CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing

Cells.Cell lines. Porcine kidney epithelial cells (PK15; ATCC, Manassas, VA, USA) were used to produce and titer PRV stocks. Human embryonic kidney cells expressing large T antigen (HEK293T cells; ATCC) were used to produce CRISPR lentiviruses, PRV mutants, and adeno-associated viruses (AAVs). All cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin (DMEM complete medium).

Primary neurons. Superior cervical ganglia (SCGs) were isolated from embryonic day 17 Sprague-Dawley rat embryos (Hilltop Labs, Scottdale, PA, USA) as previously described (35). SCG neurons were cultivated either dissociated in plain dishes or compartmented in trichamber-mounted dishes. Plastic tissue culture dishes (35 mm, Falcon) or optical plastic dishes (Ibidi) were coated with 500 μg/ml of poly-DL-ornithine (Sigma-Aldrich, St. Louis, MO, USA) and 10 μg/ml of natural mouse laminin (Invitrogen). Trichambers were installed onto these dishes for compartmentation of neurons and axons as previously described (5). Neurons were cultured for a minimum of 4 weeks prior to experiments.

Viruses. PRV Becker is a wild-type (PRV WT) strain commonly used as background for PRV mutants (36). PRV 180 expresses a monomeric red fluorescent protein (mRFP)-VP26 fusion protein in a PRV Becker background (15). PRV ΔUL13, PRV 180 eGFP-UL13, PRV 180 mTurq-VP16, and PRV 180 VP16-mTurq viruses were constructed using CRISPR/Cas9-mediated gene replacement, as described below.

Plasmids and sgRNA selection.Synthesized oligonucleotide primers (Integrated DNA Technologies, Coralville, IA, USA) corresponding to separate targets were cloned into an EspI-digested (New England BioLabs [NEB], Ipswich, MA, USA) LentiCRISPR v2 plasmid (gift from Feng Zhang; Addgene plasmid number 52961). Guide sequences were selected using CRISPOR by containing minimal mismatches to any human or PRV genomic sequences, while maximizing the presumable on-target effect (37). Selected target sites and protospacer adjustment motif (PAM) sequences are shown in Table 1. There were no potential off-target regions in the PRV genome, as determined using CRISPOR (http://crispor.tefor.net/) (19).

Donor plasmids were constructed using the HiFi DNA assembly method (NEB), as described below. All donor plasmids consisted of a pcDNA3-eGFP backbone (gift from Doug Golenbock; Addgene plasmid number 13031) with an upfront and downstream homology arm with eGFP or mTurquoise in between (Fig. 1B). Each set consisted of four fragments, which were PCR-amplified from purified PRV DNA (homology arms), pcDNA3-eGFP (vector backbone and eGFP), or pcDNA3-mTurq (mTurquoise v2) using Q5 high-fidelity polymerase (NEB) and specific primer pairs shown in Table 3 (Integrated DNA Technologies). Silent mutations (underlined) were introduced in PAM sequences of PRV 180 fusion mutants.

The backbones of AAV plasmids (CMV-eGFP-p2a-WPRE-SV40pA or CMV-eGFP-p2a-UL13-WPRE-SV40pA) were generated by cloning. Purified viral DNA, a pAAV-mTurq-p2a-WPRE-SV40pA (Engel lab) and pcDNA-eGFP vector were modified by PCR and assembled using HiFi DNA assembly (NEB). Specific fragments and primer pairs are shown in Table 3.

Lentivirus production and transduction.Lentiviruses were produced in HEK293T cells by cotransfecting LentiCRISPRv2 plasmids containing separate targets with packaging plasmids pMD2.G and psPAX2 using polyethylenimine (PEI) reagent (VWR International, Radnor, PA, USA) at a DNA:PEI ratio of 1:3 (wt/wt) following a chloroquine hydrochloride (25 μM; Sigma-Aldrich) preincubation step. Fifty-six hours posttransfection, the cell supernatant was collected, centrifuged at 900 × g, clarified by passing through a 0.45-μm polyethersulfone (PES) filter, and stored at –80°C. Lentiviral titers were determined using the QuickTiter lentivirus titer kit according to the manufacturer’s instructions (Cell Biolabs, San Diego, CA, USA).

