Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates

Human cytomegalovirus (HCMV) is a herpesvirus that is widespread in the population worldwide [1,2], where it remains latent after infection in its host for a lifetime [3–5]. While primary infection or reactivation in immunocompetent individuals usually remains subclinical and causes little or no symptoms, it can cause severe damage in immunocompromised individuals [6–8]. Prevention and treatment of HCMV-related diseases have been improved through the development of antiviral drugs such as foscarnet, ganciclovir, and letermovir, but their use is limited, as they may cause adverse effects or lose efficacy due to the development of resistance [9–14]. The development of new antiviral drugs is therefore desirable. In this context, it is important to include freshly isolated HCMV because the established laboratory strains have been genetically altered during adaptation to growth in cell cultures and hence may be less representative of HCMV in patients [15–17]. Regardless of whether such genetic alterations are due to the selection of de novo mutations or the selection of pre-existing variants, they become detectable usually after several passages in the fibroblast cell culture due to selection pressure against RL13, the UL128 locus and the UL/b’ region [15]. The UL128 gene locus encodes for the three proteins pUL128, pUL130 and pUL131, which form the pentameric envelope complex together with the glycoproteins H and L [18–20]. Mutations in the UL128 locus result in higher cell-free viral titers, and thus facilitate work with cell culture-adapted HCMV laboratory strains, but have the disadvantage of altering the glycoprotein composition of the virus [21]. The pentameric complex is also required for infection of endo- and epithelial cells, so that any disrupting mutation in the UL128 locus affects the tropism of the virus [20,22,23]. Certain research questions will therefore require the virus to be studied before these mutations appear. Work with freshly isolated HCMV is, however, difficult, since the virus usually grows strictly cell-associated, leading to restricted foci of infected cells with almost no detectable cell-free infectious virus in the supernatant [17,24–26]. Due to the lack of cell-free infectivity, synchronized infections with defined multiplicities of infection are not possible, making it difficult to study the replication cycle of clinical HCMV isolates in detail. Conclusions based exclusively on genetically altered laboratory strains, in contrast, may be misleading regarding the behavior of the virus in the human body, as when testing the effect of neutralizing antibodies. It is therefore desirable to include genetically unmodified clinical isolates that resemble more closely the virus in its host. One approach to obtaining cell-free virus from genetically intact HCMV is a variant of the laboratory strain Merlin, which was cloned as a bacterial artificial chromosome (BAC), genetically restored and then equipped with Tet operators upstream of RL13 and the UL128 gene locus [27]. The latter allows the expression of these gene regions to be temporarily silenced, resulting in cell-free virus. However, this genetically restored variant of strain Merlin is also not fully representative of recent isolates (e.g., concerning the ratio of pentamer to trimer) [ [21] and, since genetic engineering is required to insert these operators, this approach is not feasible for freshly isolated HCMV that is not yet available as a BAC clone. An alternative method to obtain cell-free viruses from genetically intact clinical isolates is siRNA-mediated transient knockdown of UL128 alone [21] or in combination with RL13 [28]. The release of cell-free infectious virus with siRNA knockdown was very reliable but is limited to a small scale and comparatively laborious. Another drawback of this method is that the knockdown lasts for only a short time, meaning that the knockdown must be repeated regularly to constantly obtain cell-free infectious virus. If fibroblasts with constitutive UL128-knockdown were available, this would allow upscaling of virus production, as such cells could be easily passaged and propagated after coculture with recent isolates. Furthermore, such cells could be inoculated with clinical samples such as urine for direct isolation of HCMV. If such isolate cultures also release cell-free infectious virus, they can be rapidly transferred to other cell types, avoiding long-term passages in fibroblasts, which might further help prevent unwanted genetic modifications. To implement this, the authors aimed to establish a lentiviral expression system for the transduction of fibroblasts that would then stably express an shRNA directed against UL128.

