane protein abundant in the endoplasmic reticulum (47), was excluded from the TEMs, indicating complete cell solubilization by CHAPS detergent. As the TEM fractions contained only ∼20% of the total plasma membrane proteins, these results indicated that the CoV receptors and priming proteases were at least 5- to 50-fold more abundant in TEMs than elsewhere on cell surfaces. While this is significant microdomain localization, we speculate that these results may be underestimating the extent of receptors and proteases naturally within TEMs, as there are upper limits to accommodating overexpressed receptors and proteases in the TEMs.

TEM localization of one CoV receptor (DPP4) and one priming protease (TMPRSS2) was validated by immunofluorescence microscopy. DPP4 and TMPRSS2 were both found near or within CD81-enriched cell surface puncta (Fig. 7). Similar partitioning of CD81 with a catalytically inactive mutant TMPRSS2_S441A was also observed (Fig. 7), indicating that enzymatic activity has no effect on subcellular localization. Thus, there are recognizable proportions of CoV receptors and priming proteases residing within TEMs.

To determine whether the isolated TEMs have virus-priming activities, we mixed them with MERS pp, inoculated the mixtures onto susceptible target cells, and then measured transduction efficiencies. To ensure that the transduction measurements reflected proteolytic priming by the TEMs, and not by endogenous target cell proteases, we suppressed target 293β5 cell priming proteases with leupeptin, a broad-spectrum protease inhibitor. Leupeptin-treated 293β5 cells were profoundly resistant to MERS pp transduction (Fig. 8A, top), indicative of requirements for the host proteases. However, MERS pp that were exposed to TEM fractions transduced the leupeptin-treated cells (Fig. 8A, top), indicating priming. Of note, bypass of leupeptin was pronounced when MERS pp were exposed to trypsin or to TEMs containing overexpressed TMPRSS2 but did not reach the levels observed in the absence of leupeptin. In addition, substantial bypass of leupeptin was not achieved by CHAPS HD fractions (data not shown), even though the HD fractions included all soluble proteins as well as ∼80% of the plasma membrane proteins (Fig. 6A). These findings indicated that MERS entry-priming activities were greatly concentrated in the TEMs. MERS pp that had been exposed to TEMs were also evaluated to assess the extent of S-protein cleavage. Western immunoblots indicated that the TEMs affected cleavage of S proteins, generating proteolytic patterns that were indistinguishable from those generated by trypsin (Fig. 8A, bottom). The apparent molecular sizes of the N-linked glycoprotein products were consistent with cleavages at three multibasic sites, one at positions 626 to 629 (RQQR) to create the minor 130-kDa fragment, one at positions 884 to 887 (RSAR) to create the major 70-kDa fragment, and one at positions 1110 to 1113 (QSKR) to create the minor 40-kDa fragment. The molecular size of the major 70-kDa fragment is equivalent to that of the previously documented S2′ (1).

Analogous experiments were completed with IAV PR8 viruses. In these assays, however, inactivation of host proteases by leupeptin was not required, as PR8 did not respond to endogenous levels of 293 cell proteases. The results of these experiments demonstrated that TEM fractions activated PR8 infectivities, nearly as much as TEMs with overexpressed HAT (Fig. 8B, top). The fact that the TEMs were isolated from IAV-resistant 293β5 cells yet were capable of priming IAV for infection are potentially explained by the significant concentration of cell proteases achieved through TEM isolation.

As with the MERS S on viral pp, PR8-associated HA proteins were analyzed for cleavage status by Western immunoblotting. Here, the TEMs effected cleavage of full-length HA0 into HA1 fragments, irrespective of whether HAT was overexpressed and equal to that achieved by trypsin (Fig. 8B, bottom). Thus, the TEM fractions harvested from 293 cells have proteases that cleave and prime both MERS-CoV S and PR8 IAV HA proteins.