Fresh HEK293T cells were transduced for 48 h with CRISPR lentiviruses at an MOI of 2.5 in the presence of 10 μg/ml polybrene (Sigma-Aldrich). After 48 h, medium was replaced daily by fresh DMEM medium containing 10 μg/ml puromycin for 5 days. Stably transfected cells were then expanded and maintained in DMEM medium containing 5 μg/ml puromycin. Monoclonal cell lines (HEK293T-UL13sgRNA, HEK293T-VP16sgRNA1, and HEK293T-VP16sgRNA2) were generated by limiting dilution in order to maintain a stable expression of sgRNAs and Cas9. Briefly, 100 μl of a cell suspension of 5 cells/ml was seeded into each well of a 96-well plate. After 7 days of incubation, the wells were visually inspected for colony formation. Wells harboring only 1 colony were selected, while those without any or with 2 or more colonies were discarded. After full expansion, Cas9 expression was evaluated using the Cas9 enzyme-linked immunosorbent assay (ELISA) kit of Cell Biolabs according to the manufacturer’s instructions. Monoclonal cell lines expressing between 150 and 200 ng/ml Cas9 were selected for downstream experiments.

Adeno-associated virus production.AAV plasmids containing either CMV-eGFP-p2a-WPRE-SV40pA or CMV-eGFP-p2a-UL13-WPRE-SV40pA were packaged into AAV-PHP.eB capsids (gift from Viviana Gradinaru, Addgene plasmid number 103005) at the Princeton Neuroscience Institute Viral Core Facility (38). AAV particles were purified by iodixanol step gradient followed by column ultrafiltration as previously described (39, 40). Infectious viral genomes were measured by Taq-Man quantitative PCR (qPCR).

CRISPR/Cas9-mediated gene editing.HEK293T-UL13sgRNA, HEK293T-VP16sgRNA1, and HEK293T-VP16sgRNA2 cells were pretreated with chloroquine hydrochloride prior to transfection with mock plasmids or donor plasmids using PEI reagent as described above. After 24 h, cells were inoculated with either mock, PRV WT, or PRV 180 at an MOI of 1. Cells were further maintained in DMEM complete medium supplemented with 10 μM SCR7 and 10 μM RS-1 (Sigma-Aldrich) to inhibit nonhomologous end joining and enhance homologous recombination, respectively. Forty-eight hours postinoculation, cells and supernatant were harvested, pooled, and stored at –80°C. Viral stocks were titrated on PK15 cells using classic plaque assay and screened for eGFP or mTurquoise expression by immunofluorescence microscopy. Fluorescent plaques were subjected to 3 rounds of plaque purification before propagating viral stocks on PK15 cells.

Quantitative PCR analysis and sequencing.Viral DNA was purified from different PRV stocks using the QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. UL13 and VP16 gene regions were amplified using primer pairs UL13 seq and VP16 seq (Table 2) using Q5 high fidelity polymerase (NEB) in the presence of 4% dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and 2.5 M betaine (Sigma-Aldrich). Sequences were confirmed by Sanger sequencing (Genewiz, South Plainfield, NJ, USA).

Genome copies per ml were determined for each virus stock by means of quantitative PCR (qPCR). Virus stocks were first treated with 100 U of DNase I for 30 min at 37°C (Thermo Fisher) followed by inactivation for 10 min at 80°C. Samples were then digested with proteinase K (NEB) in 0.5% Tween 20 for 60 min at 55°C, followed by inactivation for 10 min at 95°C. Viral genomic DNA was quantified using UL54-specific primers (Table 2), as previously published (41). A serial dilution of purified whole genome of PRV Becker virions functioned as a standard. Triplicate reaction mixtures were prepared using a KAPA SYBR FAST qPCR kit (Sigma-Aldrich). Each experiment was performed in duplicates. The qPCR was performed with an Eppendorf RealPlex Mastercycler with the following amplification conditions: preincubation at 95° for 2 min with 40 cycles of denaturation (5 s at 95°C), annealing (20 s at 55°C), and extension (10 s at 72°C). The threshold cycle (C_T) values were calculated using Mastercycler EP RealPlex 2.2 software. Sample C_T values were plotted against standard dilution values to determine exact genomic DNA concentrations. Finally, DNA concentrations were converted into viral genome copies/ml based on the total PRV genome size (141,113 bp).

Virus purification.Culture supernatants of PRV-infected PK15 cells were clarified by centrifugation at 40,000 × g for 30 min at 4°C. The virion pellet was pooled onto a discontinuous OptiPrep gradient (Sigma-Aldrich) containing 10 to 30% (wt/vol) of iodixanol and centrifuged at 100,000 × g for 2.5 h at 4°C. After centrifugation, purified opalescent virion bands were harvested at the interface of the 15% and 20% layers. Virion bands were pooled in HNE buffer (5 mM HEPES, 150 mM NaCl, 0.1 mM EDTA, pH 7.4) by the use of a 50K filter device (Millipore Corporation, Bedford, MA, USA).

