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The CD225/Dispanin superfamily contains membrane proteins that regulate vesicular transport and membrane fusion events required for neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking has remained unknown. Here we show that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline-rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins; mutations in this region disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins. Synopsis: Proteins of the CD225 superfamily regulate membrane trafficking events in diverse tissues, but the mechanism remains unclear. This study reveals that CD225 proteins contain a conserved SNARE-like motif that binds canonical SNARE proteins and regulates membrane fusion in mammalian cells. Genes in the CD225 family encode an R-SNARE-like motif. Mutations in the R-SNARE-like motif of the CD225 protein PRRT2 disrupt neuronal SNARE binding and are linked to neurological disease. Another CD225 protein, IFITM3, interacts with endosomal SNARE proteins, inhibits homotypic late endosome fusion, and promotes the delivery of endosomal cargo to lysosomes. CD225 proteins regulate membrane transport in mammalian cells by binding SNARE proteins through an R-SNARE-like motif. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane proteins (IFITMs) are upregulated by interferons. They are not only highly conserved in evolution but also structurally consistent and have almost identical structural domains and functional domains. They are all transmembrane proteins and have multiple heritable variations in genes. The IFITM protein family is closely related to a variety of biological functions, including antiviral immunity, tumor formation, bone metabolism, cell adhesion, differentiation, and intracellular signal transduction. The progress of the research on its structure and related functions, as represented by IFITM3, is reviewed. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane proteins (IFITMs) are a family of proteins which inhibit infections of various enveloped viruses. While their general mechanism of inhibition seems to be non-specific, involving the tightening of membrane structures to prevent fusion between the viral envelope and cell membrane, numerous studies have underscored the importance of viral envelope proteins in determining the susceptibility of viruses to IFITMs. Mutations in envelope proteins may lead to viral escape from direct interaction with IFITM proteins or result in indirect resistance by modifying the viral entry pathway, allowing the virus to modulate its exposure to IFITMs. In a broader context, the nature of viral envelope proteins and their interaction with IFITMs can play a crucial role in the context of adaptive immunity, leading to viral envelope proteins that are more susceptible to antibody neutralization. The precise mechanisms underlying these observations remain unclear, and further studies in this field could contribute to a better understanding of how IFITMs control viral infections. [ABSTRACT FROM AUTHOR]

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nevertheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium. [ABSTRACT FROM AUTHOR]

Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Toward improving gene transfer rates with these vectors, whole transcriptome analyses on human T lymphocytes are conducted after exposure to CAR‐encoding conventional vectors (VSV‐LV) and vectors targeted to CD8+ (CD8‐LV) or CD4+ T cells (CD4‐LV). Genes related to quiescence and antiviral restriction are found to be upregulated in CAR‐negative cells exposed to all types of LVs. Down‐modulation of various antiviral restriction factors, including the interferon‐induced transmembrane proteins (IFITMs) is achieved with rapamycin as verified by mass spectrometry (LC‐MS). Strikingly, rapamycin enhances transduction by up to 7‐fold for CD8‐LV and CD4‐LV without compromising CAR T cell activities but does not improve VSV‐LV. When administered to humanized mice, CD8‐LV results in higher rates of green fluorescent protein (GFP) gene delivery. Also in vivo CAR T cell generation is improved in kinetics and tumor control, however to a moderate extent, leaving room for improvement by optimizing the rapamycin administration schedule. The data favor multi‐omics approaches for improvements in gene delivery. [ABSTRACT FROM AUTHOR]

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane. [ABSTRACT FROM AUTHOR]

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE: Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatomaderived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses. [ABSTRACT FROM AUTHOR]

The interferon‐induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co‐factors for endosomal antiviral inhibition through high‐throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)‐localized IFITM restriction that targets infectious SARS‐CoV2 and other PM‐fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5‐trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon‐inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS‐CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell‐compartment‐specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies. Synopsis: The interferon‐induced transmembrane proteins (IFITM) can restrict entry of multiple viruses through mechanisms that are still elusive. This work shows that IFITM3‐dependent endosomal restriction of viral entry requires phosphatidylinositol (3,4,5)‐trisphosphate (PIP3) binding.Inhibition of endosomal viral entry depends on the interaction of lysines within the conserved IFITM3 intracellular loop with PIP3.PIP3 is an interferon‐inducible phospholipid that acts as a rheostat for endosomal innate immunity.An interferon‐dependent increase in PIP3 is regulated by the PI3K/Akt/mTOR pathway in a cell‐type dependent manner.Exogenous PIP3 delivery enhances inhibition of endocytic viral entry, including that of the recent SARS‐CoV2 Omicron variant. [ABSTRACT FROM AUTHOR]

