BackgroundHuman cytomegalovirus (HCMV) establishes a persistent life-long infection and increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. Breast milk is an important route of HCMV transmission in humans and we hypothesized that mammary epithelial cells could be one of the main cellular targets of HCMV infection.MethodsThe infectivity of primary human mammary epithelial cells (HMECs) was assessed following infection with the HCMV-DB strain, a clinical isolate with a marked macrophage-tropism. The impact of HCMV-DB infection on expression of p53 and retinoblastoma proteins, telomerase activity and oncogenic pathways (c-Myc, Akt, Ras, STAT3) was studied. Finally the transformation of HCMV-DB infected HMECs was evaluated using soft agar assay. CTH cells (CMV Transformed HMECs) were detected in prolonged cultures of infected HMECs. Tumor formation was observed in NOD/SCID Gamma (NSG) mice injected with CTH cells. Detection of long non coding RNA4.9 (lncRNA4.9) gene was assessed in CTH cells, tumors isolated from xenografted NSG mice and biopsies of patients with breast cancer using qualitative and quantitative PCR.ResultsWe found that HCMV, especially a clinical strain named HCMV-DB, infects HMECs in vitro. The clinical strain HCMV-DB replicates productively in HMECs as evidenced by detection of early and late viral transcripts and proteins. Following infection of HMECs with HCMV-DB, we observed the inactivation of retinoblastoma and p53 proteins, the activation of telomerase activity, the activation of the proto-oncogenes c-Myc and Ras, the activation of Akt and STAT3, and the upregulation of cyclin D1 and Ki67 antigen. Colony formation was observed in soft agar seeded with HCMV-DB-infected HMECs. Prolonged culture of infected HMECs resulted in the development of clusters of spheroid cells that we called CTH cells (CMV Transformed HMECs). CTH cells when injected in NOD/SCID Gamma (NSG) mice resulted in the development of tumors. We detected in CTH cells the presence of a HCMV signature corresponding to a sequence of the long noncoding RNA4.9 (lncRNA4.9) gene. We also found the presence of the HCMV lncRNA4.9 sequence in tumors isolated from xenografted NSG mice injected with CTH cells and in biopsies of patients with breast cancer using qualitative and quantitative PCR.ConclusionsOur data indicate that key molecular pathways involved in oncogenesis are activated in HCMV-DB-infected HMECs that ultimately results in the transformation of HMECs in vitro with the appearance of CMV-transformed HMECs (CTH cells) in culture. CTH cells display a HCMV signature corresponding to a lncRNA4.9 genomic sequence and give rise to fast growing triple-negative tumors in NSG mice. A similar lncRNA4.9 genomic sequence was detected in tumor biopsies of patients with breast cancer.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19.

A novel coronavirus, SARS-CoV-2, emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drugs with broad anti-coronavirus activity embody a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested ten small-molecules with chemical structures close to ferulic acid derivatives (FADs) (n = 8), caffeic acid derivatives (CAFDs) (n = 1) and carboxamide derivatives (CAMDs) (n = 1) for their ability to reduce HCoV-229E replication, another member of the coronavirus family. Among these ten drugs tested, five of them namely MBA112, MBA33, MBA27-1, OS4-1 and MBA108-1 were highly cytotoxic and did not warrant further testing. In contrast, we observed a moderate cytotoxicity for two of them, MBA152 and 5c. Three drugs, namely MBA140, LIJ2P40, and MBA28 showed lower cytotoxicity. These candidates were then tested for their antiviral propreties against HCoV-229E and SARS-CoV2 replication. We first observed encouraging results in HCoV-229E. We then measured a reduction of the viral SARS-CoV2 replication by 46% with MBA28 (EC50 > 200 µM), by 58% with MBA140 (EC50 = 176 µM), and by 82% with LIJ2P40 (EC50 = 66.5 µM). Overall, the FAD LIJ2P40 showed a reduction of the viral titer on SARS-CoV-2 up to two logs with moderate cytotoxicity which opens the door to further evaluation to fight Covid-19.

Human cytomegalovirus (HCMV), whose genome is around 235 kb, is a ubiquitous human herpesvirus that infects between 40% and 95% of the population. Though HCMV infection is commonly asymptomatic and leads to subtle clinical symptoms, it can promote robust immune responses and establish lifelong latency. In addition, in immunocompromised hosts, including individuals with acquired immunodeficiency syndrome (AIDS), transplant recipients, and developing fetuses it can lead to severe diseases. Immunosenescence, well-defined as the alterations in the immune system, is linked mainly to aging and has been recently gathering considerable attention. Senescence was characterized by an elevated inflammation and hence considered a powerful contributor to “inflammaging” that is measured mainly by tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) levels as well as latent viral infections, for instance, cytomegalovirus (CMV). Inflammaging resulted in a senescence-associated secretory phenotype (SASP). HCMV is markedly associated with accelerated aging of the immune system as well as several age-associated diseases that accumulate and subsequently deteriorate the immune responses, thus have been linked to mortality, declined vaccine efficacy, serious diseases, and tumors in the elderly. HCMV triggers or exacerbates immunosenescence; on the other hand, the weakened immune responses and inflammaging favor viral reactivation and highlight the role of HCMV in aging as well as viral-associated tumors. HCMV reactivation resulting in sequential lytic and latent viral cycles could contribute to HCMV genomic variability. Besides the oncomodulatory role and transforming capacities of HCMV, the immune-privileged tumor microenvironment has been considered the main element in tumor progression and aggressiveness. Therefore, the interplay between HCMV, immunosenescence, and cancer will aid in discovering new therapeutic approaches that target HCMV and act as immune response boosters mainly to fight cancers of poor prognosis, particularly in the elderly population.