Compartmented complementation assay in primary neurons.SCG neurons were infected by adding a low MOI (10^2.5 PFU) PRV 180 to the axonal (N) compartment to enable genome silencing after axonal transport. UV-inactivated PRV WT or mutants (10¹⁰ genome copies ∼10^5–6 PFU) were added at the same time into the S compartment. We chose to standardize for genome copies instead of PFU, as PRV mutant stocks might contain more noninfectious virus particles compared to PRV WT. These defective particles might still be capable of delivering their content (e.g., tegument proteins) to neurons, thereby biasing the results. AAVs were added 3 days prior to PRV inoculation to ensure stable UL13 and/or eGFP expression, but no overexpression, prior to inoculation.

Virus plaque assay.PK15 cells were grown to confluence in 6-well dishes prior to inoculation with 10⁶ genome copies (∼10 to 100 PFU) of PRV WT or PRV mutants for 1 h at 37°C. Next, cells were covered with a solution containing 50% 2× MEM (Thermo Fisher Scientific) and 50% 1.88% carboxymethylcellulose (Sigma-Aldrich). Forty-eight hours postinoculation (hpi), medium was removed, and cells were fixed in ice-cold methanol for 20 min at −20°C. To visualize virus plaques and/or single infected cells, viral immediate early (IE) 180 protein was stained for immunofluorescence as described below using a rabbit polyclonal anti-IE180 protein antibody and Alexa Fluor 488-conjugated goat anti-rabbit antibodies (42).

Viral growth kinetics.PK15 cells were grown to confluence in 24-well dishes and dissociated SCGs were cultivated for 4 weeks on optical plastic dishes prior to inoculation. Cells were inoculated with PRV WT or mutants at an MOI of 1 for 1 h at 37°C. Next, cells were briefly exposed to 40 mM citrate buffer (pH 3) to inactivate any free virus particles left from the inoculum that had not infected. After 3 washing steps with DMEM, cells were further incubated with complete medium at 37°C. At the indicated time points, supernatants were collected for virus titration and cells were fixed in ice-cold methanol (20 min at −20°C) for immunofluorescence staining.

Virus titration.PRV titrations were conducted on PK15 cells, which were incubated at 37°C for 7 days. Titers were expressed as 50% tissue culture infective dose (TCID₅₀).

Immunofluorescence staining.Purified virus particles. For immunofluorescence staining of purified virus particles, 1 μl of purified virus stocks was spotted onto glass coverslips and left to adsorb to the glass for 15 min at 37°C. Particles were permeabilized in 0.1% Triton X-100 followed by 1 h staining at 37°C with primary antibodies (rabbit anti-UL13 antibody, produced in-house, rabbit anti-VP16 antibody, produced by GenScript [Piscataway, NJ, USA] or isotype control rabbit IgGs against rabies virus nucleoprotein [43]) diluted in phosphate-buffered saline (PBS) with 10% normal goat serum (NGS) and Alexa Fluor 647-conjugated secondary antibody (Thermo Fisher Scientific). Particles were washed 3 times with PBS for 5 min per wash prior to and after secondary antibody staining.

Viral growth kinetics in PK15 cells and SCG neurons. For immunofluorescence staining of infected PK15 cells and dissociated SCGs, cells were fixed in methanol at −20°C for 20 min at the indicated times after infection. Primary antibodies (rabbit polyclonal anti-UL13, rabbit polyclonal anti-VP16, rabbit polyclonal anti-VP26, mouse monoclonal anti-Us3, and isotype controls [44]) were diluted in 10% NGS-PBS and added to cells for 1 h at 37°C followed by incubation of 1 h at 37°C with Alexa Fluor 488 (green)-, 594 (red)-, or 647 (magenta)-conjugated secondary antibodies. During the last 10 min of secondary antibody staining, DAPI (4′,6-diamidino-2-phenylindole) was added to cells to stain cell nuclei.

Chambered SCG neurons. For immunofluorescence staining of chambered SCGs during virus trafficking experiments, cells were fixed in 1% paraformaldehyde (PFA) at room temperature (RT) for 10 min, permeabilized in 0.1% Triton X-100 for 2 min at RT. Staining was performed with primary polyclonal rabbit anti-eGFP antibodies (Invitrogen) or isotype control antibodies and secondary Alexa Fluor 488-conjugated antibodies, as described above.

Live-cell imaging.All imaging was done using a previously described Nikon Ti-E inverted epifluorescence microscope (45).

