Human Cytomegalovirus UL130 Protein Promotes Endothelial Cell Infection through a Producer Cell Modification of the Virion

Abstract

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes life-long, subclinical infections ubiquitously in human populations (5). HCMV causes serious morbidity in settings of immune system immaturity or depression: it is the leading viral cause of defects at birth and generates a potentially life-threatening disease in immunocompromised patients. In patients with HCMV disease, the virus can be demonstrated in a variety of cells, including hematopoietic cells (monocytes-macrophages, dendritic cells, and neutrophils), endothelial cells (EC), epithelial cells, fibroblasts, neurons, smooth muscle cells, and hepatocytes (5, 38). Much recent work has focused on HCMV infections of EC, for several reasons: (i) arterial endothelia, along with CD34 ϩ myeloid progenitors, have been proposed as sites of HCMV persistence and latency (20); (ii) a bidirectional transmission of HCMV between EC and leukocytes can be demonstrated in vitro and may reflect a mechanism of dissemination in vivo (11,14,34,44); (iii) circulating giant EC may similarly contribute directly to dissemination (32); (iv) HCMV infections of the uterine microvasculature and cytotrophoblasts may underlie motherto-fetus transmission and compromise the placental trophic function in affected pregnancies (25, 47); and (v) HCMV may play a role in atherogenesis, postangioplasty restenosis, posttransplantation endot...

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

“…Previous reports regarding the function of pUL130 suggested that this protein might be critical for formation of the pentameric gCIII complex. The C-terminal frameshift naturally occurring in HCMV strain Towne has been reported to cause accelerated lysosomal degradation of pUL130 compared to the wild-type protein (15), and lack of the UL130 protein results in loss of the complete pentameric complex from the virion envelope (21). It was therefore tempting to speculate that the Cterminal UL130 mutations that reduced EC tropism in our analysis also affected incorporation of this complex into mutant virions.…”

“…1). The first charge cluster was omitted because amino acids 1 to 25 have been shown to be a signal peptide that is cleaved off (15 The generation of mutants was performed in E. coli by markerless replacement of the selected charged amino acids with alanines in BACs containing the genome of the highly endotheliotropic HCMV-TB40-BAC4. A complete list of the generated mutants and the respective amino acid exchanges as well as the primer sequences used for markerless mutagenesis are given in Tables 1 and 2.…”

“…As pointed out in a number of publications, extended propagation of HCMV strains on fibroblast cultures leads to a loss of EC tropism, which is regularly associated with mutations in the UL128 locus (1,2,4,8,11,15). In HCMV strain Towne, UL130 is mutated with a frameshift replacing the C-terminal 11 amino acids by 26 amino acids (8).…”

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

“…In contrast, entry into epithelial, endothelial, dendritic, and monocytic cells requires gB, gH/gL/gO, and the PC and occurs through endocytosis or macropinocytosis followed by a pH-dependent fusion event (Fig. 3B) (31-33, 35,[44][45][46][47][48][49]. The stark difference in CMV entry pathways underscores the involvement of fundamentally distinct entry mechanisms that likely extend beyond mere differences in cell-type-specific receptors.…”

The immunological underpinnings of vaccinations to prevent cytomegalovirus disease

“…The evaluation of the chimeras is now in an investigator-initiated phase I study to evaluate safety and immunogenicity properties in CMV-naive subjects. A different 'dial-up' strategy seeks to improve the AD169 LACV by repairing mutations that prevent expression of the gH pentamer complex, 108,109 with the intention of broadening cell tropism and the quality of neutralizing antibody responses. One challenge is the loss of gH pentamer expression during propagation in fibroblasts, 110,111 so the LACV candidate V160 must be propagated on the ARPE19 109,112 epithelial cell line.…”