Materials & methods

Cells

For the cultivation of HEK293T cells, the authors used minimal essential medium supplemented with GlutaMAX (Life Technologies GmbH, Darmstadt, Germany), 5% fetal calf serum (FCS; PAN Biotech GmbH, Aidenbach, Germany) and 100 μg/ml gentamicin. For cultivation of primary human foreskin fibroblasts (HFFs), they used Dulbecco’s modified Eagle medium with GlutaMAX (Thermo Fisher Scientific, MA, USA) with the addition of 10% FCS (PAN Biotech, Aidenbach, Germany) and 100 μg/ml gentamicin (Sigma-Aldrich, MO, USA).

Viruses

The diagnostic laboratory of the Institute of Virology in Ulm provided recent clinical HCMV isolates, which originated from throat washings of routine tests from patients of the Ulm University Medical Center. HFFs were inoculated with sample material, and HCMV-positive cell cultures were passaged for several weeks until the cytopathic effect indicated an infection rate of 40–60%. Infected cultures were frozen in aliquots at -80°C and tested for their cell-associated phenotype. For this, cell culture supernatants were centrifuged for 10 min at 2790× g to remove cells and debris, and the clarified supernatants were added onto HFFs, which were used as indicator cells for the detection of cell-free infectivity. After 1 day, the cells were stained for viral immediate-early (IE) antigens via indirect immunofluorescence and the number of antigen-positive cells was counted. Only if the infectivity of the supernatant did not exceed ten infected cells per 15,000 cells, the isolates were regarded as ‘cell-associated’ and used further.

To test the release of clinical isolates on UL128-shRNA-transduced cells, these cells were cocultured with clinical isolates growing in normal fibroblasts. The UL128-shRNA-transduced HFFs were seeded first. Then, the infection rate of the isolate culture was estimated based on phase contrast microscopy, and an appropriate number of cells from this culture were added to the transduced cells to achieve the desired final infection rate (0.5–5%) in the coculture. The clinical isolate was then expected to spread in the coculture and infect the transduced cells. The supernatants from such cocultures were analyzed for infectivity at various time points.

For direct isolation of HCMV on UL128-shRNA-transduced HFFs, a urine sample from the pediatric clinic in Ulm was used that had been tested HCMV-DNA-positive in the routine diagnostics of the Institute of Virology. The virus was isolated from this sample as previously reported [29].

shRNA oligonucleotide design

According to the Lenti-X™ shRNA Expression Systems user manual (Takara Bio Europe SAS, Saint-Germain-en-Laye, France), two complementary shRNA oligonucleotides were designed that contained a UL128-specific target sequence (5′-CTGCTACAGTCCCGAGAAA-3′), which has successfully been used in siRNA-mediated knockdown of pUL128 [21]. In detail, oligo number 1 contained the target sequence in sense and antisense direction separated by a hairpin loop (5′-TTCAAGAGA-3′). Upstream of this element, a 5′-GATCCG-3′ sequence was included. Downstream of the UL128-specific element, an RNA Pol III terminator sequence (6 poly[T]), an MIuI restriction site (5′-ACGCGT-3′) and a guanine residue were added. Oligo number 2 was complementary to oligo 1 except for the ends that were designed to create overhangs after annealing of the two oligos. To fit into the multiple cloning site of the pLVX vector, a BamHI overhang (5′-GATC-3′) was unique at the 5′ end of oligo number 1 and an EcoRI overhang (5′-AATT-3′) was unique at the 5′ end of oligo number 2. The shRNA oligonucleotides were purchased from a commercial provider (Sigma-Aldrich, MO, USA).

