Genetic Variability of Human Cytomegalovirus Clinical Isolates Correlates With Altered Expression of Natural Killer Cell-Activating Ligands and IFN-γ

Abstract

Human cytomegalovirus (HCMV) infection often leads to systemic disease in immunodeficient patients and congenitally infected children. Despite its clinical significance, the exact mechanisms contributing to HCMV pathogenesis and clinical outcomes have yet to be determined. One of such mechanisms involves HCMV-mediated NK cell immune response, which favors viral immune evasion by hindering NK cell-mediated cytolysis. This process appears to be dependent on the extent of HCMV genetic variation as high levels of variability in viral genes involved in immune escape have an impact on viral pathogenesis. However, the link between viral genome variations and their functional effects has so far remained elusive. Thus, here we sought to determine whether inter-host genetic variability of HCMV influences its ability to modulate NK cell responses to infection. For this purpose, five HCMV clinical isolates from a previously characterized cohort of pediatric patients with confirmed HCMV congenital infection were evaluated by next-generation sequencing (NGS) for genetic polymorphisms, phylogenetic relationships, and multiple-strain infection. We report variable levels of genetic characteristics among the selected clinical strains, with moderate variations in genome regions associated with modulation of NK cell functions. Remarkably, we show that different HCMV clinical strains differentially modulate the expression of several ligands for the NK cell-activating receptors NKG2D, DNAM-1/CD226, and NKp30. Specifically, the DNAM-1/CD226 ligand PVR/CD155 appears to be predominantly upregulated by fast-replicating (“aggressive”) HCMV isolates. On the other hand, the NGK2D ligands ULBP2/5/6 are downregulated regardless of the strain used, while other NK cell ligands (i.e., MICA, MICB, ULBP3, Nectin-2/CD112, and B7-H6) are not significantly modulated. Furthermore, we show that IFN-γ; production by NK cells co-cultured with HCMV-infected fibroblasts is directly proportional to the aggressiveness of the HCMV clinical isolates employed. Interestingly, loss of NK cell-modulating genes directed against NK cell ligands appears to be a common feature among the “aggressive” HCMV strains, which also share several gene variants across their genomes. Overall, even though further studies based on a higher number of patients would offer a more definitive scenario, our findings provide novel mechanistic insights into the impact of HCMV genetic variability on NK cell-mediated immune responses.

Deciphering the Potential Coding of Human Cytomegalovirus: New Predicted Transmembrane Proteome

“…The analysis of the generated pangenome ( Figure 2 and Figure 3 ), revealed wide differences among strains [ 13 ]. Clinical isolate VR7863 and TB40-E_UNC strains lacked a large number of genes compared to the other strains.…”

“…Our results highlight the utility of using these bioinformatic tools to gain knowledge of previously uncharacterized proteins that may be useful to select potential targets of the immune system. Our work also suggests that differences among the strains may be crucial for CMV tropism, replication, latency or the evasion of the host immune response [ 13 ]. Among the 77 identified proteins with predicted TM domains, only 39 (designated as a core TM proteome) were shared by all analyzed strains most of which were highly conserved which may have potential clinical relevance for the design of new therapeutics and preventives measures.…”

“…In addition to the 165 canonical ORFs [ 9 , 10 ], CMV genome encodes for other alternative transcripts in addition to have non-canonical translation initiation sites [ 11 , 12 ]. Furthermore, CMV encodes for a large number of genes, many of them with unknown functions that may probably be involved in key processes during host-cell interaction [ 13 ].…”

A Virus Genetic System To Analyze the Fusogenicity of Human Cytomegalovirus Glycoprotein B Variants

“…resulting in a more fusogenic gB that promotes cell-cell fusion [17]. Interestingly, three congenital HCMV isolates from Italy, described by Galitska et al [19,20], carried the same gB(S585G) variant that was found to mediate cell-cell fusion in HCMV strain VR1814 [17]. Therefore, we wondered whether highly fusogenic gB variants were frequently present in congenital HCMV isolates.…”

“…Interestingly, a study on clinical HCMV isolates from congenitally infected infants described syncytium-forming phenotypes for some of the isolates [19]. The complete viral genome sequence has recently been published for a few of these isolates [20], and it turned out that three isolates (P4, P14, and P15) contain the same gB(S585G) variant present in VR1814. This finding raised the question of whether syncytium-forming gB variants are highly prevalent in congenital HCMV isolates and whether they might be associated with pathogenicity.…”