Interferon‐induced transmembrane protein 3 (IFITM3) is an antiviral protein that alters cell membranes to block fusion of viruses. Conflicting reports identified opposing effects of IFITM3 on SARS‐CoV‐2 infection of cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with SARS‐CoV‐2 experience extreme weight loss and lethality compared to mild infection in wild‐type (WT) mice. KO mice have higher lung viral titers and increases in inflammatory cytokine levels, immune cell infiltration, and histopathology. Mechanistically, we observe disseminated viral antigen staining throughout the lung and pulmonary vasculature in KO mice, as well as increased heart infection, indicating that IFITM3 constrains dissemination of SARS‐CoV‐2. Global transcriptomic analysis of infected lungs shows upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS‐CoV‐2 infection and overall demonstrate that IFITM3 is protective in SARS‐CoV‐2 infections in vivo. Synopsis: IFITM3 limits severity of SARS‐CoV‐2 infections by restricting viral replication and spread, thereby reducing lung inflammation, immune cell infiltration, angiogenesis, and pathology. IFITM3 KO mice are a new model for studying severe SARS‐CoV‐2 infections. IFITM3 limits morbidity and mortality following SARS‐CoV‐2 infection in two mouse models.Enhanced viral replication in IFITM3 KO mice is accompanied by increased lung pathology and extrapulmonary virus dissemination.IFITM3 KO mice upregulate inflammatory, angiogenic, and pro‐thrombotic gene programs following SARS‐CoV‐2 infection.IFITM3 KO mice provide a tool for studying severe SARS‐CoV‐2 infections with likely relevance to human populations with IFITM3 deficiencies. [ABSTRACT FROM AUTHOR]

Rift Valley Fever virus (RVFV) and Toscana virus (TOSV) are two pathogenic arthropod-borne viruses responsible for zoonotic infections in both humans and animals; as such, they represent a growing threat to public and veterinary health. Interferon-induced transmembrane (IFITM) proteins are broad inhibitors of a large panel of viruses belonging to various families and genera. However, little is known on the interplay between RVFV, TOSV, and the IFITM proteins derived from their naturally infected host species. In this study, we investigated the ability of human, bovine, and camel IFITMs to restrict RVFV and TOSV infection. Our results indicated that TOSV was extremely sensitive to inhibition by all the animal IFITMs tested, while RVFV was inhibited by human IFITM-2 and IFITM-3, but not IFITM-1, and exhibited a more heterogeneous resistance phenotype towards the individual bovine and camel IFITMs tested. Overall, our findings shed some light on the complex and differential interplay between two zoonotic viruses and IFITMs from their naturally infected animal species. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 spike requires proteolytic processing for viral entry. A polybasic furin-cleavage site (FCS) in spike, and evolution toward an optimized FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show interferon-inducible restriction factors Guanylate-binding proteins (GBP) 2 and 5 interfere with furin-mediated spike cleavage and inhibit the infectivity of early-lineage isolates Wuhan-Hu-1 and VIC. By contrast, VOCs Alpha and Delta escape restriction by GBP2/5 that we map to the spike substitution D614G present in these VOCs. Despite inhibition of spike cleavage, these viruses remained sensitive to plasma membrane IFITM1, but not endosomal IFITM2 and 3, consistent with a preference for TMPRSS2-dependent plasma membrane entry. Strikingly, we find that Omicron is unique among VOCs, being sensitive to restriction factors GBP2/5, and also IFITM1, 2, and 3. Using chimeric spike mutants, we map the Omicron phenotype and show that the S1 domain determines Omicron’s sensitivity to GBP2/5, whereas the S2’ domain determines its sensitivity to endosomal IFITM2/3 and preferential use of TMPRSS2- independent entry. We propose that evolution of SARS-CoV-2 for the D614G substitution has allowed for escape from GBP restriction factors, but the selective pressures on Omicron for spike changes that mediate antibody escape, and altered tropism, have come at the expense of increased sensitivity to innate immune restriction factors that target virus entry. [ABSTRACT FROM AUTHOR]

