The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production

We hypothesized that US28, a cytomegalovirus (CMV) CC chemokine receptor homolog, plays a role in modulating the host antiviral defense. Monocyte chemotaxis was induced by supernatants from fibroblasts infected with a US28 deletion mutant of CMV (CMV Delta US28) due to endogenously produced CC chemokines MCP-1 and RANTES. However, these chemokines were sequestered from the supernatants of CMV-infected cells that did express US28. US28 was also capable of sequestering exogenously added RANTES. Surprisingly, cells infected with CMV Delta US28 transcribed and secreted increased levels IL-8, a CXC chemokine, when compared to CMV-infected cells. Finally, because chemokines are potent mediators of immune cell migration through the endothelium, we characterized the CC chemokine binding potential of CMV-infected endothelial cells. We propose that US28 functions as a 'chemokine sink' by sequestering endogenously and exogenously produced chemokines and alters the production of the CXC chemokine IL-8, suggesting that CMV could significantly alter the inflammatory milieu surrounding infected cells.     

Identification of the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G protein-coupled receptor

Human cytomegalovirus (HCMV) encodes a G protein-coupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.     

US28 Is a Potent Activator of Phospholipase C during HCMV Infection of Clinically Relevant Target Cells

Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-β in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca⁺⁺ release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca⁺⁺ release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.     

The Cytomegalovirus-Encoded Chemokine Receptor US28 Can Enhance Cell-Cell Fusion Mediated by Different Viral Proteins

The human cytomegalovirus (CMV) US28 gene encodes a functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the actual role of the protein in the CMV life cycle. Expression of US28 allows human immunodeficiency virus type 1 (HIV-1) entry into certain CD4⁺ cells and their fusion with cells expressing HIV-1 envelope (Env) proteins. Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4, which behave as CD4-associated HIV-1 coreceptors. We found that coexpression of US28 and either CXCR4 or CCR5 in CD4⁺ cells resulted in enhanced synctium formation with HIV-1 Env⁺ cells. This positive effect of US28 on cell fusion seems to be distinct from its HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also observed when US28 was expressed on the HIV-1 Env⁺ cells instead of an CD4⁺ target cells. Furthermore, US28 could enhance cell fusion mediated by other viral proteins, in particular, the G protein of vesicular stomatitis virus (VSV-G). The HIV-1 coreceptor and fusion-enhancing activities could be affected by mutations in different domains of US28. The fusion-enhancing activity of US28 seems to be cell type dependent. Indeed, cells coexpressing VSV-G and US28 fused more efficiently with human, simian, or feline target cells, while US28 had no apparent effect on fusion with the three mouse or rat cell lines tested. The positive effect of US28 on cell fusion might therefore require its interaction with a cell-specific factor. We discuss a possible role for US28 in the fusion of the CMV envelope with target cells and CMV entry.     

The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.     

Complete DNA sequence of the rat cytomegalovirus genome

We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the rep gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.     

Intracellular Signaling by the Chemokine Receptor US28 during Human Cytomegalovirus Infection

In patients with impaired cell-mediated immune responses (e.g., lung transplant recipients and AIDS patients), cytomegalovirus (CMV) infection causes severe disease such as pneumonitis. However, although immunocompetency in the host can protect from CMV disease, the virus persists by evading the host immune defenses. A model of CMV infection of the endothelium has been developed in which inflammatory stimuli, such as the CC chemokine RANTES, bind to the endothelial cell surface, stimulating calcium flux during late times of CMV infection. At 96 h postinfection, CMV-infected cells express mRNA of the CMV-encoded CC chemokine receptor US28 but do not express mRNA of other CC chemokine receptors that bind RANTES (CCR1, CCR4, CCR5). Cloning and stable expression of the receptor CMV US28 in human kidney epithelial cells (293 cells) with and without the heterotrimeric G protein α₁₆ indicated that CMV US28 couples to both Gα_i and Gα₁₆ proteins to activate calcium flux in response to the chemokines RANTES and MCP-3. Furthermore, cells that coexpress US28 and Gα₁₆ responded to RANTES stimulation with activation of extracellular signal-regulated kinase, which could be attributed, in part, to specific Gα₁₆ coupling. Thus, through expression of the CC chemokine receptor US28, CMV may utilize resident G proteins of the infected cell to manipulate cellular responses stimulated by chemokines.     

Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1 alpha, and the CX(3)C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca(2+). Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G(q/11)-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5'-O-(3-[(35)S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.     

Functional Analysis of the Human Cytomegalovirus US28 Gene by Insertion Mutagenesis with the Green Fluorescent Protein Gene

The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR). Previous studies demonstrated that recombinant US28 protein can bind the β class of chemokines (K. Neote, D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall, Cell 72:415–425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J. L. Gao and P. M. Murphy, J. Biol. Chem. 269:28539–28542, 1994). In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of the Escherichia coli gpt gene and the gene for the green fluorescent protein. The US28 gene is not required for growth in human fibroblasts (HF). HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES. In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the β-chemokine binding and induced calcium signaling in HCMV-infected cells. The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells. Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.Open reading frames (ORF) with homology to cellular seven transmembrane spanning receptors have been identified in the genomes of both beta and gamma herpesviruses (15, 37). Many cellular seven transmembrane spanning receptors have been shown to be G protein-coupled receptors (GCR) and comprise a superfamily of genes encoding the receptors for a variety of biological compounds, including neurotransmitters, hormones, odorants, and chemotactic agents. GCR link the binding of an extracellular ligand to processes within the cell by their activation of associated G proteins. G proteins can activate serine/threonine kinases, phosphatidylinositol 3-kinase, phospholipases, or Ras (9). These proteins, in turn, can stimulate mitogen-activated protein kinase or generate second messenger molecules, such as diacylglycerol and inositol triphosphate, resulting in the activation of protein kinase C and increases in intracellular Ca²⁺ levels (9). Ultimately, these processes result in the amplification of the initial signal transduced by the ligand-GCR interaction into complex cellular processes such as chemotaxis.GCR are receptors for chemokines, derived from chemotactic cytokine, a multigene family of 70- to 90-amino-acid soluble proteins that are excreted from a variety of cell types and play important roles in leukocyte trafficking and immune regulation (7). Two classes of these structurally similar proteins are defined by the first two of four conserved cysteines. In the α class (e.g., interleukin-8 [IL-8], MGSA, and GCP-2) the first two cysteines are separated by an intervening residue (C-X-C), while in the β class (e.g., RANTES, MIP-1α, MIP-1β, and MCP-1) they are adjacent (C-C). In general, the α-chemokines attract primarily neutrophils, while β-chemokines can have activity on monocytes, lymphocytes, eosinophils, and basophils (46).The human cytomegalovirus (HCMV) US28 ORF shows approximately 33% homology to the cellular β-chemokine receptor CCR-1 (35). Conserved features of viral and cellular proteins include the putative seven-membrane spanning regions and cysteines implicated in disulfide bond formation. The sequence homology between US28 and cellular GCR led to the identification of β-chemokines as the ligand for the viral receptor. Recombinant HCMV US28 protein expressed in 293 cells was shown to bind β-chemokines (35), and as with the binding of chemokines by their cellular receptors, the binding of ligand by recombinant US28 expressed in K562 cells led to an increase in intracellular calcium (21).During an acute infection, HCMV can be found in the blood as well as in numerous tissues, with the lungs, kidneys, salivary gland, and liver being commonly involved. HCMV has been identified in a wide variety of cells both in culture and in patients’ tissue, including epithelial cells, endothelial cells, fibroblasts, monocytes/macrophages, and lymphocytes (33, 47, 52). Because HCMV can infect cell types that respond to chemokines and cell types that produce chemokines, the viral GCR may mimic the functions of cellular GCR, but the role of the expression of a viral GCR in viral biology and the cell type in which it is important are not known.While researchers examined US28 function with recombinant protein in previous studies (21, 36), we have investigated the functions of the US28 gene expressed from the viral genome. We have constructed a recombinant HCMV with the US28 ORF disrupted by the genes for the green fluorescent protein (GFP) and guanine phosphoribosyl transferase (GPT) and have demonstrated that US28 is responsible for the functions of a GCR in HCMV-infected cells.     

