IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion

Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3\u27s ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes

Prospective evaluation of sexual function after open and laparoscopic surgery for rectal cancer

Sexual function may be harmed after treatment for rectal cancer. This study aimed to evaluate prospectively the incidence of sexual dysfunction after rectal cancer treatment and to compare the effects of laparoscopic and traditional open approaches in terms of postoperative sexual function. Baseline and 3-, 6-, and 12-month assessments of sexual dysfunction using the International Index of Erectile Function (IIEF) and its specific domains prospectively took place for 56 patients who underwent rectal cancer surgery (38 open vs. 18 laparoscopic procedures, 38 low anterior vs. 18 abdominoperineal resections). The preliminary results are presented. The average total IIEF and isolated IIEF response domain scores were significantly decreased after surgery (p < 0.01) except for the intercourse satisfaction and overall satisfaction scores at 12 months. An improvement in IIEF scores was observed between the 3- and 6-month assessment points (p < 0.01) except for the erectile function and orgasmic function scores. No significant differences were observed between the open and laparoscopic groups in the total IIEF and domain scores preoperatively and at the 3- and 6-month assessment points. The rates of sexual dysfunction did not differ significantly preoperatively or at 3 months postoperatively when open and laparoscopic procedures were compared, although there was a trend in favor of laparoscopic surgery at 6 months (p = 0.076). The baseline IIEF score and the baseline, 3-, and 6-month sexual desire scores were better (p = 0.035, 0.004, 0.017, and 0.061, respectively) in the low anterior resection group than in the abdominoperineal resection group. Rectal cancer resections were postoperatively associated with a significant reduction in IIEF scores and high rates of sexual dysfunction at 3 and 6 months. The IIEF and domain scores at different assessment points were comparable between the laparoscopic and open surgery groups. Extending the monitoring period and adding more patients in this ongoing prospective study will further elucidate postoperative sexual dysfunction after rectal cancer surgery

The IFITMs Inhibit Zika Virus Replication

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

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

International audienc

Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration

Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’s integration into gene-dense regions

IFITM-mediated restriction of virus-endosome fusion in different cell types.

<p>(A) IFITM3-mediated inhibition of viral fusion with different cell types. BlaM-Vpr carrying pseudoviruses (IAVpp, VSVpp and LASVpp, MOI = 1) were bound to IFITM3- or vector-transduced A549, MDCK, CV1, HeLaH1 or CHO cells in the cold. Fusion was allowed to proceed for 90 min at 37°C and was measured by the BlaM assay, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#s4" target="_blank">Materials and Methods</a>. ND, not determined. Data are means and SEM from 2 independent triplicate experiments. (B) IFITM3 expression patterns in A549, MDCK and CHO cells transduced with an empty vector (left) or IFITM3 (right). Cells were fixed, permeabilized and immunostained for IFITM3 (red), as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#s4" target="_blank">Materials and Methods</a>. The nuclear stain, Hoechst-3342, is shown in blue. (C) IFITM3 restricts fusion of influenza virus-like particles containing β-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1). Experiments were carried out as described above. Data are means and SEM from 2 independent triplicate experiments. (D) Exposure to low pH overcomes the IFITM3-mediated block of IAVpp fusion. To force pseudovirus fusion at the plasma membrane, A549 cells transduced with IFITM1, IFITM3 or an empty vector were pretreated with 50 nM BafA1 for 30 min at 37°C or left untreated. IAVpp/BlaM-Vpr pseudoviruses (MOI = 1) were bound to cells of in the cold and exposed to either a pre-warmed pH 5.0 MES-citrate buffer or neutral buffer for 10 min at 37°C and further incubated in growth medium (with or without BafA1) for 90 min at 37°C. Data are means and SEM from 2 independent triplicate experiments. ***, P<0.001 by two-tailed t-test.</p

IFITM3 blocks fusion pore formation between single influenza viruses and endosomes.

<p>Pseudoviruses bearing WSN HA and NA glycoproteins were co-labeled with HIV-1 Gag-iCherry (viral content marker, red) and YFP-Vpr (viral core marker, green). Viruses were pre-bound in the cold to A549-Vector cells (A, B) or MDCK-Vector cells (C, D) and their entry was initiated by raising the temperature. (A, C) Images of IAVpp are extended projections of 3 Z-stacks illustrating the loss of the mCherry signal (arrow) upon virus-endosome fusion. A schematic illustration between image panels A and C illustrates fusion between the YFP-Vpr (green) and Gag-iCherry (red) labeled IAVpp and an endosome (gray). (B, D) Mean mCherry and YFP fluorescence intensities obtained by tracking the particles shown in panels A and C. (E) Normalized efficiencies of IAVpp fusion (content release) with A549 and MDCK cells transduced with an empty vector or with IFITM3. The middle bar shows the lack of mCherry release in A549-Vector cells in the presence of NH₄Cl. ***, P<0.001. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#ppat.1004048.s016" target="_blank">movies S4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#ppat.1004048.s017" target="_blank">S5</a>.</p

Models for IFITM3-mediated restriction of IAV infection.

<p>Purple arrows illustrate possible mechanisms of the IAV restriction by IFITM3: direct (Pathway 1) and indirect (Pathway 2) inhibition of transition from hemifusion to full fusion at the limiting membrane of an endosomes, as well as non-productive IAV fusion with ILVs in the absence of back fusion (Pathway 3). Partial dilution/dequenching of viral vDiD upon hemifusion/fusion is shown by lighter red color and full dequenching is shown by light red glow. Alternative endosomal localizations of IFITM3 (limiting membrane vs. ILVs) are shown. Dashed black arrow illustrates possible IAV fusion pathway in cells expressing low, endogenous levels of IFITM3.</p