Construction of vectors

To generate a cell line that can constantly knock down pUL128, UL128-specific shRNA was cloned into the pLVX-shRNA1 lentivirus vector (Takara Bio Europe SAS). The pLVX vector was linearized by EcoRI and BamHI restriction enzyme digestion (New England Biolabs GmbH, Frankfurt am Main, Germany) and then purified (NucleoSpin Gel and PCR Clean-up Kit, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). The shRNA oligonucleotides were mixed at a 1:1 ratio (50 μmol/l of each oligo), and annealing was allowed by subsequent incubations at 95°C for 30 s, 72°C for 2 min, 37°C for 2 min and 25°C for 2 min, followed by storage on ice. The double-stranded oligonucleotide was then ligated into the shRNA vector using a Quick Ligation Kit (New England Biolabs GmbH) according to the manufacturer's instructions. Competent Escherichia coli (DH5-α; Thermo Fisher Scientific) were then transformed with the ligated vector according to the manufacturer's instructions, and positive clones were selected with ampicillin (100 μg/ml; Sigma-Aldrich) on lysogeny broth (LB) agar (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and an overnight incubation at 37°C. Colonies were harvested, shaken overnight at 37°C in LB medium (Carl Roth GmbH & Co. KG) containing ampicillin (100 μg/ml, Sigma-Aldrich) and then purified using the Nucleo Spin Plasmid Kit (MACHEREY-NAGEL GmbH & Co. KG). Successfully recombined plasmids were identified by previously integrated MIuI restriction site analysis (New England Biolabs GmbH) and subsequent agarose (Thermo Fisher Scientific) gel electrophoresis. The correct insertion of the shRNA cassette was confirmed by Sanger sequencing (Eurofins Genomics Germany GmbH, Ebersberg, Germany).

Lentivirus production & transduction

HEK293T cells were seeded the day before transfection, and medium without antibiotics was applied on the day of transfection. For transfection, packaging plasmids (pLP1, pLP2 and pVSVG [ViraPower™ Packaging Mix, Thermo Fisher Scientific] along with the lentivirus expression plasmid [pLVX-shRNA or pLVX]) were cotransfected into HEK293T cells using Lipofectamine™ 2000 transfection reagent (Life Technologies) according to the manufacturer's instructions and incubated overnight. The empty vector (pLVX) functioned as a control. After transfection, HEK293T cells were incubated for 2 days before the lentivirus-containing supernatant was aspirated with a syringe, filtered with 0.45 μm filters and centrifuged at 2093× g for 15 min. These supernatants were then used to transduce HFFs in minimal essential medium with 7% FCS, gentamicin (100 μg/ml) and polybrene (7.5 ng/μl; Sigma-Aldrich H9268). The lentiviral supernatants were added to the fibroblasts in a serial dilution reaching from two- to 20-fold. The medium was changed to a regular cultivation medium the next day to remove excess lentivirus. Two days after transduction, successfully transduced cells with stably integrated lentivirus, either UL128-shRNA (shUL128) or empty vector (vec), in the cellular genome were selected with 1 μg/ml puromycin (Invivogen, Toulouse, France) for at least 2 weeks with repeated refreshment of the selection medium. The lentivirus harvested from producer cultures was replication-incompetent due to the lack of structural and regulatory genes in the packaged genomes. As an additional safety measure, three different plasmids are used to express the respective viral proteins in the producer culture, rendering lentivirus production from the transduced cells extremely unlikely. Concerning infectivity remaining in the supernatant of transduced cultures, decay of infectivity over time and reduction by repeated medium exchanges would finally reduce titer by 8–9 log steps. Hence, no safety concerns remained when working with the transduced cells. Phase contrast microscopy was then used to estimate the fraction of surviving cells, under the assumption that only cells with a vector containing a puromycin resistance cassette were expected to survive. Consistent with this assumption, cell loss was complete in the control culture without lentivirus, whereas >95% of cells survived in the lentivirus-treated cultures at all dilutions tested. To obtain the highest lentivirus dose but avoid toxic effects, the highest concentration that did not cause morphological alteration of the cell layer was chosen for further experiments. After selection of the appropriate dilution level of the lentiviruses, the successfully transduced cells were passaged from this well. The medium containing puromycin was replaced regularly, and when the culture had reached the desired density, cells were frozen in aliquots at -80°C for later use.

