Nuclear aggregates induced by HSV1-XFPVP26 impair nuclear capsid egress.

<p>Vero cells were infected (inf.) with 10 PFU/cell of HSV1(17⁺)blueLox (A, wild type), HSV1(17⁺)blueLox-mRFPVP26_{Δaa<b>1</b>–7} (B), or HSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7}(C), and fixed at 9 h with PFA. Alternatively, cells were transfected (transf.) with pHSV1(17⁺)blueLox-GFPVP26_{Δaa<b>5</b>–7} (D) or pHSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7} (E), and fixed at 24 h. In addition to the intrinsic fluorescence of the XFPVP26 constructs (mRFPVP26 or GFPVP26), the subcellular localization of VP26 (α-VP26) and VP5 (MAb 5C10) were analyzed after permeabilization with TX-100 and immunolabeling by confocal fluorescence microscopy. The nuclei were stained with TO-PRO-3 (A, E). Arrows highlight cytoplasmic capsids (A–D) or incoming capsids at the nuclear rim of a neighboring cell (Ei). Scale bar: 10 µm.</p

Nuclear GFPVP26 aggregates do not sequester pUL36 and are not DNA replication compartments.

<p>Vero cells were infected (inf.) for 9 h with 10 PFU/cell of HSV1(17⁺)blueLox (A) or HSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7} (B, D), or transfected (transf.) with the pHSV1(17⁺)blueLox-GFPVP26_{Δaa<b>5</b>–7} for 24 h(C, E). The cells were fixed with PFA and permeabilized with TX-100. GFPVP26 was detected by its intrinsic fluorescence. Furthermore, the cells were labeled with anti-pUL36_{aa1408–2112} and with MAb 5C10 against VP5 (A–C), against the single-strand DNA binding protein ICP8 and VP26 (D, E), or with TO-PRO-3 to stain the DNA (A). The arrows point to cytoplasmic capsids. Scale bar: 10 µm.</p

Characterization of HSV1(17⁺)blueLox-VP26 strains.

<p>(A) Restriction digestion analysis of different BAC clones with <i>Not</i>I and <i>Eco</i>RI. Band shifts resulting from modifications in the UL35 ORF (arrowheads) and the <i>Not</i>I joint fragments containing the viral a-sequences (asterisks) are indicated. DNA sizes in kb. (B) For single-step growth kinetics, Vero cells were infected in duplicates with 10 PFU/cell, and the amount of secreted infectious virus at a given time point was determined by duplicate plaque assays. (C) Vero cells were infected at an MOI of 10 PFU/cell with HSV1(17⁺) or HSV1(17⁺)blueLox and its derivatives as indicated. After 48 h, the cells and virions secreted into the culture medium were harvested, and analyzed by SDS-PAGE and immunoblotting for expression of VP5 (α-NC-1) and VP26 (α-VP26). The signals in the molecular weight range below 25 kDa were further contrast enhanced.</p

Effect of BA on HDV infection of HuH-7/hNTCP cells.

<p>HuH-7/hNTCP cells were exposed to HDV containing supernatant in the presence or absence of 200 µM of different BA. For glycocholic acid 50 µM results are shown because the BA is toxic at 200 µM. preS1 peptide (368 nM) and cyclosporine A (25 µg/mL) served as positive controls. After 6 h cells were washed and repleted with regular media without virus, BA or drugs. After another 5 d cells were fixed and stained with a polyclonal antibody against HDV antigen (right hand images in each panel). Nuclei were counterstained with DAPI (left hand images). A representative of at least three independent experiments is shown.</p

HSV1-pUL20 is not required for directed intracellular transport in epithelial cells.

<p>Vero cells were infected with 6.4 x 10⁶ pfu/ml (10 pfu/cell) of HSV1(17⁺)Lox-mCheVP26 (Ai), -ΔUL36 (Aii), -ΔUL37 (Aiii), or -ΔUL20 (Aiv) and movies were acquired at 8–10 hpi. (A) Stills of movies in Vero cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006813#ppat.1006813.s005" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006813#ppat.1006813.s008" target="_blank">S4</a> Movies) and with representative track profiles (indicated by a colored line and the front of each track marked by an open circle). Scale bar, 5 μm. (B) Analysis of track profiles of HSV1(17⁺)Lox-mCheVP26 (parental), -ΔUL36, -ΔUL37, or –ΔUL20. (Bi) Track length and (Bii) maximum step velocity of tracks with a MSD_ex ≥ 1.2, (Biii) mean square displacement exponent (MSD_ex), with each dot representing one track. Box and whiskers with min and max.</p

HSV1-pUL36, pUL37 and pUL20 are required for efficient targeting of capsids to axons.

<p>DRG neurons were infected after 3 div with 1 x 10⁷ pfu/mL of HSV1(17⁺)Lox, Lox-ΔUL36, Lox-ΔUL37, or Lox-ΔUL20 (or mock treated, fixed and permeabilized using the PHEMO protocol at 24 to26 hpi, labeled with antibodies directed against VP26 (pAb VP26_aa95-112) and gD (mAb DL6, A-D) or VP22 (mAb 22–3, E-H), and analyzed by confocal fluorescence microscopy. The images were quantified with a semi-automated algorithm. Each bar represents the mean from one experiment, normalized to the parental values. (A-D) Labeling with anti-VP26 and anti-gD: (A) Total number of VP26 positive structures. (B) VP26 and gD double positive structures. (C) total gD positive structures (D) Size of gD-positive structures. (E-H) Labeling with anti-VP26 and anti-VP22: (E) Total VP26 positive structures. (F) Total number of structures labeled for VP26 and VP22. (G) Total number of structures labeled for gD. (D) Area of structures labelled for gD. The number of capsids, gD or VP22 structures, image numbers and total axon length are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006813#ppat.1006813.s013" target="_blank">S1 Table</a>.</p

Cytosolic capsids accumulate in the absence of pUL36 or pUL37 and pUL20 is required for secondary envelopment in neurons.

<p>DRG neurons infected with 1 x 10⁷ pfu/mL of HSV1(17⁺)Lox-CheVP26 (A), -ΔUL36 (B), -ΔUL37 (C), -ΔUL20 (D), fixed at 24 or 30 hpi and processed for conventional electron microscopy. Nucleus (N), primary enveloped virions (white stars), cytosolic capsids (white arrowheads), wrapping intermediates with capsids being closely associated with cytoplasmic membranes (black arrowheads), virions after complete secondary envelopment (black star), and extracellular virions (arrow). Scale bar is 500 nm.</p