The appearance of new dominant variants of concern (VOC) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the coronavirus disease 2019 (COVID-19) pandemic. Of these, the alpha variant (also known as B.1.1.7), which appeared initially in the United Kingdom, became the dominant variant in much of Europe and North America in the first half of 2021. The spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation adjacent to the polybasic cleavage site, which has been suggested to enhance S cleavage. Here, we show that the alpha spike protein confers a level of resistance to beta interferon (IFN-b) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN-b and context-dependent resistance to IFITMs in the alpha S. P681H reduces dependence on endosomal cathepsins, consistent with enhanced cell surface entry. However, reversion of H681 does not reduce cleaved spike incorporation into particles, indicating that it exerts its effect on entry and IFN-b downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOC may well also confer a replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCE Accumulating evidence suggests that variants of concern (VOC) of SARS-CoV-2 evolve to evade the human immune response, with much interest focused on mutations in the spike protein that escape from antibodies. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here, we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type I interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2 and enhancement by its paralogue IFITM3. The key determinant of this is a proline-to-histidine change at position 681 in S adjacent to the furin cleavage site, which in the context of the alpha spike modulates cell entry pathways of SARS-CoV-2. Reversion of the mutation is sufficient to restore interferon and IFITM2 sensitivity, highlighting the dynamic nature of the SARS CoV-2 as it adapts to both innate and adaptive immunity in the huma [ABSTRACT FROM AUTHOR]

Interferon-inducible transmembrane (IFITM) proteins are broad-spectrum antiviral factors that confer cellular resistance to virus invasion. α/β-Hydrolase domain-containing 16A (ABHD16A) has recently been identified as a novel depalmitoylase that can inhibit the antiviral activity of IFITM proteins by catalyzing the depalmitoyl reaction; this pattern may be crucial for the host to avoid damage caused by excessive immune response. However, it remains largely elusive about how host cells regulate the activity of ABHD16A. In the present study, we performed the AlphaFold2-based protein-protein interaction prediction and identified swine E3 ubiquitin ligase ring finger protein 5 (sRNF5) as a sABHD16A-interacting protein and negatively regulated the stability of sABHD16A. Using immunofluorescence and co-immunoprecipitation techniques, we uncovered that sRNF5 targeted sABHD16A for ubiquitination and degradation via the proteasomal pathway at residues K3 and K452. Furthermore, sABHD16A catalyzed the depalmitoylation of sIFITM1, which obstructed the antiviral function of sIFITM1, while sRNF5 caused ubiquitination of sABHD16A, which attenuated the depalmitoylation effect on sIFITM1, and consequently restored the antiviral activity of sIFITM1. Collectively, our findings demonstrate for the first time that sRNF5 positively regulates the antiviral function of sIFITM1 by mediating the degradation of sABHD16A, which expands the biological functions of RNF5 and ABHD16A in immune regulation. Moreover, our work highlights the well-designed interplay between RNF5, ABHD16A, and IFITM, which balances antiviral immune responses to avoid the disorders induced by excessive immune response., Importance: Interferon and interferon-stimulated genes play significant and protective roles in the host's defense against viral infection. IFITM family proteins, which can be strongly induced by interferon, have been identified as the first line of defense to prevent invasion of various viruses. Further analysis reveals the antiviral activity of IFITMs depends on palmitoylation/depalmitoylation. Recently, we reported that ABHD16A, as the first depalmitoylase of IFITMs, negatively regulated the antiviral activity of IFITMs. However, these raise crucial questions: how ABHD16A is regulated and remained in a balanced manner? Here, we show that swine RNF5 attenuates the negative regulation of sIFITM1 against virus invasion by modifying sABHD16A through ubiquitination and guiding sABHD16A for degradation. Thus, sRNF5-sABHD16A interplay plays an indispensable role in regulating immune response and avoiding the disorders induced by elevated interferon levels. Overall, our findings extend the upstream subtle regulatory molecular mechanism of IFITMs and provide potential targets for viral disease therapy.