Detection of viral IE proteins by indirect immunofluorescence

Infected cells were fixed with 80% acetone for 5 min at room temperature, washed with phosphate-buffered saline and incubated with an antibody directed against the viral IE antigen (UL122/123) for 2 h at 37°C with E13 (Argene/Biomerieux, Marcy-l′Etoile, France) or overnight at 37°C with the antibody CH160 (Virusys, MD, USA). In the next step, the secondary antibody Cy3-goat-anti-mouse Ig F(ab′)2 (Jackson ImmunoResearch, PA, USA) was added for 45–60 min at 37°C. Next, the cells were counterstained with 4′,6-diamidin-2-phenylindol (DAPI, Sigma-Aldrich) for 5 min at room temperature. With appropriate fluorescence excitation, all cells showed blue nuclear fluorescence, while infected cells displayed red nuclear fluorescence. Fluorescence microscope images and evaluation were performed with an ImageXpress Pico (Molecular Devices, Wokingham, Berkshire, UK) or an Axio Observer D1 microscope with Zen software (Zeiss, Oberkochen, Germany).

Immunoblotting

Virions were gradient purified as previously described [30]. For protein analysis, cells or virions were lysed in 2× Laemmli lysis buffer [31] at various time points after infection (up to 29 dpi), incubated for 30 min on ice and boiled for 5 min at 95°C. Precipitates were removed by centrifugation to clarify the lysates, and lysates were stored at -80°C. For immunoblotting under nonreducing conditions, the lysates were thawed and directly loaded onto 10% polyacrylamide gels. Electrophoresis was performed then in Tris-glycine SDS buffer. Proteins were transferred onto polyvinylidene membranes in Tris-glycine buffer with 15% methanol for 3 h at 15 V. The membranes were blocked for 1 h at room temperature in phosphate-buffered saline containing 0.1% Tween and 5% milk powder and then incubated with the respective antibodies at 4°C overnight. Mouse monoclonal antibodies against pp65 (clone 28–77 [32], kindly provided by W Britt), IE1 (clone 63–27 [33], kindly provided by W Britt), MCP (clone 28–4 [34], kindly provided by W Britt) and a rabbit polyclonal antibody against gL (raised against peptide CKQTRVNLPAHSRYGPQAVDAR [19], kindly provided by B Ryckman and D Johnson) were used. After washing in phosphate-buffered saline with 0.1% Tween, the membranes were incubated with horseradish peroxidase-conjugated rabbit anti-mouse-Ig (Agilent DAKO, CA, USA) or horseradish peroxidase-conjugated goat anti-rabbit-Ig (Merck, Darmstadt, Germany) as a secondary antibody. Using Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific), bound antibodies were visualized and chemiluminescence signals were quantified with FusionCapt Advance Solo (v.7, Vilber Lourmat, Eberhardzell, Germany).

Statistical analysis

Significant differences between different conditions were analyzed with one-way analysis of variance using the built-in data analysis function of OriginLab for datasets with more than two data groups. If significant differences were indicated by analysis of variance between the groups in the dataset tested, post hoc analyses (Fisher's exact tests) were performed to identify the pUL128 knockdown groups that differed from control fibroblasts treated with vec. These differences were then rated as marginally significant if p-values were <0.05 and significant if p-values were <0.01.

Results & discussion

The authors have previously shown that siRNA-mediated knockdown of UL128 allows transient release of cell-free infectivity [21]. The release of cell-free infectivity was reliable, but this method is limited to small-scale experiments in terms of cost and labor, and the effect is transient because siRNA is degraded after a few days. These factors make it difficult to produce cell-free virus in larger quantities. Therefore, the authors aimed to further facilitate the work with clinical HCMV isolates by an shRNA-mediated knockdown of pUL128. The basic idea was to stably transduce fibroblasts with lentiviruses encoding UL128-specific shRNA and use such cell lines to release cell-free virus from any clinical HCMV isolate without requiring any action other than adding the virus to the cells. Transduced cultures in which HCMV isolates are growing can be further propagated, so that the harvest of cell-free progeny can be scaled up to desired levels at almost no additional cost.

shRNA-mediated knockdown of pUL128 leads to the release of cell-free infectivity from recent HCMV isolates

