RNA binding specificity of Ebola virus transcription factor VP30

<p>The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3′-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3′-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125–80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3′-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2′-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the <i>Paramyxoviridae</i> which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.</p

rMARV_PSAPmut exhibits delayed growth kinetics.

<p>Vero E6 cells were inoculated with either rMARV_PSAPmut or rMARV_wt. Supernatants and cell lysates were collected at indicated time points p.i. and viral titres determined by TCID₅₀ assay or subjected to Western blot analysis. (<b>A</b>) Growth kinetics of rMARV_PSAPmut (grey circle) or rMARV_wt (black square) at MOI of 0.01. (<b>B</b>) Western Blot analysis of viral protein levels during an infection at MOI of 0.01. Cell lysates and culture supernatants were collected at indicated time points and were analyzed by SDS-PAGE and Western Blotting using NP- and VP40-specific antibodies. (<b>C</b>) Growth kinetics of rMARV_PSAPmut (grey circle) or rMARV_wt (black square) at MOI of 0.1. (<b>D</b>) rMARV_PSAPmut- or rMARV_wt– or mock-infected cells were analyzed for CPE formation during infection at MOI of 0.1 at 3 days p.i. (<b>E</b>) Western Blot analysis of viral protein levels during an infection at MOI of 0.1. Cell lysates and culture supernatants were collected at indicated time points and were analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004463#ppat-1004463-g001" target="_blank">Fig. 1B</a>. P-values are indicated (_*, P≤0.05; _**, P≤0.001; _***, P≤0.0001).</p

Interaction with Tsg101 Is Necessary for the Efficient Transport and Release of Nucleocapsids in Marburg Virus-Infected Cells

<div><p>Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV_PSAPmut). rMARV_PSAPmut was attenuated by up to one log compared with recombinant wild-type MARV (rMARV_wt), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV_PSAPmut-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV_wt-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV_wt-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV_PSAPmut nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.</p></div

Inclusions in rMARV_PSAPmut–infected cells are more densely packed with nucleocapsids than inclusions in rMARV_wt–infected cells.

<p>Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut. At 28 h p.i., cells were processed in two ways (i) fixed, scraped, pelleted and then embedded in Epoxy resin (A and C); or (ii) fixed and embedded in Epoxy resin on Thermanox slides (B and D). Ultrathin sections were stained with uranyl acetate and subjected to electron microscopy. (<b>A–B</b>) rMARV_wt–infected cells, (<b>C–D</b>) rMARV_PSAPmut–infected cells. Bars, 500 nm. (<b>E</b>) Morphometric analysis of inclusions. Volume density of nucleocapsids inside inclusions is shown, p-value (***, p≤0.0001). (<b>F</b>) Amount of electron dense (mature) nucleocapsids inside inclusions (see black arrows <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004463#ppat-1004463-g006" target="_blank">Fig. 6B und D</a>) determined per 2.5 µm² of inclusion at electron micrographs, p-value (*, p≤0.05).</p

Budding from filopodia is reduced in rMARV_PSAPmut-infected cells.

<p>(<b>A</b>) Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut at an MOI of 1, and fixed at 19–23 h p.i. The whole mounted cells were analyzed by electron microscopy on grids after negative staining. Graphics shows the percentage of nucleocapsids in the process of budding from the filopodia (20 micrographs were analyzed for each virus), p-value (*** P≤0.0001). (<b>B</b>) Western Blot analysis of viral proteins in cell lysates. Huh-7 cells were infected as indicated in (A), harvested at 19 h p.i., and analyzed by using NP- and VP40-specific antibodies. (<b>C</b>) Live cell imaging of Huh-7 cells were infected with rMARV_wt- or rMARV_PSAPmut. At 28 h (rMARV_PSAPmut) and 43 h p.i. (rMARV_wt), a series of 600 pictures was taken every second for a period of 10 min. Maximal projection of the picture series is displayed. Boxes in the left panels indicate areas that are shown at higher magnification in the middle panels. Trajectories of individual nucleocapsids are highlighted with white dashed lines. White asterisks indicate regions with several static rMARV_PSAPmut nucleocapsids. Right panels show the trajectories of nucleocapsids in rMARV_wt- or rMARV_PSAPmut-infected cells. Bars, 10 µm. (<b>D</b>) Length of nucleocapsid trajectories. The length of nucleocapsid trajectories was measured in rMARV_wt- or rMARV_PSAPmut-infected cells using the Leica LAS AF software, p-value (***, p≤0.0001). (<b>E</b>) Co-localization of Tsg101-Venus1/2 with MARV inclusions and nucleocapsids. Huh-7 cells were infected either with rMARV_wt or rMARV_PSAPmut and transfected with Venus1-Tsg101 and Venus2-Tsg101 plasmids. Cells were fixed at 22 h p.i. and subjected to immunofluorescence analysis with a NP-specific antibody. The Tsg101-Venus1/2 signal is displayed in green and the NP signal in red. Merged pictures show the overlay. The grey boxes indicate marginal region of cells, which are shown at higher magnification in the right panels. Arrows indicate nucleocapsids (approximately 1 µm in length).</p

