HHV-6A/6B infection of NK cells modulates the expression of miRNAs and transcriptional factors potentially associated to impaired NK activity

Objectives: Natural killer (NK) cells have a critical role in controlling virus infections, and virus presence can result in alterations of their intracellular environment finally leading to an impairment of their functions, as observed for several viruses as a part of their immune evasion mechanisms. Since we recently showed that HHV-6 in vitro infection is accompanied by the modulation of many miRNAs in human T cells and thyrocytes, possibly associated to alterations in immune response, the present work was aimed to study the impact of HHV-6A and -6B acute infection on the intracellular factors that might be directly correlated to NK functionality. Methods: To this aim, we infected a human NK cell line (NK-92, ATCC CRL-2407) with cell-free HHV-6A or 6B inocula, and analyzed daily both the expression of transcription factors and miRNA for one week after infection. Virus infection was verified by analyzing virus DNA, RNA and antigen expression inside infected cells. In parallel, human transcription factors and miRNAs expression were studied by quantitative PCR microarrays and individual assays. Results: The results confirmed that both virus species can promptly infect NK cells, where establish a productive cycle. Virus DNA was detectable at all days post infection (d.p.i.), as well as virus lytic transcripts and antigen expression. As hypothesized, both viruses induced significant alterations in the expression of NK miRNAs, with clearly distinct early and late effects and species differences. In particular, we observed a 6-fold down modulation of miRNAs known for their role in NK development and maturation (miR-let-7, miR-181, miR-155), a further 10-fold decrease of miRNAs not yet directly associated to NK functions but with recognized roles in immune response (miR-23, miR-340), and a 12-fold increase of inhibitory miRNAs (miR-301, miR-223, miR-146)(p<0.001, Student t test). Similarly, also the expression of transcription factors was significantly influenced by virus infection, with an increase of factors impairing NK functions and a simultaneous of factors promoting NK cell activation. In particular, HHV-6B mainly induced an early 8-fold increase of ATF3 and FOXO1, whereas HHV-6A induced a 15-fold decrease of POU2AF1 and a 4-fold decrease of ESR1. Conclusions: Our data show that HHV-6A and -6B infections have a powerful effect on the intracellular signaling based on expression of miRNAs and transcription factors. This might induce NK function impairment, which could be important for HHV-6A/6B escaping and potentially affect viruses-related pathologies.