The human breast cancer cell lines MCF-7, SK-BR-3 and MDA-MB-231 were provided by Dr Musheng Zeng (State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China), and the MDA-MB-468 cell line was obtained from Dr Xiaoming Xie (Sun Yat-sen University Cancer Center, Guangzhou, China). The Vero cell line (Cercopithecus aethiops kidney cells) was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell types were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), which was supplemented with 4.5 g/l glucose and 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO₂.

The plasmid pRB4871, containing the γ34.5-MyD116 chimaera was kindly provided by Professor Bernard Roizman (Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, Chicago, IL, USA) (19). The pG47Δ-BAC backbone plasmid and the pVec91 shuttle plasmid were obtained from Professor Samuel D. Rabkin (Molecular Neurosurgery Laboratory, Massachusetts General Hospital, Boston, MA, USA), and the pG47Δ-BAC backbone plasmid was created by a two-step replacement procedure, in which G47Δ DNA was cloned into a BAC vector for propagation in Escherichia coli, as described previously (20). The γ34.5-MyD116 chimaera fragment from pRB4871 was ligated into the BamHI-StuI site of pVec91 [containing LacZ, loxP, and FRT sites; multiple cloning sites; lambda stuffer sequences; and a cytomegalovirus (CMV) promoter] to generate pVec91-116.

The recombinant oHSV-1 vectors were constructed using a BAC and Cre/loxP and FLP/FRT recombinase systems, as reported previously (20,21). Mixtures of the pG47Δ-BAC plasmid (2 µg) and pVec91-116 or pVec91 (200 ng) were incubated with Cre recombinase (New England BioLabs, Ipswich, MA, USA) at 37°C for 30 min and then electroporated into E. coli DH10B using a Gene Pulser Xcell™ (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The bacteria were streaked onto LB plates containing chloramphenicol (15 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and kanamycin (30 µg/ml; Sigma-Aldrich; Merck KGaA), and positive clones containing the pG47Δ-BAC-116 recombinant and pG47Δ-BAC-empty plasmids were selected. Next, the recombinant plasmids were digested with HindIII and separated by electrophoresis on 0.75% agarose gels in TBE buffer (Tris-base, boric acid and EDTA) at 2.5 cm/V for 12 h with a 1 kb DNA extension ladder (Invitrogen; Thermo Fisher Scientific, Inc.). Co-transfection of pG47Δ-BAC-116 or pG47Δ-BAC-empty and a FLP recombinase expression plasmid, pOG44 (Invitrogen; Thermo Fisher Scientific, Inc.), into Vero cells was performed with Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following this, the removal of the FRT-flanked BAC sequences resulted in the generation of recombinant viruses. The progeny viruses were further selected by limiting dilution and were finally designated ‘GD116’ and ‘GD-empty’.

Viral DNA was isolated using the Hirt Supernatant DNA Purification kit (GenMed Scientifics Inc., Shanghai, China), and polymerase chain reaction (PCR) was performed using recombinant Taq DNA polymerase (Takara Bio, Inc., Otsu, Japan). Primers (CMV forward 5′-ACTGCTTACTGGCTTATCG-3′ and reverse, 5′-GTCTAACTCGCTCGTCTC-3′) were used to amplify a specific 113 bp fragment for detection of the CMV sequence. Primers (γ34.5-MyD116 forward, 5′-GCGGCTCAGATTGTTCAA-3′ and γ34.5-MyD116 reverse, 5′-CGGACTGTGGAAGAGATG-3′) were used to amplify a 284 bp product that was specific for the γ34.5-MyD116 chimaera. The samples were initially denatured for 5 min at 94°C; followed by 35 cycles of denaturation at 94°C for 30 sec, re-naturation at 52°C for 30 sec and extension at 72°C for 20 sec; with a final extension at 72°C for 10 min. pVec91 or pRB4871 plasmids and PBS were used as the positive and blank controls, respectively. The PCR products were separated by size in 1% agarose gels.