To stably knock down pUL128 by a lentivirus-mediated shRNA approach, the authors first generated a UL128-specific oligonucleotide sequence based on a UL128-specific siRNA successfully applied in previous work (Figure 1A) [21]. Two complementary oligonucleotides were designed to form dsDNA with overhangs suited for integration into the multiple cloning site of a lentiviral vector (pLVX), resulting in an shRNA expression vector (pLVX-shRNA) that can insert into the cellular genome upon transduction. Two days after transfection of pLVX-shRNA or pLVX along with the appropriate packaging plasmids into HEK293T cells, lentiviruses containing UL128-shRNA or vec could be harvested in the supernatant and were used to transduce fibroblasts. Successfully transduced cells were then selected with puromycin, resulting in cell lines containing the UL128-shRNA-expression cassette (shUL128) or vec, respectively. Clinical isolates could then be cocultured with the transduced cells and the supernatants assayed for infectivity. Regularly, such assays revealed that supernatants of shUL128 knockdown cells induced more IE-positive cells than supernatants from vec controls, indicating higher cell-free infectivity in the former (Figure 1B).

The authors first wanted to check if the shUL128-transduced fibroblasts were able to reduce pUL128 levels in recent clinical HCMV isolates. For that, they cocultured a clinical isolate together with the shUL128-transduced fibroblasts and used the vec-transduced fibroblasts as a control. The transduced cells were seeded first, and the fibroblasts infected with clinical isolate were added at a cell number adjusted to achieve the desired initial infection rate (0.5–5%). At various time points, cell lysates were prepared from these cocultures, which were then analyzed for their part of the pentamer levels by nonreducing western blot using an antibody directed against the viral protein gL, with equivalent cell numbers loaded in each lane (Figure 2A). As reduction of pUL128 will result in reduction of the entire pentameric complex, detection of the covalently linked components of the pentamer (gH/gL/pUL128) will directly reflect the expression of pUL128. The amount of pentamer in cell lysates is affected both by the number of infected cells and by the expression level in individual infected cells. While care was taken to normalize the number of infected cells at the beginning of the experiment, the efficiency of virus spread was expected to increase with the knockdown of UL128, resulting in increased numbers of infected cells, which could result in increased total amounts of pentamer despite downregulation on the level of individual cells. To compensate for this factor, the authors also detected the viral IE protein pUL123 (IE1) as a reference value reflecting the number of infected cells in the culture. The ratio of pentamer signals/IE signals was assumed to reflect the amount of pentamer per infected cell.

As expected, western blot analyses showed increased IE1 levels for shUL128-transduced cells compared with vec controls (Figure 2A), indicating increased viral spread in the former, a finding that would be consistent with downregulated pUL128. Fitting with this notion, the comparison of pentamer/IE1 ratios as an indicator of UL128 expression per infected cell showed that the shUL128-transduced cells significantly reduced pentamer levels to approximately 50% compared with vec control cells. This proved that the shRNA against pUL128 was effective (Figure 2B). At 10 days, the levels could not be quantified because the signal intensities were too weak, whereas a clear reduction became obvious at 14 days after coculture and persisted until 29 days, with a marginally significant difference in three repeated experiments (p = 0.013).

As increased levels of IE-Ag in the western blot had suggested increased viral growth in shUL128-transduced cells, the authors reexamined this issue using indirect immunofluorescence as a readout where the number of IE-Ag-positive cells directly indicates the efficiency of viral spread. Consistent with the western blot data, the knockdown of pUL128 significantly increased the spread of the isolate (hereafter referred to as isolate 1) in the culture within 1 week of incubation by 50% as compared with the vec control (Figure 2C; p = 0.0018).

Since the immunoblotting analyses had demonstrated reduced pentamer levels in shUL128-transduced cells, and increased IE-Ag-signals had indicated that this alteration was functionally relevant, the authors next addressed the central question of this study, whether the knockdown of UL128 resulted in relevant amounts of cell-free virus in the supernatant of these isolate cultures. Three different isolates were cocultured either with shUL128-transduced cells or with vec-transduced cells. Because the authors wanted to test whether the initial number of infected cells influenced the efficiency of virus release, they normalized the fraction of infected cells in the coculture either to 0.5% or to 2.5–5%. They then collected supernatants at several time points after coculture and analyzed them for their cell-free infectivity (Figure 3A & B).

