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

Quantification of nuclear aggregate formation.

<p>Vero cells were infected with 10 PFU/cell of HSV1(17⁺)blueLox (B) or HSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7}-gDmRFP (C), and the cells were fixed at 4, 6, 8, 10, or 12 h. After permeabilization, HSV1(17⁺)blueLox infected cells were labeled with an antibody directed against VP26. According to their intranuclear VP26 phenotype, the cells were classified into “none”, “single”, “grainy” and “aggregate” (A). The numbers above the columns describe the number of nuclei analyzed for each time point.</p

HSV1-VP26 constructs.

<p>(A) 1^st column: HSV1 constructs in which the SCP VP26 has been tagged with different fluorescent protein domains. 2^nd column: Genomic organization of the UL35 region approximately drawn to scale. The gene UL35 coding for VP26 has been disrupted by replacing it with lacZ or an rpsLneo cassette out of frame. Some constructs lack a 65 bp region upstream of UL35 (D65 bp) including the first seven N-terminal codons of VP26 (Daa1–7), while others lack only four (Δaa1–4) or just one (Daa1) codon. For the present study, the fluorescent protein tag was inserted between VP26 residues 4 and 8 (Daa5–7). Due to the mutagenesis, some strains contain additional linkers (*, AW; **, NSS; ***, HST). 3^rd column: Propensity of the fluorescent protein (FP) to dimerize <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044177#pone.0044177-Espagne1" target="_blank">[76]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044177#pone.0044177-Campbell1" target="_blank">[80]</a>. 4^th column: Ability of the construct to replicate and to form plaques (+++, similar to wild type; ++ attenuated, but robust growth; − strongly attenuated, tiny plaques; −−, single fluorescent cells, no plaques). 5^th column: Propensity of the construct to induce nuclear aggregates (+++, large irregular shaped aggregates; ++, large aggregates early after infection or transfection; +, aggregates late in infection; − aggregates in less than 2% of cells even late in infection). 6^th column: References. (B) Nucleotide (upper lines) and amino acid (lower lines) sequences of the UL34/UL35 (pUL34/VP26) intergenic region. The 3¢ end of the UL34 ORF until the 5¢ start of the UL35 ORF are shown for wild type HSV-1, the GFPVP26_{Äaa<b>5</b>–7} (Äaa5–7) and GFPVP26_{Äaa<b>1</b>–7} (Äaa1–7) mutants. Additional nucleotides inserted during mutagenesis are shown in bold capitals, and the GFP amino acids are shown in italics. Putative Inr late promoter elements are underlined with the element perfectly matching the consensus sequence being underlined and in italics. The original amino acids encoded by UL35 are shown in bold capitals, the inserted GFP residues in italic capitals and the additional linker residues in normal script capitals.</p

Characterization of HSV1-GFPVP26 nuclear aggregates.

<p>Vero cells were infected with HSV1(17⁺)blueLox-GFPVP26_{Daa<b>1</b>–7} at an MOI of 10 PFU/cell and fixed at 9 h, or transfected with pHSV1(17⁺)blueLox-GFPVP26_{Daa<b>5</b>–7} or -GFPVP26_{Daa<b>1</b>–7} and fixed at 24 h. The cells had been fixed with PFA unless indicated otherwise, and subsequently labeled with different antibodies. MeOH, methanol fixation; PHEMO, PHEMO fixation; –, not detected.</p

Oligonucleotides used for BAC-mutagenesis.

<p>Homology to the <i>galK-kan</i> selection cassette is underlined.</p><p>Coding sequence of the SV40NLS is bold, Start sites are bold and underlined.</p><p>Duplications are indicated by small letters.</p><p>Oligonucleotides used for BAC-mutagenesis.</p

N-terminal mutants of pUL31.

<p>N-terminal mutants of pUL31.</p

Detailed analysis of Lox-UL31-hbpmp1mp2.

<p>To analyze the subcellular localization of nucleocapsids, Vero cells were infected with HSV1(17⁺)Lox or Lox-UL31-hbpmp1mp2 using an MOI of 1 and analyzed at 12 hpi by IF using antibodies against VP5 (mAb 8F5) in combination with anti-pUL31 antibodies (A) or anti-ICP8 antibodies (B) followed by Alexa 555- (pUL31, ICP8) and Alexa 488- (VP5) conjugated secondary antibodies. To evaluate the degree of co-localization, fluorescence signals were measured along the indicated blue arrows and plotted as green (VP5) and red (pUL31, ICP8) values against the length of the measured line using Image J (right). The scale bar corresponds to 5 μm. (C) To determine the subcellular localization of VP5 and pUL25, Vero cells were infected at an MOI of 1 with HSV1(17⁺)Lox or Lox-UL31-hbpmp1mp2 and analyzed at 12 hpi by IF using anti-VP5 and anti-pUL25 antibodies followed by secondary antibodies. The scale bar corresponds to 10 μm. For analysis, confocal microscopy was applied.</p

The N-terminal domain of pUL31 contains a classical bipartite NLS.

<p>(A) Hep2 cells were transfected with plasmids encoding the EYFP-pUL31-bp1bp2, EYFP-pUL31-bp1, EYFP-pUL31-bp2, or EYFP-SV40NLS fusion proteins; EYFP lacking an NLS was used as negative control. Twenty hours post transfection (hpt), EYFP was detected by confocal fluorescence microscopy. (B) Interaction of pUL31, pUL31-mp1mp2, pUL31-mp1, pUL31-mp2 or a control protein (Ctrl) with α importins KPNA2, KPNA4, or KPNA5 was tested by Y2H using the HIS3 reporter gene activation. Grey squares represent positive and white squares negative results. (C) Hep2 cells were transfected with plasmids encoding myc-tagged pUL31, pUL31-mp1, pUL31-mp2, pUL31-mp1mp2, pSV40NLS-UL31-mp1mp2, or pUL31-hbpmp1mp2, at 20 hpt cells were fixed and analyzed by indirect immunofluorescence microscopy (IF) using primary antibodies directed against the myc-tag on pUL31 and Alexa 594-conjugated secondary antibodies. (D) HeLa cells were transfected with plasmids encoding myc-tagged pUL31, pUL31ΔN, or pSV40NLS-UL31ΔN, at 20 hpt the cells were fixed and analyzed as described in (C). (E) To determine whether pUL31 contains nuclear export activity, the NEX-TRAP assay was applied. HeLa cells were co-transfected with the plasmids pCR3-N-HA-UL10/gM-FKBP and pEYFP-FRB-UL31. Twenty hpt, cells were incubated with anisomycin in absence or presence of rapamycin (-/+ Rap), and processed for IF using anti-gM antibodies followed by Alexa 594-conjugated secondary antibodies, while EYFP was visualized directly. (A, C, D and E) Nuclei were visualized by DAPI, confocal microscopy was applied for analysis. Each scale bar corresponds to 10 μm.</p