A total of 5×10⁶ Vero cells were mock treated or treated with viruses at an MOI (multiplicity of infection) of 2 for 24 h, and then cells were lysed using a Whole Cell Lysis kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A total of 30 µg protein (quantified using the BCA protein quantification kit; Nanjing KeyGen Biotech Co., Ltd.) was separated by 15% SDS-PAGE and transferred to a 0.22-µm thick nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat dry milk in TBS-Tween-20 (TBS-T, 0.1% Tween) for 1 h at room temperature, the membrane was incubated overnight at 4°C with primary antibodies against ICP34.5 (amino terminus) (diluted 1:500, rabbit polyclonal; cat no. HX5191; TSZ Biosciences, San Fransisco, CA, USA; http://www.tsz-biological.com/) or β-actin (diluted 1:1,000; rabbit monoclonal; cat no. 4970; Cell Signaling Technology, Inc., Danvers, MA, USA). The membrane was then washed and blotted with a horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (diluted 1:6,000; cat no. 7074; Cell Signaling Technology, USA) for 1 h at room temperature. The Protein antibody complexes were visualized with the chemiluminescent HRP substrate (EMD Millipore) using a chemiluminescence imaging system (Tanon-5200; Tanon, Guangzhou, China).

Cells were seeded in 12-well plates at 1×10⁵ cells per well and infected at 70–80% confluence with GD116 or GD-empty at a MOI of 0.1 (Vero, MCF-7, SK-BR-3 and MDA-MB-468) or 0.3 (MDA-MB-231). The inoculum was removed after 2 h and replaced with DMEM supplemented with 1% heat-inactivated FBS (iFBS). The cells and the supernatant were harvested at the indicated times (24 and 48 h) post-infection, processed with three freeze/thaw cycles, and virus titres were determined by plaque assays on Vero cells. In brief, Vero cells were seeded in 12-well plates at 1×10⁵ cells/well and infected with 500 µl virus dilute (10⁻⁴/ml, 10⁻⁵/ml, 10⁻⁶/ml or 10⁻⁷/ml) at 100% confluence. Then, the infected Vero cells were cultured at 37°C in 5% CO₂ for an additional 48 h prior to subsequent X-gal staining for plaque counting. The X-gal staining was performed using the in situ β-galactosidase staining kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. The virus titres were calculated using the following equation: Virus titres=plaque numbers/0.5 × dilution ratio. Experiments were repeated at least three times.

Cells were seeded in 48-well plates at 8,000 cells per well. After 16 h, the cells were infected with either GD116 or GD-empty at MOIs of 0.01 and 0.1 (MCF-7, SK-BR-3 and MDA-MB-468) or at MOIs of 0.1 and 0.3 (MDA-MB-231). Cells were incubated for up to 5 days in DMEM medium supplemented with 1% iFBS at 37°C. The cell viability was assessed daily with an MTT assay and expressed as a percentage of the mock-infected control. Formazan crystals were dissolved using 400 µl DMSO and the visualization wavelength was 490 nm. Meanwhile, infection was monitored using β-galactosidase activity via X-gal staining at the indicated times (1, 2, 3, 4 and 5 days post-infection). Experiments were repeated at least three times for each condition in quadruplicate.

Student's t-test was used for statistical analyses. P<0.05 were considered to be statistically significant. Data are presented as the mean ± standard deviation. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for all of the statistical analyses.

The genomic structure of the mutants is depicted in Fig. 1A. The pRB4871 plasmid was cleaved using the BamHI and StuI restriction endonucleases, and then a 1.51 kb fragment containing the γ34.5-MyD116 chimaera was purified from an agarose gel and directionally cloned into the pVec91 vector to generate pVec91-116. To identify the recombinant plasmid pVec91-116, pRB4871 and pVec91-116 were digested with BamHI and StuI, and the products were separated on a 1% agarose gel. As shown in the map of double restriction endonuclease digestions (Fig. 1B), the desired 1.51 kb bands on an agarose gel confirmed the successful construction of the recombinant pVec91-116 vector.

The generation of two novel oHSV-1 mutants was performed by a previously described two-step replacement procedure (22). In the first step, the shuttle plasmid pVec91-116 or pVec91-empty were integrated into pG47Δ-BAC using Cre recombinase in a tube and an integrated BAC clone (pG47Δ-BAC-Vec91-116 or pG47Δ-BAC-empty) was isolated in E. coli by selection with chloramphenicol and kanamycin. The integrated plasmids were collected and the DNA structures of the plasmids were confirmed by gel analyses following Hind III restriction endonuclease digestion (Fig. 1C).

