Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

Abstract

The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a chargecluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.Human cytomegalovirus (HCMV) is a widespread betaherpesvirus that causes lifelong persistent infections with occasional reactivations. While HCMV infection is usually clinically unapparent in the immunocompetent host, it can cause severe disseminated infections under conditions of immunosuppression, with manifestations in the lung, retina, and gastrointestinal tract, among others (12). Various cell types support viral replication, including epithelial cells and endothelial cells (ECs), smooth muscle cells, fibroblasts, and cells of hematopoietic origin (13,14,18,19,25,26,37). Among these target cells, endothelial cells are assumed to contribute particularly to hematogenous dissemination of HCMV (24).While recent clinical HCMV isolates are characterized by this broad cell tropism, the target cell range becomes restricted during long-term propagation on fibroblasts (28, 33). The underlying mechanism for this cell culture adaptation is a modulation within the viral genes UL128, UL130, and UL131A (8, 11). These three genes have been shown to be essential for infection of granulocytes, dendritic cells, epithelial cells, and endothelial cells but are dispensable for infection of fibroblasts (1, 9, 11, 34, 35). The encoded proteins pUL128, pUL130, and pUL131A were reported to form a complex with the viral glycoproteins gH and gL that is distinct from the glycoprotein complex gCIII (gH/gL/gO) (35). Whereas poorly endotheliotropic HCMV strains bear just the gH/gL/gO complex in their envelopes, highly endotheliotropic strains bear both gCIII variants: gH/gL/gO and gH/gL/pUL128-131A. Deletion of any of the three genes UL128-131A results in loss of EC tropism (11), most likely because only a complete complex of gH/gL and pUL128, pUL130, and pUL131A can efficiently function in endocytic entry in ECs (21). However, functional sites within the proteins (e.g., mediating binding to the viral complex partners or interaction with a putative cellular rece...

Scanning Mutagenesis of Human Cytomegalovirus Glycoprotein gH/gL

“…This was a surprising result and stands in stark contrast to the reports that similar CCTA mutagenesis of the UL128-131 proteins greatly disrupted gH/gL/UL128-131 (54)(55)(56). Given that gH/gL/UL128-131 likely binds cell type-specific receptors to facilitate infection (21,22), these observations suggest that the receptor-binding surfaces lie on UL128-131 themselves and gH/gL may serve as a scaffold or platform for presentation of the UL128-131 proteins.…”

Mutational Mapping of pUL131A of Human Cytomegalovirus Emphasizes Its Central Role for Endothelial Cell Tropism

“…The finding that virtually all parts of the UL131A protein are relevant for endothelial cell infection is supported by comparison of the amino acid sequence of pUL131A in clinical isolates of HCMV from different geographical regions that showed that the pUL131A sequence of 11 of 12 clinical isolates is absolutely identical (data not shown). In contrast, comparison of the amino acid sequence of pUL130 in the same clinical isolates showed variation in 12 amino acid positions, mainly in the N-terminal and central part of pUL130, and the function for EC tropism was mapped to the C-terminal third of pUL130, which was again highly conserved (22).…”

“…Interestingly, a peptide containing charge cluster pUL131A [91][92][93][94][95][96][97][98] has recently been successfully used to induce neutralizing antibodies in rabbits (20), hence arguing against extended mutation of this epitope for vaccine development. However, as we have previously defined similar mutations with partial effects on the EC tropism in UL128 and UL130 (22,23), combinations of mutations in different genes are also feasible and may result in an immunogenic mutant that is robust against spontaneous genetic reversion and displays strongly reduced but not completely abrogated replication potential in endothelial cells.…”

“…Anti-gB served as a control antibody. Wild-type HCMV-TB40-BAC4 and a mutant lacking the complete UL128-131A gene region (22) served as positive-and negative-control viruses, respectively. sentative mutants with regard to the total number of virus particles binding to HFF and HUVEC and with regard to their ability to translocate toward the nucleus of the respective cell type.…”

“…Each of the three genes within the UL128-131 locus is essential for EC tropism, most likely because only a complete pentameric gcIII complex can efficiently mediate endocytic entry in ECs (10,19). We have previously defined tropism-relevant peptides within UL128 and UL130 by charge-cluster-to-alanine (CCTA) mutagenesis (22,23), and we report here the completion of this analysis with functional mapping of UL131A.…”