Interferon-induced transmembrane protein 3 (IFITM3) plays a substantial role in the immune system by repressing viral entry into host cells and restricting virus replication. Recent research suggests that specific polymorphisms of the IFITM3 gene, rs34481144 and rs12252, contribute to susceptibility to viral infections across populations dependent on their population frequencies, which needs further clarification. This population-based study determined the prevalence of two regulator SNPs in the Turkish population and evaluated genotype and allele frequencies in individuals stratified into groups based on a pilot survey conducted during the COVID-19 pandemic. Tetra-Primer Arms PCR assays and Sanger sequencing methods were used to genotype rs34481144 and rs12252; all participants (n = 200) answered a questionnaire on individual experiences (e.g., disease severity, vaccine side effects) during the COVID-19 pandemic. Distributions of genotype frequencies and pairwise linkage disequilibrium correlations were calculated and compared to publicly available data from worldwide populations. The minor allele frequencies for rs12252-G and rs34481144-T were 0.128 and 0.324, respectively, in the total sample. Our preliminary survey data gave no concrete evidence of a correlation between the analyzed SNPs and COVID-19 severity or vaccine side effects yet pinpointed a trend for an association between rs12252-G and symptom burdens, which merits further investigation. Overall, our results present genotype and allele distributions of two IFITM3 polymorphisms in the Turkish population and provide preliminary data on previously suggested correlations of genotype with COVID-19 in our population. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane (IFITM) proteins as host restriction factors are known to inhibit the replication of several viruses. In this study, transient IFITM expression vectors were used to investigate whether IFITMs inhibit feline foamy viral (FFV) replication and which step of viral replication is inhibited. In our studies, viral production was significantly reduced when cells were infected with FFV at almost same times such as −3, 0, or 3 h post-transfection with IFITM vector. However viral production was not reduced even though cells were infected with FFV at 3 or 6 days post-transfection when production of IFITM proteins was maximized. Considering that IFITM expression was maximized at 3 days post-transfection, the stage of viral replication inhibited by IFITM appears to be the late step of viral replication. Moreover, the viral Gag proteins detected in the virus-infected cell lysates were proportionally correlated with viral titer of the culture supernatants. Therefore, it is likely that IFITMs can restrict production of FFV at the late step of viral replication.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. [ABSTRACT FROM AUTHOR]

The interferon-induced transmembrane (IFITM) gene family performs multiple functions in immunity, including inhibition of virus entry into cells. The IFITM repertoire varies widely between species and consists of protein-coding genes and pseudogenes. The selective forces driving pseudogenization within gene families are rarely understood. In this issue, the human pseudogene IFITM4P is characterized as a virus-induced, long noncoding RNA that contributes to restriction of influenza A virus by regulating mRNA levels of IFITM1, IFITM2, and IFITM3. [ABSTRACT FROM AUTHOR]

Canine influenza virus (CIV) is an emerging virus that is associated with major hidden hazards to the canine population and public health. Until now, how canine uses its innate immunity to restrict CIV replication is seldomly investigated. Recently, studies on interferon-inducible transmembrane (IFITM) of several major hosts of influenza virus (human, chicken, duck, pig) indicated it can potently restrict the viral replication. Here, the gene locus of five previously annotated canine IFITM (caIFITM) genes was determined on chromosome 18 using multiple bioinformatics strategies, provisionally designated as caIFITM1, caIFITM2a, caIFITM2b, caIFITM3, and caIFITM5. An analysis on protein sequences between caIFITM and its homologs indicated they shared the same conserved amino acids important for the antiviral activity. Expression profile analysis showed that caIFITM was constitutively expressed in tissues and MDCK cell line. After treatment with interferon or infection with influenza virus, the expression level of caIFITM increased with different degrees in vitro. An animal challenge study demonstrated CIV infection resulted in upregulation of caIFITM in beagles. caIFITMs had a similar subcellular localization to their human homologs. caIFITM1 was present at the cell surface and caIFITM3 was present perinuclearly and colocalized with LAMP1-containing compartments. Finally, we generated A549 cell lines stably expressing caIFITM and challenged them with influenza virus. The result demonstrated caIFITM1, caIFITM2a, caIFITM2b, and caIFITM3 had a potent antiviral activity against influenza virus. Our study will help better understand the evolutional pattern of IFITM and its role in the host’s defense against virus infection.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/ or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. [ABSTRACT FROM AUTHOR]

