Turnover rate of NS3 proteins modulates bluetongue virus replication kinetics in a host-specific manner

Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate

The sheep tetherin paralog oBST2B blocks envelope glycoprotein incorporation into nascent retroviral virions

Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the “tethering” mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A

Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles

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

Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs.

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

Genetic elements swapping indicates the mode of virion assembly as a dominant determinant in the virus susceptibility to IFITMs.

<p>Glycoproteins of IFITM-resistant viruses were used to pseudotype viral cores of IFITM-susceptible viruses (as indicated). HIV-1 virions presenting the HCV E1/E2 glycoproteins were produced in the presence of IFITMs by co-transfection of the respective DNAs (1:1 ratio). VSV pseudoparticles (VSVpp) incorporating the RVFV GnGc in the presence of IFITMs were produced in HEK293T transfected with DNAs coding for GnGc along with IFITMs (0.7:1 ratio), following challenge with a viral stock of ΔG-VSV virus that had been previously complemented with the G protein to allow its entry into cells. After entry, absence of G allows the production of novel virion particles that assembled in the presence of IFITMs and of the RVFV GnGc glycoprotein. Residual input virus was neutralized by incubation with an anti-G neutralizing antibody. Virion particles were purified from the supernatant of virus-producing cells, normalized and used to challenge target cells in a classical single round of infection prior to flow cytometry analysis. The panels present typical results, while the graphs present averages and SEM of 3 to 5 different experiments. *, p≤0.05 after a Student t test.</p

Silencing of endogenous IFITMs increases the infectivity of virions of different viruses and increases EBOV spread in primary macrophages.

<p>A) Endogenous IFITM1, 2 and 3 were silenced by shRNA-mediated lentiviral transduction along with shRNA-control silenced cells and then treated according to the scheme provided. B) Expression and extracellular release properties of endogenously-expressed IFITMs in the uninfected cell types used here (cells and sup., respectively). The basal expression levels of IFITM proteins were measured in the cell types mentioned above with a pool of anti-IFITM1, 2 and 3 antibodies in cell lysates and supernatants purified by ultracentrifugation through sucrose, as viral particles in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.g002" target="_blank">Fig 2</a>. The same amounts of cells used elsewhere for viral production and in the case of primary cells, the same donors were used. C) In the case of primary cells, primary blood lymphocytes were stimulated with 1 μg/ml PHA and 150 U/ml of Interleukin 2 (IL2) for twenty-four hours, then challenged with HIV-1 viral vectors expressing either control (Luciferase), or IFITMs-specific shRNAs and enriched in knockdown cells following a three-day selection in Puromycin (resistance coded by the shRNA vector). Kd-cells were then challenged with the indicated viruses at MOI comprised between 0.1 and 0.5 to obtain virus-producing cells, prior to extensive cell washing to remove input virus. Newly produced virion particles were collected 1 to 2 days afterwards (5 days for HIV-1), purified and normalized prior to WB and infectivity analyses. The infectivity of virions purified from kd-cells was measured on HeLaP4 (HeLa cells bearing the HIV-1 receptors and an LTR-driven promoter driving β-galactosidase expression, used for HIV-1 and VSV and analyzed twenty-four and sixteen hours after challenge by β-gal assay and FACS, respectively) or Vero/hSLAM (MeV, flow cytometry). Primary macrophages were challenged with shRNA-coding vectors in the presence of Vpx-containing virion-like particles (VLPs-Vpx) to increase the efficiency of silencing and then challenged with EBOV at an MOI of 0.3. Pictures of infected cultures were collected with a Leica DM IRB inverted microscope. The graphs present averages and SEM of 4 to 5 independent experiments with cells obtained from different donors. *, p≤0.05 after a Student t test.</p

CCR5 usage relieves the negative effects of IFITM3 on HIV-1 replication and on its ability to decrease the virion particles infectivity.

<p>A) Human CCR5 was introduced in the IFITM3-stable SupT1 cells used before, by retroviral-mediated gene transduction and cells were challenged with the indicated viruses. HIV spreading was assessed by exo-RT activity over time (day 0 through 7). The panels and the histogram overlay present the patterns of expression obtained for IFITM3 and CCR5 following WB and flow cytometry analyses. The graph presents normalized data obtained in 2 to 3 independent experiments. B) Virions obtained at late times after infection were harvested, normalized and used to infect HeLaP5 cells that contain a β-galactosidase reporter gene under the control of the HIV-1 LTR.</p

IFITM3 is a <i>bona fide</i> virion-associated protein.

<p>Virion particles produced as described above were then analyzed by immuno-gold electron microscopy. Briefly, unfixed viral preparations purified by ultracentrifugation and produced in the presence or absence of IFITM3 were incubated with anti-Flag antibodies, followed by incubation with a gold-conjugated secondary antibody (arrows). Representative pictures are shown here. The graph displays the number of gold particles counted on a per virion basis.</p