IQGAP1 is co-localized with inclusions and nucleocapsids and supports MARV release.

<p>(<b>A</b>) Co-localization of Tsg101 and IQGAP1 in NP inclusions. Huh-7 cells were co-transfected with plasmids encoding NP_wt or NP_PSAPmut and VP40, mCherry-Tsg101 and IQGAP1-YFP. Cells were fixed 24 h p.tr., stained with NP-specific antibody and subjected to immunofluorescence analysis. White arrow shows co-localization of mCherry-Tsg101 (red), IQGAP1-YFP (green) with NP inclusions (blue) in the merge picture. In NP_PSAPmut transfected cells only mCherry-Tsg101 and IQGAP1-YFP are co-localized (arrowhead). (<b>B</b>) Co-localization of Tsg101 and IQGAP1 in MARV infected cell. Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut and co-transfected with mCherry-Tsg101 and IQGAP1-YFP encoding plasmids. Cells were fixed 24 h p.i. stained with NP-specific antibody and analyzed by CLSM. Lower panels show higher magnification of boxed area and white arrow indicates co-localization of nucleocapsid with mCherry-Tsg101 and IQGAP1-YFP in wild type infected cells whereas mutant nucleocapsids did not show any co-localization. (<b>C</b>) IQGAP1 depletion of infected cells. MARV-infected Huh-7 cells (MOI of 1) were transfected with IQGAP1-specific siRNA or control siRNA at 1 h p.i. Cells and culture supernatants were harvested at 48 h and 72 h p.i. Lysates and supernatants collected at 72 h p.i. were subjected to Western Blot analysis. (<b>D</b>) Virus titers in the supernatants of MARV infected cells transfected with IQGAP1-specific or control siRNA were determined by TCID₅₀ titration, p-value (_*, P≤0.05). (<b>E</b>) Phenotype of IQGAP1-knockdown in MARV-infected cells at 48 h p.i. Huh-7 cells grown on cover slips were infected with MARV_wt and treated with IQGAP1 specific or control siRNA and subjected to immunofluorescence analysis using NP-specific antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrow indicates accumulation of nucleocapsids upon IQGAP1 knockdown at cell periphery marked with dashed line. Bars, 10 µm.</p

Tsg101 knockdown in MARV-infected cells results in reduced particle release.

<p>MARV-infected Huh-7 cells (MOI of 1) were transfected twice with Tsg101-specific siRNA or control siRNA (1 h and 18 h p.i.). Cells and virus particles were harvested at 48 h p.i. (<b>A</b>) Quantification of Tsg101 protein level was performed by Western Blot in cells transfected with Tsg101-specific siRNA and control siRNA. Tsg101 levels in cells transfected with control siRNA (ctr.) were set to 100%. (<b>B</b>) Lysates and supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were subjected to Western Blot and analyzed for the presence of viral proteins NP and VP40 and Tsg101. (<b>C</b>) The 65 kDa form of Tsg101 is ubiquitinated. HEK293 cells were transfected with Tsg101-Flag and HA-Ubiquitin expression plasmids. At 48 h p.tr., cell lysates were subjected to immunoprecipitation with anti-HA-agarose. Cell lysates and precipitates were separated by SDS-PAGE and analyzed by Western Blot using HA- and Tsg101-specific antibodies. The position of the ubiquitinated Tsg101 (Ub-Tsg101) band is indicated by an arrow between 55 and 70 kDa. (<b>D</b>) Virus titers in the supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were determined by TCID₅₀ titration, p-value (_*, P≤0.05). (<b>E</b>) <b>P</b>henotype of Tsg101 knockdown in MARV-infected cells. Huh-7 cells were infected with MARV and treated with Tsg101 specific or control siRNA and subjected to immunofluorescence analysis using a guinea pig anti-NP and secondary goat anti-guinea pig FITC-conjugated antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrows indicate accumulation of nucleocapsids upon Tsg101 knockdown. Bars, 10 µm. (<b>F</b>) Western blot analysis of Tsg101 knockdown. Cells transfected with Tsg101-specific or control siRNA were analyzed by Western Blot using Tsg101- and tubulin-specific antibodies.</p

rMARV_PSAPmut-infected cells contain more nucleocapsids in the cytoplasm and at early steps of budding than rMARV_wt-infected cells.

