Identification of Residues Controlling Restriction versus Enhancing Activities of IFITM Proteins on Entry of Human Coronaviruses

Abstract

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes. The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.

GILT restricts the cellular entry mediated by the envelope glycoproteins of SARS-CoV, Ebola virus and Lassa fever virus

“…Plasmids expressing mutant GILT protein with point mutations were constructed by overlap extension PCR [12,31]. By using the template of pcDNA3/ ACE2-C9 reported previously [32], human ACE2 with C-terminally C9 tag was subcloned into retroviral vector pCX 4 bsr vector between BamH I and Not I sites. LentiCRISPRv2/gilt plasmids were constructed using methods previously described [33].…”

“…Plasmids and procedures used to generate the variety of viral envelope protein-pseudotyped lentiviruses bearing luciferase reporter genes were previously reported [32,34]. Also, VSV G protein-pseudotyped retroviruses expressing wild-type GILT or human ACE2 were packaged in GP2-293 cells with previously described methods [35].…”

Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1

“…A number of studies have firmly established that IFITMs target a large spectrum of distinct viral families , although viruses that resist IFITMs, or use them as cofactors, such as human coronavirus OC43, have been identified (6,(34)(35)(36).…”

Construction, expression and antiviral activity analysis of recombinant adenovirus expressing human IFITM3 in vitro

“…IFITMs have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralization, and mediating germ cell development [16]. Recently, IFITMs were identified as broad-spectrum antiviral proteins by inhibiting the early step of virus infection, however different IFITMs has different antiviral activity [17][18][19][20][21]. Furthermore, there is almost no species specificity in antiviral activity of IFITMs [22,23].…”