Identification of UL69 Gene and Protein in Cytomegalovirus-Transformed Human Mammary Epithelial Cells

Abstract

A growing body of evidence addressing the involvement of human cytomegalovirus (HCMV) in malignancies had directed attention to the oncomodulation paradigm. HCMV-DB infected human mammary epithelial cells (HMECs) in culture showed the emergence of clusters of rapidly proliferating, spheroid-shaped transformed cells named CTH (CMV-Transformed HMECs) cells. CTH cells assessment suggests a direct contribution of HCMV to oncogenesis, from key latent and lytic genes activating oncogenic pathways to fueling tumor evolution. We hypothesized that the presence of HCMV genome in CTH cells is of pivotal importance for determining its oncogenic potential. We previously reported the detection of a long non-coding (lnc) RNA4.9 gene in CTH cells. Therefore, we assessed here the presence of UL69 gene, located nearby and downstream of the lncRNA4.9 gene, in CTH cells. The HCMV UL69 gene in CTH cells was detected using polymerase chain reaction (PCR) and sequencing of UL69 gene was performed using Sanger method. The corresponding amino acid sequence was then blasted against the UL69 sequence derived from HCMV-DB genome using NCBI Protein BLAST tool. A 99% identity was present between the nucleotide sequence present in CTH cells and HCMV-DB genome. UL69 transcript was detected in RNA extracts of CTH cells, using a reverse transcription polymerase chain reaction (RT-PCR) assay, and pUL69 protein was identified in CTH lysates using western blotting. Ganciclovir-treated CTH cells showed a decrease in UL69 gene detection and cellular proliferation. In CTH cells, the knockdown of UL69 with siRNA was assessed by RT-qPCR and western blot to reveal the impact of pUL69 on HCMV replication and CTH cell proliferation. Finally, UL69 gene was detected in breast cancer biopsies. Our results indicate a close link between the UL69 gene detected in the HCMV-DB isolate used to infect HMECs, and the UL69 gene present in transformed CTH cells and tumor biopsies, further highlighting a direct role for HCMV in breast tumor development.

Keywords: CTH cells; UL69; cytomegalovirus - HCMV; latency; oncogenesis.

Figure 1

Detection of UL69 gene in CTH cells. (A) Screening strategy used to detect HCMV UL69 sequences in CTH cells using PCR assay. (B) UL69 amplicons detection in CTH cells using conventional PCR. Uninfected HMECs and MRC5 cells were used as negative controls; HCMV-DB viral stock and infected HMECs were considered as positive controls. β-globin was used as an internal control. Agarose gels show results representative of three independent experiments. The complete primer sets used are described in Supplementary Table 1 . (C) Detection of nucleotide point mutations in HCMV-DB UL69 sequence present in CTH cells compared to wild-type HCMV-DB virus.

Figure 2

Detection of UL69 transcript and pUL69 protein in CTH cells. (A) RT-PCR assay to detect the presence of UL69 transcript in RNA extracts of CTH cells in the presence (+) and absence (-) of reverse transcriptase (RT). Infected HMECs were used as a positive control and no template control (NTC) as a negative control. (B) Detection of pUL69 protein in CTH cells by western blot. (C) Two residue point mutations present in pUL69 in CTH cells when compared to the original pUL69 from HCMV-DB isolate.

Figure 3

Phylogenetic analysis of UL69 gene and pUL69 protein present in CTH cells. Phylogenetic analysis of UL69 gene (A) and pUL69 protein (B) present in CTH cells compared to the original HCMV-DB strain in addition to other clinical and laboratory-adapted HCMV strains. This analysis was performed over three independent experiments.

Figure 4

Ganciclovir treatment of CTH cells decreases UL69 viral load, cell count, and cellular proliferation in culture and reduces colony formation in soft agar. (A) Untreated and ganciclovir-treated (20 µM for 4 days) CTH cells were compared for detection of UL69 gene by qPCR, (B) cell counting under the microscope (total magnification of 200x), (C) cellular proliferation as measured by Ki67 antigen quantification by flow cytometry, and (D) colony formation in soft agar.

Figure 5

pUL69 favors HCMV replication and cellular proliferation in CTH cells. (A, B) Knockdown of UL69 transcript and protein in CTH cells was monitored by RT-qPCR assay (A) and western blot (B) respectively. CTH cells were treated with UL69 siRNA and a scramble control. RT-qPCR assay and western blot were performed as specified in Methods. (C) Knockdown of UL69 resulted in decreased HCMV replication in CTH cell cultures as measured by detection of IE1 in culture supernatants by qPCR assay. (D) UL69 knockdown decreased CTH cell proliferation as measured by Ki67Ag quantification (MFI, mean fluorescence intensity) by flow cytometry. (E) Overexpression of pUL69 favors the proliferation of HMECs and CTH cells. Increased cellular proliferation as measured by Ki67 antigen quantification (MFI) by flow cytometry in HMEC and CTH cells transfected with pUL69 plasmid. Empty pcDNA3.1 transfected cells were used as control. Results are means (± SD) of three independent experiments.

Figure 6

Detection of UL69 DNA in human breast cancer biopsies. (A) Percentage of UL69-positive samples (Ct<50) in breast tumor biopsies (n=30), including luminal (n=15) and basal-like (n=15) tumors, and in adjacent healthy paired breast tissue (n=29) using UL69 quantitative PCR measurement. (B) Quantification of HCMV UL69 DNA in breast tumor biopsies (n=30) compared to healthy adjacent paired breast tissue (n=29), and in patients with basal-like breast cancer (n=15) compared to patients with luminal breast cancer (n=15), as measured by quantitative PCR UL69 Ct value. * A statistically significant difference at p-value < 0.05. Histogram data are represented as mean ± SD of two independent experiments.