Canine influenza virus (CIV) is an emerging virus that is associated with major hidden hazards to the canine population and public health. Until now, how canine uses its innate immunity to restrict CIV replication is seldomly investigated. Recently, studies on interferon-inducible transmembrane (IFITM) of several major hosts of influenza virus (human, chicken, duck, pig) indicated it can potently restrict the viral replication. Here, the gene locus of five previously annotated canine IFITM (caIFITM) genes was determined on chromosome 18 using multiple bioinformatics strategies, provisionally designated as caIFITM1, caIFITM2a, caIFITM2b, caIFITM3, and caIFITM5. An analysis on protein sequences between caIFITM and its homologs indicated they shared the same conserved amino acids important for the antiviral activity. Expression profile analysis showed that caIFITM was constitutively expressed in tissues and MDCK cell line. After treatment with interferon or infection with influenza virus, the expression level of caIFITM increased with different degrees in vitro. An animal challenge study demonstrated CIV infection resulted in upregulation of caIFITM in beagles. caIFITMs had a similar subcellular localization to their human homologs. caIFITM1 was present at the cell surface and caIFITM3 was present perinuclearly and colocalized with LAMP1-containing compartments. Finally, we generated A549 cell lines stably expressing caIFITM and challenged them with influenza virus. The result demonstrated caIFITM1, caIFITM2a, caIFITM2b, and caIFITM3 had a potent antiviral activity against influenza virus. Our study will help better understand the evolutional pattern of IFITM and its role in the host's defense against virus infection. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane proteins (IFITMs) are a family of interferon-inducible proteins that inhibit a broad range of viruses by interfering with viral-tocellular membrane fusion. The antiviral activity of IFITMs is highly regulated by several posttranslational modifications and by a number of protein domains that modulate steady-state protein levels, trafficking, and antiviral effectiveness. Taking advantage of the natural diversity existing among IFITMs of different animal species, we have compared 21 IFITMs for their ability to inhibit HIV-1 at two steps, during virus entry into cells (target cell protection) and during the production of novel virion particles (negative imprinting of virion particles' infectivity). We found a high functional heterogeneity among IFITM homologs with respect to both antiviral modalities, with IFITM members that exhibit enhanced viral inhibition, while others have no ability to block HIV-1. These differences could not be ascribed to known regulatory domains and could only be partially explained through differential protein stability, implying the existence of additional mechanisms. Through the use of chimeras between active and inactive IFITMs, we demonstrate that the cross talk between distinct domains of IFITMs is an important contributor of their antiviral potency. Finally, we identified murine IFITMs as natural variants competent for target cell protection, but not for negative imprinting of virion particles' infectivity, suggesting that the two properties may, at least in principle, be uncoupled. Overall, our results shed new light on the complex relationship between IFITMs and viral infection and point to the cross talk between IFITM domains as a novel layer of regulation of their activity. IMPORTANCE IFITMs are broad viral inhibitors capable of interfering with both early and late phases of the replicative cycle of many different viruses. By comparing 21 IFITM proteins issued from different animal species for their ability to inhibit HIV-1, we have identified several that exhibit either enhanced or impaired antiviral behavior. This functional diversity is not driven by differences in known domains and can only be partly explained through differential protein stability. Chimeras between active and inactive IFITMs point to the cross talk between individual IFITM domains as important for optimal antiviral activity. Finally, we show that murine IFITMs are not capable of decreasing the infectivity of newly produced HIV-1 virion particles, although they retain target cell protection abilities, suggesting that these properties may be, in principle, disconnected. Overall, our results shed new light on the complex layers of regulation of IFITM proteins and enrich our current understanding of these broad antiviral factors. [ABSTRACT FROM AUTHOR]

The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-β), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus. • IFITM1 and IFITM2 inhibit the entry and replication of senecavirus A. • IFITM1 and IFITM2 promote the type I interferon antiviral response mediated by RIG-I. • IFITM1 and IFITM2 promote the expression of RIG-I and its mediated IFNs in a dose-dependent manner. • IFITM1 and IFITM2 inhibit the replication of senecavirus A by positive feedback with RIG-I signaling pathway. [ABSTRACT FROM AUTHOR]

ABSTRACTDiverse SARS-like coronaviruses (SL-CoVs) have been identified from bats and other animal species. Like SARS-CoV, some bat SL-CoVs, such as WIV1, also use angiotensin converting enzyme 2 (ACE2) from human and bat as entry receptor. However, whether these viruses can also use the ACE2 of other animal species as their receptor remains to be determined. We report herein that WIV1 has a broader tropism to ACE2 orthologs than SARS-CoV isolate Tor2. Among the 9 ACE2 orthologs examined, human ACE2 exhibited the highest efficiency to mediate the infection of WIV1 pseudotyped virus. Our findings thus imply that WIV1 has the potential to infect a wide range of wild animals and may directly jump to humans. We also showed that cell entry of WIV1 could be restricted by interferon-induced transmembrane proteins (IFITMs). However, WIV1 could exploit the airway protease TMPRSS2 to partially evade the IFITM3 restriction. Interestingly, we also found that amphotericin B could enhance the infectious entry of SARS-CoVs and SL-CoVs by evading IFITM3-mediated restriction. Collectively, our findings further underscore the risk of exposure to animal SL-CoVs and highlight the vulnerability of patients who take amphotericin B to infection by SL-CoVs, including the most recently emerging (SARS-CoV-2).

