Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

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 (GP1,2) 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

Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry

Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors

MACV glycoprotein determinants for hTFR1 binding and cell entry.

<p>Top panel, structure of the MACV GP1-hTfR1 complex (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021398#pone.0021398-Abraham2" target="_blank">[25]</a>) with the cell surface orientated to the bottom (PDB ID number: 3KAS). The TfR1 protease-like, helical, and apical domains are colored blue, gold, and magenta, respectively. MACV GP1 is colored in dark grey. Mutated residues are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021398#pone-0021398-g001" target="_blank">Fig. 1</a>. Bottom panel, enlargement of the TfR1∶MACV GP1 contact sites. MACV residues important for TfR1 binding are labeled and colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021398#pone-0021398-g001" target="_blank">Fig. 1</a>. TfR1 residues are labeled and colored in magenta.</p

Cell transduction efficiency of eGFP-expressing retrovirus pseudotyped with wild-type MACV GPC, or variants thereof (grey bars, HeLa cells; black bars, Vero cells).

<p>This assay was performed twice in triplicates yielding similar results. Bars indicate average of triplicates in one representative experiment. A representative western blot analysis of the various MoMLV pseudotypes is shown using an antibody against MACV GP2. The numbers to the left of the blots indicate relative molecular mass in kDa.</p

Binding of MACV GP1Δ variants to hTfR1 and the surface of MACV-susceptible cells.

<p>A, expression of wild-type MACV GP1Δ, mutants thereof, and Fc control. The numbers to the left of the blots indicate relative molecular mass in kDa. B, ability of wild-type MACV GP1Δ and variants thereof to co-immunoprecipitate hTfR1. Shown is a representative western blot from two independent experiments. C, ability of these proteins to bind to the surface of MACV-permissive (Vero) cells as analyzed by flow cytometry. This assay was performed twice in duplicates yielding similar results. Bars indicate average of duplicates in one representative experiment. Results were normalized by subtracting measurements with secondary antibody only. D, far-UV circular dichroism (CD) of wt GP1Δ and mutants D123A, D159A, and N178A.</p