IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle. IFITMs are interferon-regulated proteins that inhibit a broad range of viruses. Until recently, IFITMs had been described to arrest incoming viral particles in target cells, by inducing their retention in endosomal vesicles. More recently in the case of HIV-1, ours and other laboratories have highlighted the existence of an additional antiviral mechanism with which IFITMs could act in virus-producing cells, leading to the production of virion particles of reduced infectivity. In the present study, we assessed whether the negative imprinting of the virion particles infectivity was a conserved antiviral property of IFITMs by examining a panel of fourteen different DNA or RNA viruses. Our results indicate that a wide spectrum of viruses is susceptible to this antiviral mechanism of inhibition, although some are able to resist it. Swapping of elements between susceptible and resistant viruses strongly suggests that specificities in the mode of virion assembly and not the viral glycoprotein are the dominant factor in the susceptibility of a given virus to this inhibition. However, we also show that HIV-1 strains that engage the CCR5 co-receptor display a notable resistance towards IFITM3, indicating that at least in the case of HIV-1, co-receptor usage is likely to add an additional layer of complexity in the relationship established between IFITMs and the virus, that may or may not extend to other viral families as well. In the context of spreading infections, the results of this study highlight that the overall antiviral effect of IFITMs is mechanistically caused by a previously unappreciated dual mode of action in which they act both in target cells and in virus-producing cells, by respectively forcing endosome trapping of incoming viruses and by commandeering the formation of new virion particles of reduced infectivity. Overall, the results presented here indicate that the negative imprinting of viral particles is a largely conserved antiviral feature of IFITMs and point to IFITMs as a novel paradigm of innate defense proteins capable of interfering with viral replication at two distinct steps of a virus life cycle.

CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55Gag^HIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV¹) or West Nile virus Kunjin (WNV^Kun). The two resulting chimeric proteins, DIII-DENV¹-RIgE and DIII-WNV^Kun-RIgE, were addressed to the plasma membrane, exposed at the surface of human and insect cells, and incorporated into extracellular VLPs when coexpressed with Pr55Gag^HIVin insect cells. The DIII domains were accessible at the surface of retroviral VLPs, as shown by their reactivity with specific antibodies, and notably antibodies from patient sera. The DIII-RIgE proteins were found to be incorporated in VLPs made of SIV, MLV, or chimeric MLV-HIV Gag precursors, indicating that DIII-RIgE could pseudotype a wide variety of retroviral VLPs. VLP-displayed DIII were capable of inducing specific neutralizing antibodies against DENV and WNV in mice. Although the neutralization response was modest, our data confirmed the capability of DIII to induce a flavivirus neutralization response, and suggested that our VLP-displayed CD16-RIgE-based platform could be developed as a vaccine vector against different flaviviruses and other viral pathogens.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA ^GFP-CFTR ) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR). Both microvesicles and exosomes had the capacity to package and deliver the GFP-CFTR glycoprotein and mRNA ^GFP-CFTR to target cells in a dose-dependent manner. Homologous versus heterologous EV-to-cell transfer was studied, and it appeared that the cellular uptake of EVs was significantly more efficient in homologous transfer. The incubation of CF15 cells, a nasal epithelial cell line homozygous for the ΔF508 CFTR mutation, with microvesicles or exosomes loaded with GFP-CFTR resulted in the correction of the CFTR function in CF cells in a dose-dependent manner. A time-course analysis of EV-transduced CF cells suggested that CFTR transferred as mature glycoprotein was responsible for the CFTR-associated channel activity detected at early times posttransduction, whereas GFP-CFTR translated from exogenous mRNA ^GFP-CFTR was responsible for the CFTR function at later times. Collectively, this study showed the potential application of microvesicles and exosomes as vectors for CFTR transfer and functional correction of the genetic defect in human CF cells.

In rheumatoid arthritis (RA), the proliferation of fibroblast-like synoviocytes (FLS) is the cause of chronic inflammation in joints and of joint damage. Delivery of the pro-apoptotic gene PUMA to FLS via human adenovirus type 5 (HAdV5) vectors has been tested as a therapeutic approach, but efficiency is hampered by low transduction, as FLS do not express HAdV5 receptors on the cell surface. Here we show that efficient transduction of PUMA in FLS can be achieved by conjugating HAdV5 to a baculovirus, which binds to the cell surface via the envelope glycoprotein Gp64. Intra-articular injection in an adjuvant-induced rat model of RA induces apoptosis of FLS, leading to significant decrease in joint inflammation, joint damage, and bone loss with improvement in joint function and mobility. Our results demonstrate the therapeutic potential of PUMA gene therapy as a local treatment in various forms of arthritis in which abnormal FLS proliferation is implicated.

Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.