Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus

Abstract

Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2)) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

Figure 1. MARV and EBOV GP_1,2-mediated entry is restricted by IFITM1, 2, and 3.

(A) A549, (C) Vero E6, (E) HUVEC, or (G) 293T cells transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone were infected with MLV-GFP pseudotyped with the entry proteins of EBOV, MARV, IAV, MLV, or MACV, as indicated. Two days later, pseudovirus infection was determined by flow cytometry. Relative infectivity represents the percentage of GFP-positive cells, normalized to that of cells transduced with vector alone. Numbers underneath figures indicate percentage of infected cells in vector-transduced cells. Differences in pseudovirus entry between vector alone and IFITM expressing cells are significant (P<0.05) for all MARV, EBOV, IAV pseudoviruses excepting H7(FPV) in 293T cells expressing IFITM1 (P<0.1). IFITM protein expression in (B) A549, (D) Vero E6, (F) HUVEC, or (H) 293T cells was measured by western blot with an anti-c-myc antibody (9E10), using aliquots of the same cells assayed in (A), (C), (E), and (G), respectively. β-actin was included as a loading control. (I) Experiment similar to (A) except that HeLa cells were treated with 5000 U/ml IFN-β or maintained in growth medium for 48 hours before infection with the indicated pseudoviruses. Differences in pseudovirus entry between MLV or MACV and MARV, EBOV, or IAV are statistically significant (P<0.05), as are all differences in pseudovirus entry between IFN-β-treated and untreated cells. (J) In parallel, aliquots of the same cells assayed in (I) were used to determine the expression of IFITM proteins. IFITM proteins were analyzed by western blot and probed with the indicated anti-IFITM1 or anti-IFITM2/3 antibody. Each panel of the figure represents at least three experiments with similar results.

Figure 2. Replication of infectious MARV and EBOV is restricted by IFITM1, 2, and 3.

Vero E6 cells transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone were incubated with infectious (A) MARV or (B) EBOV at indicated MOIs for 1 hour and then maintained in growth medium. After 72 hours, culture supernatant was harvested and viral titer was assayed using quantitative RT-PCR. A single asterisk indicates a significant difference with controls cells (P<0.05); double asterisks indicate P<0.1. (C) IFITM protein expression in Vero E6 cells was assayed by western blot using anti-c-myc antibody. (D) and (E) are experiments similar to those in (A) and (B), except A549 cells transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone were used. (F) IFITM protein expression in A549 cells was assayed by western blot using anti-c-myc antibody. Each panel represents two experiments with similar results.

Figure 3. SARS-CoV S infection is restricted by IFITM1, 2, and 3.

(A) A549 or (D) Vero E6 cells transduced to express ACE2 were subsequently transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone. Two days later, cells were infected with indicated pseudoviruses. Pseudovirus infection was determined by flow cytometry, and normalized to that of cells transduced with vector alone. Differences in pseudovirus entry between vector alone and IFITM expressing cells are significant (P<0.05) for all SARS-CoV and IAV pseudoviruses. In parallel, cell-surface expression of ACE2 was assayed using aliquots of the same (B) A549 or (E) Vero E6 cells analyzed in (A) and (D), respectively. Cells were labeled with Alexa-488 conjugated S protein RBD of SARS-CoV and analyzed by flow cytometry. ACE2 expression is shown as mean fluorescence intensity. Expression of c-myc-tagged IFITM proteins was assayed by western blot using aliquots of the same (C) A549 or (F) Vero E6 cells analyzed in (A) and (D), respectively. Each panel represents at least three experiments with similar results. (G) Vero cells transduced to express indicated c-myc-tagged IFITM proteins or with vector alone were incubated in duplicate with infectious SARS-CoV at a MOI of 0.1 for 1 hour. Supernatants were harvested 1, 6, 12, 18, 24, or 30 hours later and viral titers were measured by plaque assay. (H) Expression of c-myc-tagged IFITM proteins was assayed by western blot using aliquots of cells analyzed in (G).

Figure 4. Depletion of IFITM proteins differentially enhances infection mediated by MARV, EBOV, SARS-CoV, and IAV entry proteins.

(A) HeLa cells were transduced to express control shRNA or shRNA targeting IFITM1, 2, 3, or IFITM1 and 3 (IFTIM1/3), and selected by puromycin. HeLa cells were then treated with 1000 U/ml IFN-β or with medium alone for 48 hours. Expression of IFITM proteins in control or IFITM depleted HeLa cells was assayed by western blot using the indicated anti-IFITM1 or anti-IFITM2/3 antibody. β-actin was included as a loading control. (B) Aliquots of the cells used in (A) were infected with MLV-GFP pseudotyped with the indicated entry proteins. Pseudovirus infection was measured by flow cytometry, and normalized to infection of cells expressing control shRNA in the absence of IFN-β. Differences in pseudovirus entry between IFN-β-treated and untreated cells for MARV, EBOV, or IAV are significant (P<0.05), except for IAV entry into HeLa cells expressing IFITM3 or IFITM1/3 shRNA. (C) Experiment similar to (B) except HeLa cells stably expressing indicated shRNA were assayed in the absence of IFN-β. (D) HeLa cells expressing indicated shRNA were transduced to express ACE2 and infected with indicated pseudoviruses. Differences in pseudovirus entry between cells expressing control and IFITM3 shRNA in (C) and (D) are significant (P<0.05) for IAV pseudoviruses only. (E) Experiment similar to (A) except that K562 cells were transduced to express control shRNA or shRNA targeting IFITM1, and selected by puromycin. (F) Experiment similar to that in (B) except that infectivity of the indicated pseudoviruses was measured in K562 cells stably expressing the indicated shRNA. (G) Experiment similar to (D) except that infectivity of the indicated pseudoviruses was measured in ACE2- and shRNA-expressing K562 cells. Differences in pseudovirus entry between cells expressing control and IFITM1 shRNA in (F) and (G) were significant (P<0.05) for MARV, SARS-CoV, and IAV pseudoviruses. Each panel of the figure represents at least two experiments with similar results.