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-a (IFN-a) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases. [ABSTRACT FROM AUTHOR]

SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes. [ABSTRACT FROM AUTHOR]

The CD225 superfamily regulates vesicular membrane fusion events essential to neurotransmission, immunity, development, and metabolism. Its importance to physiology is reinforced by the identification of polymorphisms associated with disease. This article highlights the shared features that drive the function of CD225 proteins such as interferon-inducible transmembrane proteins 3 (IFITM3) and proline-rich transmembrane protein 2 (PRRT2) and is intended to catalyze efforts towards characterizing the lesser-known family members. [ABSTRACT FROM AUTHOR]

Pregnancy complications occur frequently and are particularly prevalent during the first trimester. They are caused by a multitude of factors, including karyotypic, genetic or environmental conditions, congenital infections and inflammation. The molecular mechanisms leading to placental complications under inflammatory conditions remain unclear. In this review, we discuss how uncontrolled inflammation, triggered by viral infections or other diseases can lead to placental complications. We first highlight the importance of syncytins, ancestral retroviral genes co-opted by mammals including humans, millions of years ago for the process of placenta formation. We then focus on recent advances elucidating how interferon-induced transmembrane (IFITM) proteins, antiviral proteins rendering cells refractory to viral infections, interfere with placental development. [ABSTRACT FROM AUTHOR]

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction. SYNOPSIS: Interferon‐induced transmembrane proteins (IFITMs) are generally restrictive of SARS‐CoV‐2 infection of cells. While IFITM3 inhibits endocytic entry of the virus, it enhances virus fusion at the plasma membrane. IFITM1, 2, and 3 from mice and humans generally decrease infection by SARS‐CoV‐2.Human IFITM3 inhibits infection at endosomes via a palmitoylation‐independent and amphipathicity‐dependent mechanism.Human IFITM3 enhances virus fusion at the plasma membrane via an amphipathicity‐independent mechanism.TMPRSS2 protease expression can shift the balance of IFITM3 activities toward enhancement of infection. [ABSTRACT FROM AUTHOR]

Described as the “proteasome of the membrane” or the “scissors in the membrane,” γ-secretase has notoriously complicated biology, and even after decades of research, the full extent of its regulatory mechanism remains unclear. γ-Secretase is an intramembrane aspartyl protease complex composed of four obligatory subunits: Nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (Pen-2), and Anterior pharynx-defective-1 (Aph-1). γ-Secretase cleaves numerous type 1 transmembrane substrates, with no apparent homology, and plays major roles in broad biological pathways such as development, neurogenesis, and cancer. Notch and the amyloid precursor protein (APP) and are undoubtedly the best-studied γ-secretase substrates because of their role in cancer and Alzheimer’s disease (AD) and therefore became the focus of increasing studies as an attractive therapeutic target. The regulation of γ-secretase is intricate and involves the function of multiple cellular entities. Recently, γ-secretase modulatory proteins (GSMPs), which are non-essential subunits and yet modulate γ-secretase activity and specificity, have emerged as an important component in guiding γ-secretase. GSMPs are responsive to cellular and environmental changes and therefore, provide another layer of regulation of γ-secretase. This type of enzymatic regulation allows for a rapid and fine-tuning of γ-secretase activity when appropriate signals appear enabling a temporal level of regulation. In this review article, we discuss the latest developments on GSMPs and implications on the development of effective therapeutics for γ-secretase-associated diseases such as AD and cancer.

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline-rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, Amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

ABSTRACT Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3. IMPORTANCE The viral envelope glycoprotein, known as “Env” in Retroviridae, is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.

Abstract The InterFeron Induced TransMembrane (IFITM) proteins are interferon stimulated genes that restrict many viruses, including HIV-1. SAMHD1 is another restriction factor blocking replication of HIV-1 and other viruses. Some lentiviruses evolved Vpx/Vpr proteins to degrade SAMHD1. However, this viral antagonism can be perturbed by host mechanisms: a recent study showed that in interferon (IFN) treated THP1 cells, Vpx is unable to degrade SAMHD1. In the present work, we designed an Interferon Stimulated Genes (ISGs)-targeted CRISPR knockout screen in order to identify ISGs regulating this phenotype. We found that IFITM proteins contribute to the IFNα-mediated protection of SAMHD1 by blocking VSV-G-mediated entry of the lentiviral particles delivering Vpx. Consistent with this, IFNα treatment and IFITM expression had no effect when the A-MLV envelope was used for pseudotyping. Using an assay measuring viral entry, we show that IFNα and IFITMs directly block the delivery of Vpx into cells by inhibiting VSV-G viral fusion. Strikingly, the VSV-G envelope was significantly more sensitive to this IFNα entry block and to IFITMs than HIV-1’s natural envelope. This highlights important differences between VSV-G pseudotyped and wild-type HIV-1, in particular relative to the pathways they use for viral entry, suggesting that HIV-1 may have evolved to escape restriction factors blocking entry.