Virus particle trafficking. Virus particle trafficking was assessed using live-cell imaging as described previously (46). Briefly, chambered SCGs grown onto Ibidi glass dishes were placed inside a stage top incubator system at 37°C and 5% (vol/vol) CO₂ (Live Cell Instrument/Quorum Scientific), prior to inoculation with 10⁶ PFU of virus. Movies were acquired either with phase, green, red, or cyan fluorescence filters using a 60× Plan Fluor Ph3 objective (Nikon) and an iXon 895 back-thinned electron multiplying charge-coupled device (EM-CCD) camera (Andor, Belfast, Northern Ireland). The imaging window was maintained at a fixed height of 100 pixels and a width of 300 pixels. The fluorescence exposure was set to 200 ms, with the EM Gain filter set to 300. The acquisition rate was approximately 4 frames per second, on average. For the dual fluorescent movies, the exposure was set to 300 ms and the acquisition rate was approximately 1 frame per second. Movies were created and virus particles were manually tracked in infected axons using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Escape from silencing. Escape from silencing was assessed on tiled images of the entire S compartment. These images were captured using the Nikon NIS Elements software, a Cool Snap ES2 camera (Photometrics), and a 4× magnification objective (Nikon). Color thresholds were set manually, prior to converting images to 8-bit gray-scale images using ImageJ. Next, the relative amount of red fluorescence (viral production) was divided by the amount of green fluorescence (only those neurons that project their axons to the N compartment) to determine the percentage of susceptible neurons that escaped from silencing. Since the majority of neurons sent axons through the N compartment (>90%), only the percentage of red fluorescence was monitored (viral production) upon AAV inoculation.

Colocalization image analysis. Colocalization of red (capsids) and green/cyan (UL13/VP16) puncta was verified by image analysis using ImageJ. Briefly, RGB images were converted to 8-bit images and subjected to conservative thresholding. Next, binary image multiplication was used to determine the overlap of puncta. The results of 5 microscopic fields were added to obtain a total value.

Colocalization of UL13 with eGFP, and VP16 with mTurquoise, respectively, in infected cells was verified by image analysis using ImageJ. Briefly, RGB images were converted to 8-bit images. Next, binary image subtraction was used to determine the overlap of colors.

Western blot analysis.Virion protein content of newly constructed PRV mutants was characterized by Western blotting. For this, 10¹² genome copies of purified virus stocks were mixed with 5× Laemmli buffer and heated at 95°C for 5 min. Samples were then loaded onto 10% NuPAGE BisTris gels (Invitrogen) and run for 15 min at 100V and 45 min at 200V in MOPS (morpholinepropanesulfonic acid) buffer. Proteins were transferred to nitrocellulose membranes using semidry transfer at 18V for 90 min. Next, membranes were washed 2 times with ultrapure water for 2 min and incubated in antigen pretreatment solution (Invitrogen) for 10 min at RT. After 5 rinses with ultrapure water, membranes were incubated in 5% nonfat dry milk in phosphate-buffered saline supplemented with 0.1% Tween 20 (PBST) solution for 1 h at RT. Following three 5-min washing steps in PBST, membranes were stained with primary antibodies diluted 1:1,000 in primary antibody diluent (Invitrogen) overnight at 4°C. Secondary antibodies (goat anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase [HRP] [Thermo Fisher Scientific]) were diluted 1:20,000 in 1% milk-PBST solution and added to the membranes for 30 min at RT after and prior to a thorough washing step (4 times 5 min washing in PBST). Membranes were incubated with chemiluminescent substrates (Supersignal Dura, Thermo Fisher scientific). Protein bands were visualized by exposure on HyBlot CL (Denville Scientific) blue X-ray films. Primary antibodies used for Western blot were anti-VP5 mouse monoclonal antibody (MAb) (gift of H. J. Rziha, Federal Research Center for Viruses Diseases for Animals, Tubingen, Germany), anti-Us3 mouse MAb (44), and anti-UL13 polyclonal rabbit sera (produced in-house). VP5 (capsid) protein functioned as an internal loading control to determine the relative amount of proteins present in different purified virus stocks. This was determined through plot profiling in ImageJ.

Statistical analyses.Significant differences (P < 0.05) between different CRISPR gene editing methods or between mock-inoculated and different PRV mutant-inoculated cells were identified by analysis of variance (ANOVA) followed by a Tukey post hoc test. If homoscedasticity of the variables was not met, as assessed by Levene’s test, the data were log transformed prior to ANOVA. The normality of the residuals was verified using the Shapiro-Wilk test. If the variables remained heteroscedastic or normality was not met after log transformation, a Kruskal-Wallis test followed by a Mann-Whitney post hoc test were performed. All analyses were conducted in IBM SPSS Statistics for Windows, version 25.0 (IBM Corp., Armonk, NY, USA).

Ethics statement.All animal work was performed in accordance with the Princeton Institutional Animal Care and Use Committee (protocols 1947–16). Princeton personnel are required to adhere to applicable federal, state, local, and institutional laws and policies governing animal research, including the Animal Welfare Act and Regulations (AWA), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training, and the Health Research Extension Act of 1985.

Data availability statement.The raw data supporting the conclusions of the manuscript will be made available by the authors, without undue reservation, to any qualified researcher.