In the second step, the integrated plasmid pG47Δ-BAC-Vec91-116 or pG47Δ-BAC-empty and the FLP expression plasmid were co-transfected into Vero cells, and the BAC backbone and lambda stuffer sequences were excised by FLP recombinase. As a result, the novel oHSV-1 mutants were generated and were designated GD116 and GD-empty.

To verify the insertion of the target sequences into the novel mutants, specific PCR analyses were conducted using pVec91 and pRB4871 plasmids as the positive controls. The PCR products were checked by electrophoresis on 1% agarose gels. GD116 and GD-empty produced the desired 113 bp band on an agarose gel when used as templates to amplify the CMV sequence (Fig. 1D). However, only GD116 generated the expected size band of 284 bp when γ34.5-MyD116-specific PCR products were detected (Fig. 1D). To determine the expression of the γ34.5-MyD116 protein, Vero cells were treated with PBS, GD-empty or GD116. Western blot analysis revealed that the expression of γ34.5-MyD116 was shown only in the GD116-treated group (Fig. 1E). In addition, the two mutants could form blue plaques on Vero cells by X-gal staining 48 h after infection, which confirmed the insertion and expression of the LacZ gene (data not shown).

Viral replication is one of the key determinants of oHSV-1 efficacy against tumour cells. The replication of the two mutants was compared in Vero cells and a panel of human breast cancer cell lines at 24 and 48 h after infection (Figs. 2A-E). Compared with GD-empty, GD116 had a significantly increased viral yield in breast cancer MCF-7 and MDA-MB-231 cell lines at 24 and 48 h (Fig. 2B and E). Additionally, the viral yields of GD116 in MDA-MB-468 cells were greater than that of GD-empty at 48 h (Fig. 2D). However, the insertion of γ34.5-MyD116 did not significantly increase the replication rate of oHSV-1 in SK-BR-3 cells (Fig. 2C), and even reduced the viral replication in Vero cells (Fig. 2A).

Next, the oncolytic activity of these two mutants was examined in human breast cancer cell lines. The breast cancer cells MCF-7, SK-BR-3 and MDA-MB-468 were infected with GD116 or GD-empty at MOIs of 0.01 and 0.1, whereas MDA-MB-231 cells were infected with these two mutants at MOIs of 0.1 and 0.3. In addition, cells infected with the virus could express LacZ and thus were stained blue by X-gal; staining of MCF-7 and SK-BR-3 cells with X-gal was presented in Fig. 3A and B. Similar to what was observed with virus replication, GD116 was more effective at inhibiting the growth of MCF-7 cells at MOIs of 0.01 and 0.1 (Fig. 3C). On the fifth day after infection with GD116 at MOIs of 0.01 and 0.1, 49.2 and 82.8% of MCF-7 cells were killed, respectively, which was higher than 31.3 and 69.6%, respectively, with GD-empty infection (Fig. 3C). However, only a slightly increased efficacy was observed with GD116 than with GD-empty in SK-BR-3 cells (74.2 vs. 69.0%, respectively) on day 3 at an MOI of 0.1 (Fig. 3D). Aside from that effect, no significant difference in cytotoxicity was observed between the two mutants at the other indicated times (Fig. 3D).

X-gal staining of MDA-MB-468 and MDA-MB-231 cells was presented in Fig. 4A and B. As shown in Fig. 4C, GD116 had a similar cytotoxic effect to GD-empty in MDA-MB-468 cells. However, the insertion of the γ34.5-MyD116 chimaera could significantly increase the oncolytic activity of GD116 on MDA-MB-231 cells (Fig. 4D). On day 5 after infection, 35.0 and 50.2% of MDA-MB-231 cells were killed by GD116 at MOIs of 0.1 and 0.3, respectively; however, the killing activity of GD-empty at the different MOIs was only 24.3 and 37.8%, respectively (Fig. 4D). In vitro, the spread of oHSV-1 was associated with cytotoxicity; therefore, X-gal staining visually showed the difference in oncolytic activity between GD116 and GD-empty in different breast cancer cell lines (Figs. 3A and B, and 4A and B).