Inactivated Wild-Type Cytomegalovirus (CMV, AD-169)

Human cytomegalovirus (HCMV) establishes a persistent lifelong infection, and increasing evidence suggests that HCMV infection can modulate signaling pathways associated with carcinogenesis. Here, researchers found that the clinical strain HCMV-DB can replicate efficiently in HMECs, as demonstrated by the detection of early and late viral transcripts and proteins. After infection of HMECs with HCMV-DB, retinoblastoma and p53 proteins were inactivated, telomerase activity was activated, proto-oncogenes c-Myc and Ras were activated, Akt and STAT3 were activated, and cyclin D1 and Ki67 antigens were upregulated. Colony formation was observed in soft agar seeded with HCMV-DB-infected HMECs. These data suggest that key molecular pathways associated with tumorigenesis are activated in HCMV-DB-infected HMECs, ultimately leading to the transformation of HMECs in vitro and the emergence of CMV-transformed HMECs (CTH cells) in culture. CTH cells displayed HCMV signatures corresponding to the lncRNA4.9 genomic sequence and generated rapidly growing triple-negative tumors in NSG mice. Similar lncRNA4.9 genomic sequences were detected in tumor biopsies from breast cancer patients.

Here, HMECs were infected with HCMV-DB and AD169, respectively, and HMECs were seeded in soft agar medium on day 1 post infection for 14 days. At the same time, uninfected cells and cells infected with heat-inactivated HCMV (Inactivated Wild-Type Cytomegalovirus) and UV-treated HCMV were seeded as negative controls, and tumor MCF-7 cells and MDA-MB-231 cells were seeded as positive controls. As an additional control, the viral supernatant was filtered through a 0.2μm filter and used to infect HMECs. In addition, HMECs were treated with ganciclovir and then HCMV infection was performed. After 14 days of culture, the researchers observed the formation of colonies in the soft agar inoculated with HMECs infected with HCMV-DB (Figure 1). Colonies of established representative breast cancer cell lines such as MCF-7 cells and MDA-MB-231 cells were also observed (Figure 1). No colonies were observed to form in soft agar inoculated with HMEC infected with AD169 (Figure 1) or MRC5 cells infected with HCMV-DB or AD169. In addition, DNA was isolated from soft agar colonies at day 15 post infection (after 14 days of culture in soft agar) and the presence of HCMV DNA, i.e., the major immediate early promoter (MIEP) sequence, was assessed using a PCR assay. Surprisingly, the researchers did not detect HCMV MIEP DNA in colonies grown in soft agar inoculated with HMEC infected with HCMV-DB 14 days earlier.

Figure 1. Colony formation in soft agar seeded with HMECs infected with HCMV-DB. (Kumar A, et al., 2018)