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 regulates interaction with pUL34.

<p>(A) To determine the subcellular localization of pUL34 in absence (left) or presence of pUL31, pUL31-mp1mp2, pSV40NLS-UL31-mp1mp2, pUL31ΔN, or pSV40NLS-UL31ΔN (right), Strep-tagged pUL34 was expressed alone or together with myc-tagged pUL31 versions in Hep2 cells. IF analysis was performed at 20 hpt with anti-myc and anti-pUL34 antibodies followed by Alexa 594- and Alexa 488-conjugated antibodies, respectively. (B) Plasmids encoding myc-tagged pUL31, pUL31ΔN, pSV40NLS-UL31ΔN, or MBP-tagged pUL34 were transiently expressed in HeLa cells (left), at 20 hpt IF analysis was performed using anti-myc and anti-MBP antibodies followed by Alexa 488- and Alexa 555-conjugated antibodies, respectively. Furthermore, MBP-pUL34 was co-expressed with myc-tagged pUL31, pUL31ΔN or pSV40NLS-UL31ΔN and analyzed as described before (right). (A, B) Nuclei were visualized by DAPI, for analysis, confocal microscopy was applied. The scale bars correspond to 10 μm. (C) Interaction of pUL34 with pUL31, pUL31-mp1mp2, pUL31ΔN, pUL31-N, pUL31ΔNΔCR1 or a control protein (Ctrl) was tested by Y2H using the HIS3 reporter gene activation. Grey squares represent positive, white squares negative results. (D) To determine the ability of pUL31ΔN to interact with pUL34, HeLa cells were transfected with plasmids encoding MBP-pUL34 and myc-tagged pUL31 or pUL31ΔN (right). For control, cells were transfected with a single plasmid encoding either myc-tagged pUL31 or pUL31ΔN (left). Cells were lysed 24 hpt and processed for affinity-purification using Amylose resin. Total cell lysates (TCL) and affinity-precipitated proteins (AP) were subjected to SDS-PAGE followed by Western blotting using anti-MBP- and anti-myc antibodies followed by peroxidase-conjugated secondary antibodies. pUL31 and pUL31ΔN are indicated by arrows and arrowheads, respectively.</p

The N-terminal domain of pUL31 harboring basic patches is essential for HSV-1 propagation.

<p>(A) The ability of the pHSV1(17⁺)Lox mutants described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004957#ppat.1004957.g004" target="_blank">Fig 4</a> to form plaques was tested by transfecting Vero cells with BAC DNA and scoring cytopathic effects at 3 dpt. To visualize transfected cells, IF analysis using anti-ICP0 antibodies followed by Alexa 555-conjugated secondary antibodies was performed. Insets contain magnifications of individual ICP0-positive cells. The scale bar corresponds to 50 μm. (B) To compare the growth properties of mutant viruses HSV1(17⁺)Lox-UL31-mp1, Lox-UL31-mp2, and Lox-UL31-hbpmp1mp2 to the parental virus Lox, Vero cells were infected at an MOI of 0.1, the supernatant was harvested at 48 hpi and titrated on Vero cells in triplicates.</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

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

Oligonucleotides used for general cloning and site-directed mutagenesis.

<p>Nucleotides altered by site directed mutagenesis are underlined.</p><p>Oligonucleotides used for general cloning and site-directed mutagenesis.</p

Functional analysis of the N-terminal domain of pUL31.

<p>(A) A schematic diagram of the pHSV1(17⁺)Lox genome as well as the strategy to replace the non-overlapping coding region of UL31 (Nucleotides 9 to 865) with the <i>galK-kan</i> selection cassette resulting in ΔUL31/<i>galk</i> are depicted. (B) To rescue the ΔUL31/<i>galk</i> intermediate, the <i>galK-kan</i> cassette was replaced by the wt UL31 sequence. (C) To generate the mutants Lox-UL31ΔN, Lox-SV40NLS-UL31ΔN, Lox-UL31-mp1,-mp2,-mp1mp2 and-hbpmp1mp2, the <i>galK-kan</i> cassette was replaced by the respective mutant sequences. (D) Lox-UL31-mp1mp2 was reverted to Lox-UL31-mp1mp2 <i>rev</i> by a two-step process first replacing the mutant sequence by the <i>galK-kan</i> cassette which in turn was replaced by a sequence encoding pUL31. (E) To insert the SV40NLS coding sequence at the 5´end of UL31, the first 8 bp of UL31 overlapping with UL32 were duplicated while at the same time the original start codon of UL31 was mutated without affecting the 3´coding sequence of pUL32. Insertion of the SV40NLS-UL31-mp1mp2 coding sequence led to Lox-SV40NLS-UL31-mp1mp2. (F) Reversion of the mutant described in (E) to a wt situation was performed in a two-step process as described in (D). Note that this revertant carries an 8 bp duplication of the 5´ UL31 region as well as the mutated original start codon of pUL31.</p

Domain organization and N-terminal mutants of pUL31.

<p>(A) Graphic depiction of HSV-1 pUL31 composed of the N-terminal variable domain carrying a putative nuclear localization signal (NLS) and the C-terminal domain with conserved regions CR1, CR2, CR3, CR4 and a pUL34-binding site (pUL34-BD). (B) Sequence of the first 44 amino acids of HSV-1 pUL31. Basic amino acids are highlighted in red, the bipartite NLS (residues 21–42) is shaded in grey and its basic patches bp1 and bp2 are underlined. (C) Mutants of the N-terminal domain of pUL31 generated in this study. Exchanged residues are shown in red, the SV40NLS is indicated in bold letters, (x) indicate numbers of interspersed residues. Mutated basic patches (mp) and the hyperbasic patches (hbp) can be deduced.</p

BAC mutagenesis.

<p>BAC mutagenesis.</p