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. 2017 Jul 31;13(7):e1006546.
doi: 10.1371/journal.ppat.1006546. eCollection 2017 Jul.

The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases

James T Earnest  1 , Michael P Hantak  1 , Kun Li  2 , Paul B McCray Jr  2 , Stanley Perlman  2   3 , Tom Gallagher  1
Affiliations

The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases

James T Earnest et al. PLoS Pathog. .

Abstract

Infection by enveloped coronaviruses (CoVs) initiates with viral spike (S) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound S proteins, which prompts S protein-mediated virus-cell membrane fusion. Infection therefore requires close proximity of receptors and proteases. We considered whether tetraspanins, scaffolding proteins known to facilitate CoV infections, hold receptors and proteases together on cell membranes. Using knockout cell lines, we found that the tetraspanin CD9, but not the tetraspanin CD81, formed cell-surface complexes of dipeptidyl peptidase 4 (DPP4), the MERS-CoV receptor, and the type II transmembrane serine protease (TTSP) member TMPRSS2, a CoV-activating protease. This CD9-facilitated condensation of receptors and proteases allowed MERS-CoV pseudoviruses to enter cells rapidly and efficiently. Without CD9, MERS-CoV viruses were not activated by TTSPs, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. Thus, we identified DPP4:CD9:TTSP as the protein complexes necessary for early, efficient MERS-CoV entry. To evaluate the importance of these complexes in an in vivo CoV infection model, we used recombinant Adenovirus 5 (rAd5) vectors to express human DPP4 in mouse lungs, thereby sensitizing the animals to MERS-CoV infection. When the rAd5-hDPP4 vectors co-expressed small RNAs silencing Cd9 or Tmprss2, the animals were significantly less susceptible, indicating that CD9 and TMPRSS2 facilitated robust in vivo MERS-CoV infection of mouse lungs. Furthermore, the S proteins of virulent mouse-adapted MERS-CoVs acquired a CD9-dependent cell entry character, suggesting that CD9 is a selective agent in the evolution of CoV virulence.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. CoV-S mediated entry into tetraspanin KO cells.
(A) Western blot analysis of 293T and HeLa clonal cell lines. Actin and the tetraspanin CD63 are used as loading controls. (B) Immunofluorescent analysis of HeLa clonal cell lines. Unpermeabilized cells were incubated with primary antibodies against CD9, CD81 or CD63 as indicated. 293T WT or CD9KO cells were transfected with the appropriate receptors and CD9 where indicated. These cells were transduced with viruses pseudotyped with S proteins from MERS (C), 229E (D), SARS (E), or MHV (F). Pseudovirus transduction was measured by luciferase assay.
Fig 2
Fig 2. Association of CoV entry factors with CHAPS-resistant membranes in the presence or absence of CD9 or CD81.
293T WT, CD9KO, or CD81KO cells were transfected with the CoV receptors DPP4 (A), APN (B), ACE2 (C), CEACAM (D), or the protease TMPRSS2 (E). KO cells were also complemented with the appropriate tetraspanin. Cell-surface proteins were biotinylated before cells were lysed in cold CHAPS and cleared lysates were subjected to ultracentrifugation. Cell surface proteins were isolated by streptavidin pulldown and analyzed in high density (HD) and low density (LD) fractions by western blot.
Fig 3
Fig 3. Proximity ligation assay of DPP4 and TMPRSS2 in CD9KO cells.
(A-F) HeLa CD9KO cells were transfected with the indicated genes and a GFP reporter before being mounted on microscopy slides. Proximity ligation assay was performed using primary antibodies against hDPP4 and hTMPRSS2. Red foci indicate close proximity of the two proteins. (G) The average number of foci/cell in GFP+ cells in each group was quantified. (H) MERSpp transduction of HeLa cells overexpressing the indicated proteins.
Fig 4
Fig 4. Protease sensitivity of MERS transduction in tetraspanin KO cells.
(A) WT or KO cells were transfected with DPP4 and either an empty vector or the complementing tetraspanin as indicated. The cells were pretreated with camostat before transduction with MERSpps. MERSpp entry was measured by luciferase assays, and the percent transduction into camostat -treated cells was plotted relative to untreated cells (dotted line). * p<0.05. (B) WT or KO cell lines were transfected with DPP4 and either an empty vector or the complementing tetraspanin as indicated. The cells were pretreated with bafilomycin or E64D before transduction with MERSpp. MERSpp entry was measured by luciferase assay.
Fig 5
Fig 5. Analysis of Adenovirus knockdown of MERS entry factors.
(A) LET-1 cells were transduced with an adenovirus carrying a GFP gene or adenoviruses carrying hDPP4 and either an empty vector, the TMPRSS2 gene, or a U6-driven shRNA against TMPRSS2 or CD9. After 3 days, cells were lysed and analyzed by western blot for the indicated proteins. (B) Quantitative rtPCR analysis of Tmprss2 transcripts in cells transduced with rAd5-hDPP4-EV and rAd5-hDPP4-shTmprss2. (C) LET-1 cells were transduced with the indicated Ad5 vector before transducing with VSV-MERSpp. Transduction was measured by luciferase assay. (D) The indicated Ad5-DPP4 vectors were installed intranasally in C57/Bl6 mice. 5 days later, mice were infected with MERS-CoV. Lungs were isolated at 2 dpi and viral titers were measured by plaque assay.
Fig 6
Fig 6. CD9 utilization of high virulence MERS viruses.
(A)LET-1 cells were transduced with rAd5-hDPP4-Empty (black bars) or rAd5-hDPP4-shCd9 (dashed bars) before transduction with VSV-pps carrying the indicated MERS S proteins. The entry kinetics of MERSpps carrying cell-culture adapted (B), MA1 (C), or MA2 (D) S proteins was measured in LET-1 cells previously transduced with rAd5-hDPP4-Empty (solid lines) or rAd5-hDPP4-shCd9 (dashed lines). Virus entry was calculated relative to a non-inhibitor treated condition. *p<0.05, **p<0.01, ***p<0.001
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