A Leopard Can Change Its Spots: How HCMV Genetic Variability Impacts Viral Fitness and NK Ligands Immunomodulation

Human cytomegalovirus (HCMV) is the leading cause of congenital infection resulting in severe morbidity and mortality among newborns worldwide; however, mechanisms contributing to HCMV pathogenesis remain unclear. To successfully establish a persistent infection, the virus evolved multiple strategies to avoid host immune recognition. For example, HCMV infected cells exhibit remarkable resistance to natural killer (NK) cytolysis via encoding immunomodulatory proteins. Furthermore, HCMV demonstrates an exceptionally high degree of variability, particularly in viral genes contributing to immune evasion, contradicting the expectation that, as a large dsDNA virus, it should maintain high genome stability.Against this background, our aim was to determine whether and to what extent the differences in genetic composition affects viral fitness and its ability to modulate NK response. Here we report extraordinary genetic and phenotypic diversity of HCMV clinical isolates obtained from a cohort of 15 pediatric patients with HCMV congenital infection. We evaluated the degree of genetic polymorphism of HCMV clinical strains by genotypic and phylogenetic analyses, primarily focusing on viral genes encoding proteins with potent NK immunomodulatory functions (UL16, US18, UL40, UL141-142, US18-21, US9). Growth analysis of isolates in several cellular models revealed different patterns of replication and dissemination (“aggressiveness” of the strain): fast-, intermediate-, and slow-replicating strains. The analysis of NK cell activating ligands (MICA, MICB, ULBP1, ULBP2/5/6, ULBP3, PVR, B7-H6), as well as HLA class I at both RNA and protein level, demonstrated that HCMV strains affect NK ligands to different degrees. For instance, all viruses downmodulate ULBP2/5/6, with the strongest effect observed with the phenotypically more aggressive isolates. Moreover, there was a strong up-regulation of PVR observed with fast-replicative isolates. Finally, to assess whether HCMV isolates affect NK recognition, we performed a NK cytotoxicity assay. Likewise, we observed a great variation between strains, indicating that the genetic variability actually reflects variability in functionality.