Described as the "proteasome of the membrane" or the "scissors in the membrane," γ-secretase has notoriously complicated biology, and even after decades of research, the full extent of its regulatory mechanism remains unclear. γ-Secretase is an intramembrane aspartyl protease complex composed of four obligatory subunits: Nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (Pen-2), and Anterior pharynx-defective-1 (Aph-1). γ-Secretase cleaves numerous type 1 transmembrane substrates, with no apparent homology, and plays major roles in broad biological pathways such as development, neurogenesis, and cancer. Notch and the amyloid precursor protein (APP) and are undoubtedly the best-studied γ-secretase substrates because of their role in cancer and Alzheimer's disease (AD) and therefore became the focus of increasing studies as an attractive therapeutic target. The regulation of γ-secretase is intricate and involves the function of multiple cellular entities. Recently, γ-secretase modulatory proteins (GSMPs), which are non-essential subunits and yet modulate γ-secretase activity and specificity, have emerged as an important component in guiding γ-secretase. GSMPs are responsive to cellular and environmental changes and therefore, provide another layer of regulation of γ-secretase. This type of enzymatic regulation allows for a rapid and fine-tuning of γ-secretase activity when appropriate signals appear enabling a temporal level of regulation. In this review article, we discuss the latest developments on GSMPs and implications on the development of effective therapeutics for γ-secretase-associated diseases such as AD and cancer. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane (IFITM) proteins as host restriction factors are known to inhibit the replication of several viruses. In this study, transient IFITM expression vectors were used to investigate whether IFITMs inhibit feline foamy viral (FFV) replication and which step of viral replication is inhibited. In our studies, viral production was significantly reduced when cells were infected with FFV at almost same times such as −3, 0, or 3 h post-transfection with IFITM vector. However viral production was not reduced even though cells were infected with FFV at 3 or 6 days post-transfection when production of IFITM proteins was maximized. Considering that IFITM expression was maximized at 3 days post-transfection, the stage of viral replication inhibited by IFITM appears to be the late step of viral replication. Moreover, the viral Gag proteins detected in the virus-infected cell lysates were proportionally correlated with viral titer of the culture supernatants. Therefore, it is likely that IFITMs can restrict production of FFV at the late step of viral replication. [ABSTRACT FROM AUTHOR]

Sexual transmission of HIV-1 consists of processes that exert either positive or negative selection pressure on the virus. The sum of these selection pressures lead to the transmission of only one specific HIV-1 strain, termed the transmitted founder virus. Different dendritic cell subsets are abundantly present at mucosal sites and, interestingly, these DC subsets exert opposite pressure on viral selection during sexual transmission. In this review we describe receptors and cellular compartments in DCs that are involved in HIV-1 communication leading to either viral restriction by the host or further dissemination to establish a long-lived reservoir. We discuss the current understanding of host antiretroviral restriction factors against HIV-1 and specifically against the HIV-1 transmitted founder virus. We will also discuss potential clinical implications for exploiting these intrinsic restriction factors in developing novel therapeutic targets. A better understanding of these processes might help in developing strategies against HIV-1 infections by targeting dendritic cells.

HIV-1 is a complex retrovirus that is adapted to replicate in cells of the immune system. To do so, HIV-1, like other viruses, developed strategies to use several cellular processes to its advantage, but had also to come to terms with an arsenal of cellular innate defense proteins, or antiviral factors, that target more or less efficiently, virtually every step of the virus replicative cycle. Among antiviral restriction factors, the family of interferon-induced transmembrane proteins (IFITMs) has emerged as a crucial component of cellular innate defenses for their ability to interfere with both early and late phases of viral replication by inhibiting cellular and viral membranes fusion. Here, we review the enormous advances made since the discovery of IFITMs as interferon-regulated genes more than thirty years ago, with a particular focus on HIV-1 and on the elements that modulate its susceptibility or resistance towards members of this family. Given the recent advances of the field in the elucidation of the mechanism of IFITM inhibition and on the mechanism(s) of viral resistance, we expect that future years will bring novel insights into the definition of the multiple facets of IFITMs and on their possible use for novel therapeutical approaches.