<p>Huh-7 and Vero E6 cells were infected with rMARV_PSAPmut- or rMARV_wt, fixed at 26 h p.i and embedded in epoxy resin. Cells were analyzed by thin section transmission electron microscopy (<b>A–D</b>) or electron tomography (<b>E–J</b>). (<b>A–B</b>) rMARV_wt-infected Huh-7 cell displaying free virions (black arrows) and a nucleocapsid in the cytoplasm near the plasma membrane (white arrow in A). (<b>C–D</b>) rMARV_PSAPmut-infected Huh-7 cells displaying fully protruded (grey arrows) or partially protruded (blue arrow in D) virus buds, and nucleocapsids bound to the plasma membrane (light blue arrows in C) or in the cytoplasm near the plasma membrane (white arrow in D). (<b>E</b>) 10 nm digital z-slice of an electron tomogram showing several nucleocapsids in the process of budding or in fully protruded virus buds in the periphery of rMARV_PSAPmut-infected Huh-7 cell. (<b>G</b>) 9 nm digital z-slice of an electron tomogram showing accumulated nucleocapsids in the cytoplasm of rMARV_PSAPmut-infected Huh-7 cell. (<b>F, H</b>) 3D surface representations of nucleocapsids (blue) and cytoplasm (yellow, semi-transparent) in the full tomograms for which z-slices are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004463#ppat-1004463-g007" target="_blank">Fig. 7E and 7G</a>, respectively. Bars, 500 nm. (<b>I–J</b>) Quantification of the nucleocapsid distribution in tomograms from 300 nm thick sections of rMARV_wt- or rMARV_PSAPmut-infected Huh-7 or Vero E6 cells. Intracellular nucleocapsids (including cytoplasmic and those bound to plasma membrane, or partially extruded nucleocapsids) and fully extruded nucleocapsids were counted in a set (5 or more) of representative tomograms (p-value, *P≤0.05).</p

Infection with MARV_PSAPmut results in compact inclusion bodies and accumulation of nucleocapsids in the periphery of cells.

<p>Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut, fixed at 24 h p.i. and subjected to immunofluorescence staining using NP-specific antibodies. Samples were then analyzed by confocal laser scanning microscopy. Left panels: rMARV_wt infection. Right panels: rMARV_PSAPmut infection. Grey boxes in the upper pictures indicate different regions of the same cell that are shown in higher magnification below. (<b>A</b>) and (<b>B</b>) periphery of cells. (<b>C</b>) and (<b>D</b>) inclusion bodies. Arrows indicate nucleocapsids.</p

rMARV_PSAPmut particles incorporate less Tsg101 and display similar infectivity.

<p>(<b>A</b>) Tsg101 incorporation into MARV particles. Vero E6 cells were infected with rMARV_wt or rMARV_PSAPmut and virus particles released into the supernatant were pelleted through a 20% sucrose cushion at 48 h p.i. Virus pellets and cell lysates were subjected to SDS-PAGE and Western Blot analysis using Tsg101-specific antibody. (<b>B</b>) Detection of ubiquitinated form of Tsg101 in viral particles. Vero E6 cells were infected with rMARV_wt or rMARV_PSAPmut and subsequently transfected with HA-Ub expression plasmid. Virus particles were pelleted from the supernatants and analyzed by SDS-PAGE and Western Blot analysis using anti-Tsg101 and anti-HA specific primary antibodies and secondary antibodies for detection with the Odyssey imaging system (see merge image). (<b>C, D</b>) Comparison of virus infectivity. (<b>C</b>) Equal amounts of TCID₅₀ units of rMARV_PSAPmut and rMARV_wt stock viruses were pelleted through 20% sucrose cushion, separated by SDS-PAGE and analyzed by Western Blot using NP- and VP40-specific antibodies. (<b>D</b>) Huh-7 cells grown on glass cover slips were inoculated with rMARV_PSAPmut and rMARV_wt normalized to nucleoprotein amount, fixed at 17 h p.i. and stained with DAPI and NP-specific antibody for detection of infected cells by immunofluorescence assay.</p