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1.
Fig 3

Fig 3. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

Western blot analyses. RK13 cells were harvested 20 h after infection with pPrV-ΔgGG, pPrV-ΔgGGΔgH K, or virus rescuants expressing the indicated gH mutants at an MOI of 5. Proteins were separated by SDS-PAGE and blots were analyzed with monospecific antisera against gH (A) or capsid protein pUL38 (B). Molecular mass markers are indicated on the left.

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.
2.
Fig 6

Fig 6. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

Penetration kinetics of PrV gH mutants. After synchronized virus adsorption at 4°C, RK13 cells were incubated for 0, 5, 10, 20, or 40 min at 37°C prior to acid inactivation of nonpenetrated virus. Numbers of plaques after 2 days were compared to those obtained without inactivation. Mean percentages of two independent experiments are shown.

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.
3.
Fig 4

Fig 4. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

EGFP autofluorescence illustrating gH function. (A) Cell-to-cell spread was analyzed 1 day after infection of RK13 cells with pPrV-ΔgGG, gH deletion mutant pPrV-ΔgGGΔgHK, or virus recombinants possessing the indicated gH mutations (gHC571S, gHC573S, gHE555C, gHE555C/V630C). (B) Induction of syncytium formation was analyzed 3 days after cotransfection of RK13 cells with an EGFP reporter plasmid and expression plasmids for PrV gB, gD, and gL, as well as for wild-type (pcDNA-gH) or mutated gH (gHC547S, gHC571S, gHS556C, gHS556C/V631C) or the empty expression vector (pcDNA3).

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.
4.
Fig 7

Fig 7. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

In vitro fusion activity of mutated gH. RK13 cells were cotransfected with expression plasmids for EGFP, gB, gD, gL, and different gH mutants or the empty expression vector (pcDNA3). After 3 days, the number of polykaryocytes in 20 fields of view (A) and the number of nuclei in 20 syncytia per plasmid set (B) were determined. Mean values and standard deviations are indicated. Statistically significant differences from the numbers obtained with wild-type gH are labeled with asterisks (*, P < 0.05; **, P < 0.01).

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.
5.
Fig 5

Fig 5. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

In vitro replication of PrV gH mutants. (A) For analysis of one-step growth kinetics, RK13 cells were infected at an MOI of 5 and harvested together with the supernatant after 3 h, 12 h, 24 h, or 36 h. Mean progeny virus titers of two experiments are shown. (B) For determination of plaque sizes, infected RK13 cells were incubated for 2 days under semisolid medium. Mean areas of 30 plaques per virus were calculated as percentages of wild-type (pPrV-ΔgGG) sizes. Standard deviations (vertical lines) and statistically significant differences from wild-type size are indicated (*, P < 0.05; **, P < 0.01).

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.
6.
Fig 1

Fig 1. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

Structure-based mutagenesis of PrV gH domain IV. (A) Structure of the PrV gH core fragment (5) is shown as space-filled model (left). The amino (N) and carboxy (C) termini of the protein, the three resolved domains (II, III, IV), and the putative position of the membrane (arrow) are indicated. The flap region is highlighted in blue, and the two disulfides clamping its ends (SS3 and SS4) are shown in yellow. A view of domain IV obtained by 90-degree rotation toward the viewer (middle and right) shows that the flap region (displayed as space-filled and wire models, respectively) covers an extensive surface area of the domain. (B) Residues forming the hydrophobic patches of domain IV (shown as space-filled model) are highlighted in cream, the glycosylation site at N627 in light blue, and disulfide bonds SS3 and SS4 in yellow. The residues mutated in this study are underlined. For clarity, the flap region is shown as wire model, with the residues mutated in this study colored in purple. C573 is not visible in the space-filled model due to its buried location. The expected location of L634 (*) is indicated, although it was not resolved in the PrV gH crystal structure. (C) Novel cysteine residues (yellow) were introduced by amino acid substitutions in the flap (C555 and C556, shown as wire model) and in the underlying hydrophobic patch (C630 and C631, shown as surfaces) in order to lock the flap region by additional disulfides (SS5a and SS5b).

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.
7.
Fig 2

Fig 2. From: Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus.

Plasmids and virus mutants. Open reading frames (pointed rectangles), transcripts (dotted arrows), and relevant restriction sites are indicated. (A) The ca. 140-kbp genome of PrV-Ka consists of unique (UL, US) and inverted repeat sequences (IR, TR). The UL22 (gH) and US4 (gG) genes were targets of mutations. (B) An EGFP expression cassette containing the HCMV immediate-early promoter (PHCMV) and a polyadenylation signal (A+), as well as a mini-F plasmid vector (pMBO131), were inserted at the 5′ end of US4 to permit BAC cloning of the PrV genome. Thereby, expression of the major 3′ part of US4 (crossed) was prevented. (C) For generation of gH-deleted PrV, codons 233 to 637 of UL22 were replaced by a kanamycin resistance gene (KanR). Virus rescuants were generated by homologous recombination with the plasmid-cloned wild-type or mutated gH gene in RK13 cells. Locations of direct repeat sequences (vertical lines) and an origin of replication (OriL) are indicated. (D) Amino acid sequence of the C-terminal part of PrV gH. Residues forming the flap (bold), hydrophobic patches, and the transmembrame domain (bold italics), as well as an N-glycosylation site (underlined), are highlighted. Native and novel disulfide bonds (SS) and amino acid substitutions introduced by site-directed mutagenesis are also indicated.

Walter Fuchs, et al. J Virol. 2012 August;86(15):8002-8013.

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