As expected, cocultures with vec-transduced cells released only small or undetectable amounts of cell-free infectious virus, indicating that the vector itself had no relevant effect on virus release. In contrast, cell-free infectivity was clearly detectable in cocultures of all three isolates with shUL128-transduced cells, and virus titers were around 1000-fold higher than the titers achieved with vector only (10⁴–10⁵ IU/ml vs 10¹–10² IU/ml). Remarkably, the maximum virus titers obtained during the 42 days of the experiment were similar, regardless of whether the infection was started at 0.5 or 2.5–5% (Figure 3C). To exclude the possibility that the isolates had generally adapted to cell-free growth, cell-free infectious supernatants were transferred to normal fibroblasts and tested for their phenotype in cells without UL128-shRNA. As expected, they switched back to cell-associated growth with confined foci and lack of infectivity in the supernatant (Supplementary Figure 1), indicating that the release of cell-free virus depended on knockdown of UL128, and the isolate phenotype was preserved during the 42 days of coculture. Genetic analysis of samples taken from isolate 3 before and 29 days after coculture indicated that more variants in the UL128 locus were selected in the coculture with the vec control as compared with shUL128-transduced cells and that the consensus sequence remained unchanged in the UL128-shRNA sample despite release of cell-free infectivity (Supplementary Figure 2).

Downstream applications of shUL128-transduced fibroblasts

The increased release of cell-free infectivity observed in clinical isolates with shUL128-transduced cells may greatly facilitate the handling of clinical isolates and enable their use in applications, which have been restricted to cell culture-adapted laboratory strains.

One example of such an application is the direct isolation of virus from patient material on these shUL128-transduced cells, which allows for avoiding the selective pressure against the UL128 locus during the initial cell-associated growth in normal fibroblasts. To test whether the transduced cells are suitable for isolation of HCMV directly from clinical specimens, the authors chose a urine sample from a pediatric patient in whom routine diagnostics had detected a high HCMV-DNA load. The sample was sterile-filtered and added to shUL128-transduced fibroblasts with centrifugal enhancement at 300× g for 30 min, incubated further for 30 min, washed with medium and then further incubated with regular monitoring for cytopathic effects in a phase contrast microscope. After 3 days, numerous rounded cells were detected in the inoculated culture, indicating successful cultivation of the virus from the clinical sample. The new isolate was then passaged weekly by coculture with uninfected shUL128-transduced cells and analyzed for its phenotype by immunofluorescence detection of viral IE antigen. Initially, the foci of IE-Ag-positive cells in the coculture appeared well confined, and no infectivity was detected in the supernatant when tested on uninfected indicator cells. Seven weeks after isolation, cell-free infectivity became detectable in the isolate culture, and the virus was then transferred via cell-free supernatant to epithelial and endothelial cell cultures as well as normal fibroblasts, where it again grew in a cell-associated fashion (Figure 4A).

A major issue in the production of these shRNA cells was the desire to harvest higher amounts of virus than possible with the siRNA process [21]. The authors expected that upscaling of virus production would be easier with stably shRNA-transduced cells because the cells could be easily propagated along with the isolate growing in them, whereas siRNA would have to be added repeatedly during the propagation process. This would yield more virus-containing supernatant, which could then be used, for example, to purify sufficient virions from clinical HCMV isolates to enable downstream applications such as immunoblotting analyses. To address whether this assumption applied, the authors cocultured a clinical isolate with shUL128- or vec-transduced cells in a T75 flask and cultured the cells in a T175 flask within 29 days. Cell-free supernatant was then harvested from both samples and virions were purified from these supernatants using a glycerol-tartrate gradient (Figure 4B). In the glycerol-tartrate gradient, they could detect distinct bands for virions, which were noticeably more intense in shUL128 than in the gradient from the vec-transduced cells. This was not surprising, since significantly more infectivity was released into the supernatant with shUL128 knockdown than with the vec-transduced control (Figure 2D). Virions were then collected from these bands and lysed with appropriate buffer to analyze several proteins of interest via western blot (Figure 4B). Four different indicator proteins/complexes were analyzed: MCP as an indicator for the viral capsid, gH/gL/gO (trimer) and gH/gL/pUL128 (the covalently linked part of the pentamer) as indicators for the viral envelope and pp65 as a part of the viral tegument. Viral IE1 antigen was examined as a nonstructural control protein, which should not be detectable in virions. As expected, no signal for IE1 was observed in the lysates of purified virions from the vector only and the shUL128 cell culture, whereas all other proteins were detected. Consistent with the difference in band intensities observed in the gradient, the immunoblotting signals of all structural proteins were less intense for vector-only control than for shUL128. More importantly, this result demonstrated that shUL128 cells indeed allowed the harvest of purified cell-free virions from clinical isolates at levels that permitted the detection of various structural proteins of interest.