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.     

Accessory human cytomegalovirus glycoprotein US9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells.

Human cytomegalovirus (CMV) encodes accessory glycoproteins that are dispensable for virus growth in nonpolarized cells in culture. We report that CMV deletion mutants lacking the gene for accessory glycoprotein US9 in the unique short component of the viral genome are impaired in plaque formation in polarized human retinal pigment epithelial (ARPE-19) cells. Comparison of CMV deletion mutants in US9 with herpes simplex virus type 1 deletion mutants lacking glycoproteins gE and gI showed that both of these mutants are impaired in altering junctional complexes and increasing paracellular permeability in polarized ARPE-19 cells cultured on permeable filter supports. Results of functional studies indicate that CMV US9 and homologs of gE have analogous roles in promoting virus spread across lateral membranes of polarized epithelial cells.     

Functional genetic analysis of rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor

Rhesus cytomegalovirus (RhCMV) is an emerging model for human cytomegalovirus (HCMV) pathogenesis that facilitates experimental CMV infection of a natural primate host closely related to humans. We have generated a library of RhCMV mutants with lesions in genes whose HCMV orthologues have been characterized as nonessential for replication in human fibroblasts, and we characterized their replication in rhesus fibroblasts and epithelial cells. The RhCMV mutants grew well in fibroblasts, as predicted by earlier studies with HCMV. However, mutations in four genes caused replication defects in rhesus retinal pigment epithelial cells: Rh01 (an HCMV TRL1 orthologue), Rh159 (HCMV UL148), Rh160 (HCMV UL132), and Rh203 (HCMV US22). Growth of the Rh01-deficient mutant was examined in detail. After entry into epithelial cells, the mutant expressed representative viral proteins, accumulated viral DNA, and generated infectious virus, but it failed to spread efficiently. We conclude that Rh01 is a cell tropism determinant that has the potential to dramatically affect virus spread and pathogenesis.     

Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.     

Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28

Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-kappaB signaling via G(q/11) protein-dependent pathways. Whereas phospholipase C activation is mediated via Galpha(q/11) subunits, the activation of NF-kappaB strongly depends on betagamma subunits with a preference for the beta(2)gamma(1) dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX(3)C chemokine fractalkine acts as a partial inverse agonist with IC(50) values of 1-5 nm. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.     

CXCL10 production from cytomegalovirus-stimulated microglia is regulated by both human and viral interleukin-10

Glial cells orchestrate immunocyte recruitment to focal areas of viral infection within the brain and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since recruitment of T lymphocytes plays a critical role in resolving cytomegalovirus (CMV) infection, we investigated the production of a T-cell chemoattractant, CXCL10 (gamma interferon-inducible protein 10) in response to viral infection of human glial cells. Infection with CMV was found to elicit the production of CXCL10 from primary microglial cells but not from astrocytes. This CXCL10 expression was not dependent on secondary protein synthesis but did require the phosphorylation of p38 mitogen-activated protein (MAP) kinase. In addition, migration of activated lymphocytes toward supernatants from CMV-stimulated microglial cells was partially suppressed by anti-CXCL10 antibodies. Since regulation of central nervous system inflammation is essential to allow viral clearance without immunopathology, microglial cells were then treated with anti-inflammatory cytokines. CMV-induced CXCL10 production from microglial cells was suppressed following treatment with interleukin-10 (IL-10) and IL-4 but not following treatment with transforming growth factor beta. The IL-10-mediated inhibition of CXCL10 production was associated with decreased CMV-induced NF-kappa B activation but not decreased p38 MAP kinase phosphorylation. Finally, CMV infection of fully permissive astrocytes resulted in mRNA expression for the viral homologue to human IL-10 (i.e., cmvIL-10 [UL111a]) in its spliced form and conditioned medium from CMV-infected astrocytes inhibited virus-induced CXCL10 production from microglial cells through the IL-10 receptor. These findings present yet another mechanism through which CMV may subvert host immune responses.