New findings on how HHV-6A/B U94 blocks angiogenesis

The HHV-6 latency gene U94 has been found to block angiogenesis, but the mechanisms behind this phenomenon have been unclear. A team lead by Roberta Rizzo and Elisabetta Caselli in Italy shed light on this process, opening the door to new potential molecular targets to pursue in treating diseases marked by improper vascularization.

New data reveals that HLA-G molecules, which are involved in inhibiting the cellular immune response and are taken advantage of by HHV-6 to evade immune recognition (Caselli 2015), may be pivotal in the process. Through the U94 gene and protein, HHV-6A and-B were found to upregulate the human transcription factor ATF3, which activates the promoter of HLA-G molecules and blocks formation of vessels. The antiangiogenic effects of U94 may have implications for conditions in which vascularization is a key factor, including malignancies and organ transplantation; these new results may help elucidate the roles that HHV-6A/B may play in these types of conditions.

Human primary endothelial cells (HUVECs) were infected with either HHV-6A or HHV-6B and allowed to proceed to latency, which occurred after 7 days of infection. Both viruses induced expression of HLA-G after only 24 hours of infection. While HHV-6A induced HLA-G1, G3, G4, G5, and G6 isoforms, HHV-6B induced only HLA-G1, a membrane-bound HLA-G, and G5, a soluble isoform. Likewise, both viral species quickly induced production of membrane-bound HLA-G1 proteins, which reached similar levels for both HHV-6A and –B-infected cells. Soluble HLA-G protein levels, mainly comprised of HLA-G5/G6, were also increased, although HHV-6B induced this secretion more strongly than HHV-6A at all time points.

Transfection of endothelial cells with only the U94 gene or the recombinant U94 protein had the same effects, and the level of HLA-G membrane-bound and soluble protein expression varied in accordance with U94 expression; after an initial increase, HLA-G protein expression decreased after 72 hours, corresponding with reduced U94 expression in the cells. Further testing indicated that U94 was not acting entirely through direct activation of the HLA-G promoter, so the investigators assessed the induction of 80 human transcriptional factors by each virus or U94 by microarray and found that only ATF3 was highly upregulated by both viruses and the U94 gene/protein. By cotransfection of endothelial cells with a reporter plasmid containing the HLA-G promoter and a plasmid with ATF3, a luciferase reporter assay showed that ATF3 was able to activate the HLA-G promoter and may be behind the virus-induced HLA-G expression.

To investigate how this translated into effects on angiogenesis, endothelial cells infected with HHV-6A or –B, transfected with a U94 or HLA-G expressing plasmid, or treated with U94 or HLA-G protein, were seeded on basal membrane extracts. Unlike the control cells treated with VEGF/FGF, HHV-6-infected, U94+, and HLA-G+ cells were not able to form capillary-like structures. Anti-HLA-G-antibody almost completely restored the angiogenic capacity of the U94+ cells.

ATF3 shows involvement in activation of p53, maintenance of genome stability, and tumor suppression (Wang 2018). Notably, it is also involved in the unfolded protein response and ER stress (Schmitz 2018), which has been studied in the context of HHV-6A/B infections and was the subject of a recent publication (Romeo 2018).

Another recently published study found U94 transcripts in 11 of 20 glioma tissues with HHV-6 infection (Gu 2018). Interestingly, U94 overexpression was found to inhibit glioma cell proliferation and colony formation, as well as migration, invasion, and ex vivo angiogenesis. It was also determined that expression of the gene could cause S phase cell cycle arrest in glioma cells, and U94+ cells exhibited a greater extent of apoptosis than control cells. Because of the potential to limit malignancy, the authors of the study suggested that U94 might be a potential target for glioma therapy.

Find the full paper here: Rizzo 2018.