In conclusion, shUL128-transduced fibroblasts allowed the isolation of HCMV directly on these cells, to produce cell-free virus at titers of up to 10⁵ IU/ml and the up-scaling of the total amount of harvest, thus facilitating structural analyses of virions from isolates. The finding that UL128-shRNA transduced fibroblasts enable the release of cell-free infectious virus from otherwise strictly cell-associated growing recent clinical HCMV isolates has practical implications, as it will further facilitate the application of standard virological procedures to such isolates.

Several reports have indicated that the UL128 gene region plays an important role in the cell-associated phenotype of HCMV. When HCMV isolates were adapted to growth in fibroblast culture, disruptions in at least one of the reading frames UL128, UL130 and UL131A were regularly detected, and these mutations were associated with an increase in cell-free infectious titers [15]. Further evidence was provided by analysis of BAC-cloned variants of the Merlin strain, which showed that an intact UL128 locus reduced cell-free infectivity by about three orders of magnitude compared with viruses mutated in UL128 [27]. Recently, the authors of the present study found that siRNA-mediated partial knockdown of UL128 was sufficient to release infectivity from cell-associated HCMV isolates, suggesting that a certain level of pUL128 is required to keep the virus cell-associated [21]. While this approach led to reliable release of cell-free infectious virus in the supernatants, it also had some drawbacks such as its limitation to a small scale and the high costs of siRNAs. These limitations are overcome by shRNA-mediated knockdown of pUL128 by fibroblast cell lines stably transduced with an appropriate retroviral vector. Both after coculture with HCMV isolates growing in normal fibroblasts and after direct inoculation with HCMV-containing clinical specimens, cell-free infectivity can be released over a period of several weeks, which would require repeated treatments with the siRNA approach. Regarding knockdown efficiency (40–50% reduction in pUL128 levels) and the titers of infectious virus harvested (up to 10⁵ IU/ml), the shRNA approach was comparable to the authors' previous results with siRNA treatment [21], which can be explained by the fact that the same part of the UL128 sequence was targeted in both procedures. As seen with the siRNA approach, virus released from shUL128-transduced cells switched back to the cell-associated phenotype when transferred to normal fibroblasts and retained the tropism for epithelial and endothelial cells.

Hence, direct inoculation of shUL128-expressing cell lines and timely transfer of released progeny to endo-/epithelial cell lines will allow isolation of HCMV from clinical samples without selection pressure on the UL128 gene locus. It will be tempting to see which of the other mutations associated with cell culture adaptation can also be avoided by this procedure. The fact that it regularly took several weeks until cell-free virus became detectable in the supernatant of infected shUL128 cells suggests that other factors are necessary in addition to UL128 knockdown to overcome the restriction to cell-associated growth. While considerations about additional factors regulating the cell-free versus cell-associated phenotype remain speculative at the moment, they fit with a recent report that transcriptional silencing of the UL128 locus in fibroblast cultures was, on the one hand, sufficient to increase the cell-free infectivity but, on the other hand, did not decrease cell-to-cell spread [35]. In the same report, overexpression of RL13 appeared to inhibit both spread modes in fibroblasts and reduced both the number and infectivity of cell-free virus particles. This is in line with the authors' previous finding that knockdown of RL13 can increase cell-free infectivity and makes it a candidate to further accelerate the process by including an additional shRNA against this gene. Unfortunately, RL13 is highly polymorphic [36–38] with ten different genotypes [39], and the authors have previously failed to identify a suitable target region shared by all genotypes [28]. To solve this issue, either a combination of shRNAs would be necessary or the function of RL13 might be inhibited by a suitable drug, which, however, remains to be identified.