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HAmediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K582I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells. [ABSTRACT FROM AUTHOR]

Sexual transmission of HIV-1 consists of processes that exert either positive or negative selection pressure on the virus. The sum of these selection pressures lead to the transmission of only one specific HIV-1 strain, termed the transmitted founder virus. Different dendritic cell subsets are abundantly present at mucosal sites and, interestingly, these DC subsets exert opposite pressure on viral selection during sexual transmission. In this review we describe receptors and cellular compartments in DCs that are involved in HIV-1 communication leading to either viral restriction by the host or further dissemination to establish a long-lived reservoir. We discuss the current understanding of host antiretroviral restriction factors against HIV-1 and specifically against the HIV-1 transmitted founder virus. We will also discuss potential clinical implications for exploiting these intrinsic restriction factors in developing novel therapeutic targets. A better understanding of these processes might help in developing strategies against HIV-1 infections by targeting dendritic cells. [ABSTRACT FROM AUTHOR]

Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses.

Interferon-induced transmembrane proteins (IFITMs) are a family of small proteins that localize in the plasma and endolysosomal membranes. IFITMs not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. Not surprisingly, some viruses can evade the restriction of IFITMs and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. Similar to many other host defense genes that evolve under the selective pressure of microorganism infection, IFITM genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (SNPs) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection (e.g., influenza A virus). Here, we review the function and potential impact of genetic variation for IFITM restriction of viral infections. Continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among IFITMs and viruses in an effort to determine their pathobiological roles in the context of viral infections in vivo.

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells. [ABSTRACT FROM AUTHOR]

Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are wellestablished immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus-cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

S-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. S-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. In this review, we focus on S-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. We discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. We highlight recent data on protein S-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. The role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed.

Avian Tembusu virus (ATMUV) is a highly pathogenic flavivirus that causes significant economic losses to the Chinese poultry industry. Our previous experiments demonstrated that ATMUV infection effectively triggered host innate immune response through MDA5 and TLR3-dependent signaling pathways. However, little information is available on the role of interferon-stimulated genes (ISGs) in defending against ATMUV infection. In this study, we found that ATMUV infection induced robust expression of type I and type III interferon (IFNs) in duck tissues. Furthermore, we observed that expression of interferon-inducible transmembrane proteins (IFITMs) was significantly upregulated in DEF and DF-1 cells after infection with ATMUV. Similar results were obtained from in vivo studies using ATMUV-infected ducklings. Importantly, we showed that knockdown of endogenous IFITM1 or IFITM3 by specific shRNA markedly enhanced ATMUV replication in DF-1 cells. However, disruption of IFITM2 expression had no obvious effect on the ATMUV replication. In addition, overexpression of chicken or duck IFITM1 and IFITM3 in DF-1 cells impaired the replication of ATMUV. Taken together, these results reveal that induced expression of avian IFITM1 and IFITM3 in response to ATMUV infection can effectively restrict the virus replication, and suggest that increasing IFITM proteins in host may be a useful strategy for control of ATMUV infection.

The interferon-inducible transmembrane (IFITM) proteins 1, 2 and 3 inhibit the host cell entry of several enveloped viruses, potentially by promoting the accumulation of cholesterol in endosomal compartments. IFITM3 is essential for control of influenza virus infection in mice and humans. In contrast, the role of IFITM proteins in coronavirus infection is less well defined. Employing a retroviral vector system for analysis of coronavirus entry, we investigated the susceptibility of human-adapted and emerging coronaviruses to inhibition by IFITM proteins. We found that entry of the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is sensitive to inhibition by IFITM proteins. In 293T cells, IFITM-mediated inhibition of cellular entry of the emerging MERS- and SARS-CoV was less efficient than blockade of entry of the globally circulating human coronaviruses 229E and NL63. Similar differences were not observed in A549 cells, suggesting that cellular context and/or IFITM expression levels can impact inhibition efficiency. The differential IFITM-sensitivity of coronaviruses observed in 293T cells afforded the opportunity to investigate whether efficiency of entry inhibition by IFITMs and endosomal cholesterol accumulation correlate. No such correlation was observed. Furthermore, entry mediated by the influenza virus hemagglutinin was robustly inhibited by IFITM3 but was insensitive to accumulation of endosomal cholesterol, indicating that modulation of cholesterol synthesis/transport did not account for the antiviral activity of IFITM3. Collectively, these results show that the emerging MERS-CoV is a target of the antiviral activity of IFITM proteins and demonstrate that mechanisms other than accumulation of endosomal cholesterol can contribute to viral entry inhibition by IFITMs.

S-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. S-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. In this review, we focus on S-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. We discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. We highlight recent data on protein S-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. The role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed. [ABSTRACT FROM AUTHOR]