Figure 5. Murine and chicken IFITM orthologs differentially restrict infection mediated by MARV, EBOV, and IAV entry proteins.

A549 cells were transduced to express the indicated c-myc-tagged (A) human, (C) mouse, or (E) chicken IFITM orthologs. Two days later, cells were infected with MLV-GFP pseudotyped with the indicated viral entry glycoproteins. Pseudovirus infection was measured by flow cytometry, and normalized to that of cells transduced with vector alone. Expression of (B) human, (D) mouse, or (F) chicken IFITM protein orthologs in A549 cells assayed in (A), (C), and (E), respectively was measured by western blot using the anti-c-myc antibody (9E10). All differences in pseudovirus entry between control and IFITM-expressing cells are significant (P<0.05) except for H5(Thai) entry into murine Ifitm6- and chicken Ifitm5-expressing cells, and for H7(FPV) entry into murine Ifitm1- and Ifitm5-expressing cells. Each panel of the figure represents at least two experiments with similar results.

Figure 6. Trypsin treatment bypasses IFITM restriction of entry mediated by SARS-CoV S protein.

(A) Vero E6 cells transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone were subsequently transduced to express ACE2. Two days later, cells were spin-inoculated at 4°C with indicated pseudoviruses and then treated with 5 µg/ml trypsin or phosphate-buffered saline (PBS) at 37°C for 13 minutes. Infected cells were maintained in growth medium and pseudovirus infection was determined by flow cytometry two days later. Relative infectivity was shown as the percentage of GFP-positive cells, normalized to that of cells transduced with vector alone and treated with PBS. Differences in SARS-CoV pseudovirus entry between trypsin-treated and untreated cells are significant (P<0.5) for all IFITM-expressing cells. (B) In parallel, ACE2 cell-surface expression was measured on aliquots of Vero E6 cells used in (A). Vero E6 cells were labeled with Alexa 488-conjugated S-protein RBD of SARS-CoV and analyzed by flow cytometry. (C) IFITM protein expression in ACE2-expressing Vero E6 cells was measured by western blot with an anti-c-myc antibody (9E10), using aliquots of the same cells assayed in (A). Figs. (A)–(C) are representative of three experiments with similar results. (D) Cathepsin L activity in Vero E6 cells transduced with vector alone or stably expressing indicated IFITM proteins was measured fluorometrically and is shown as mean fluorescence. Cells treated with a cathepsin L inhibitor were used as a control. No statistically significant differences were observed between vector-transduced and IFITM-expressing cells. (E) Vero E6 cells transduced with vector alone or stably expressing the indicated IFITM proteins were labeled with MR-(FR)₂ or with MR-(RR)₂, which bind to the active forms of cathepsin L or B, respectively. Cells were labeled for 1 hour, fixed with formaldehyde, and analyzed by flow cytometry. In vector-transduced cells, mean fluorescence for MR-(FR)₂ was 1175.7 and for MR-(RR)₂ was 454.7. The modest enhancement for cathepsin B activity in IFITM2 and 3 expressing cells is significant (P<0.05), whereas no significant differences were observed in cathepsin L activity. Experiments in (D) and (E) are representative of two experiments with similar results.

Figure 7. IFITM proteins do not interfere with virion access to acidic cellular compartments.

Infectious influenza A/PR/8/34 (H1N1) virus was labeled with Alexa 488-conjugated murine anti-NA (N1) IgG2a (NA-112-S2.4) at 4°C for 16 hours (green). Vero E6 cells transduced to express the indicated IFITM proteins or with vector alone were spin-inoculated with labeled influenza A/PR/8/34 (H1N1) virus (M.O.I. = 100) at 4°C, washed twice with PBS, and incubated with medium containing 100 nM LysoTracker Red DND-99 (red), which labels low pH compartments, at 37°C for 100 minutes. Cells were then washed twice with PBS, fixed with formaldehyde, and imaged by confocal microscopy. Bottom panels show a control in which Vero E6 cells transduced with vector alone were pretreated with 1 U/ml bacterial neuraminidase (NA) for 24 hours before influenza A/PR/8/34 (H1N1) virus infection. Leftmost figures show differential interference contrast (DIC) and rightmost figures show the merged images of labeled virions and LysoTracker labeled cells.