As intended, the shRNA approach facilitated upscaling to enable the recovery of larger amounts of cell-free virus for certain applications, such as the analysis of the protein composition of virions from recent isolates. Already, one 175 cm² flask was sufficient to yield a prominent virion band in the density gradient and to obtain strong signals when using the virion lysate in an immunoblotting analysis. Further upscaling can be easily achieved by propagating the infected shUL128 cultures for any desired number of cell doublings. It is noteworthy that virions were also recovered from the vec-transduced control cells containing the same isolate, although the supernatant of this culture contained almost no infectivity. With the limited set of proteins tested here, the main difference between the two conditions was in the intensity of the bands in the immunoblot, reflecting the visual difference in band density in the gradient, whereas the ratios of the different proteins appeared similar at first glance. Remarkably, one additional band was only detectable in the shUL128 virions, but this could be due to the detection limit, given the small amount of virions from the control culture. For a more accurate comparison, the control cultures would need to be further propagated, but this carries the risk of mutations occurring in the UL128 region before sufficient virions are produced. A solution to this problem could be to propagate the virus at high levels in shUL128 cultures, where the selection pressure is reduced, then use the supernatant to infect large numbers of vec-transduced cells to produce virions under conditions of normal UL128 expression. These virions could then be compared with the same amount of virions harvested from shUL128 cells, in order to reveal true differences in the composition of noninfectious versus infectious virions and to understand how exactly the level of UL128 expression contributes to the switch between strictly cell-associated growth and cell-free virus spread.

While a comprehensive comparison of virions released from infected cells with normal or reduced pUL128 levels was far beyond the scope of this methodological project, it was surprising to see that the highly infectious virions released from shUL128 cells resembled the noninfectious control virions regarding the signal intensity of pentamer in comparison with trimer, MCP and pp65. This could mean either that relatively small changes in the pentamer content have a strong impact on the infectivity of virions or that the reduction of pUL128 in the infected cell indirectly affects other yet unidentified factors in the released virions, which are more relevant to their infectivity than the pentamer itself. The surprisingly strong pentamer signals together with the preserved endothelial cell tropism suggest that such cell-free virus isolate preparations may represent a relatively good approximation of wild-type HCMV for the development of neutralizing antibodies and other antiviral agents.

For practical purposes, it may be relevant that the release of cell-free infectivity appeared insensitive to differences in the initial infection rate in the coculture of a clinical isolate with shUL128-transduced cells, indicating that this ratio does not need to be optimized. Considering possible explanations, the authors assume two competing factors: On the one hand, a higher proportion of isolate culture provides more infected cells, which could increase the titer produced in coculture with the shUL128-transduced cell line. On the other hand, this also increases the number of uninfected normal fibroblasts that will compete with the shRNA-expressing cells during further propagation of the virus. Apparently, these two factors roughly balance each other, so that a similar release of cell-free virus occurs after several passages, regardless of the ratio in the original coculture.

Conclusion

In conclusion, knockdown of UL128 using cells transduced with an shRNA-expressing lentiviral vector was as effective as our previously reported siRNA approach in releasing cell-free infectivity from otherwise strictly cell-associated recent HCMV isolates. These stably shUL128-expressing fibroblasts are suitable for isolation of HCMV directly from clinical samples, and the yield of infectious progeny can be easily scaled up to levels desired for downstream applications that require cell-free infectivity, including transfer to other cell types, neutralization assays and purification of virions for various purposes.

Future perspective

The presented approach enables the release of cell-free infectivity from recent HCMV clinical isolates. This allows the consideration of clinical isolates for experiments requiring cell-free infectious virus, which can contribute significantly to the development of new antiviral strategies. For example, new neutralizing antibodies can now be tested for their effect on clinical isolates as potential candidates for antiviral treatment.