Identification of a novel signaling complex containing host chemokine receptor CXCR4, Interleukin-10 receptor, and human cytomegalovirus US27.

Human cytomegalovirus (HCMV) is a widespread herpesvirus that establishes latency in myeloid cells and persists by manipulating immune signaling. Chemokine receptor CXCR4 and its ligand CXCL12 regulate movement of myeloid progenitors into bone marrow and out into peripheral tissues. HCMV amplifies CXCL12-CXCR4 signaling through viral chemokine receptor US27 and cmvIL-10, a viral cytokine that binds the cellular IL-10 receptor (IL-10R), but precisely how these viral proteins influence CXCR4 is unknown. We used the proximity ligation assay (PLA) to examine association of CXCR4, IL-10R, and US27 in both transfected and HCMV-infected cells. CXCR4 and IL-10R colocalized to discrete clusters, and treatment with CXCL12 and cmvIL-10 dramatically increased receptor clustering and calcium flux. US27 was associated with CXCR4 and IL-10R in PLA clusters and further enhanced cluster formation and calcium signaling. These results indicate that CXCR4, IL-10R, and US27 form a novel virus-host signaling complex that enhances CXCL12 signaling during HCMV infection.

Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms.

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes lifelong infection in the host. Virus persistence is aided by extensive manipulation of the host immune system, particularly cytokine and chemokine signaling pathways. The HCMV UL111A gene encodes cmvIL-10, an ortholog of human interleukin-10 that has many immunomodulatory effects. We found that cmvIL-10 increased signaling outcomes from human CXCR4, a chemokine receptor with essential roles in hematopoiesis and immune cell trafficking, in response to its natural ligand CXCL12. Calcium flux and chemotaxis to CXCL12 were significantly greater in the presence of cmvIL-10 in monocytes, epithelial cells, and fibroblasts that express CXCR4. cmvIL-10 effects on CXCL12/CXCR4 signaling required the IL-10 receptor and Stat3 activation. Heightened signaling occurred both in HCMV-infected cells and in uninfected bystander cells, suggesting that cmvIL-10 may broadly influence chemokine networks by paracrine signaling during infection. Moreover, CXCL12/CXCR4 signaling was amplified in HCMV-infected cells compared to mock-infected cells even in the absence of cmvIL-10. Enhanced CXCL12/CXCR4 outcomes were associated with expression of the virally encoded chemokine receptor US27, and CXCL12/CXCR4 activation was reduced in cells infected with a deletion mutant lacking US27 (TB40/E-mCherry-US27Δ). US27 effects were Stat3 independent but required close proximity to CXCR4 in cell membranes of either HCMV-infected or US27-transfected cells. Thus, HCMV encodes two proteins, cmvIL-10 and US27, that exhibit distinct mechanisms for enhancing CXCR4 signaling. Either individually or in combination, cmvIL-10 and US27 may enable HCMV to exquisitely manipulate CXCR4 signaling to alter host immune responses and modify cell trafficking patterns during infection.IMPORTANCE The human chemokine system plays a central role in host defense, as evidenced by the many strategies devised by viruses for manipulating it. Human cytomegalovirus (HCMV) is widespread in the human population, but infection rarely causes disease except in immunocompromised hosts. We found that two different HCMV proteins, cmvIL-10 and US27, act through distinct mechanisms to upregulate the signaling activity of a cellular chemokine receptor, CXCR4. cmvIL-10 is a secreted viral cytokine that affects CXCR4 signaling in both infected and uninfected cells, while US27 is a component of the virus particle and impacts CXCR4 activity only in infected cells. Both cmvIL-10 and US27 promote increased intracellular calcium signaling and cell migration in response to chemokine CXCL12 binding to CXCR4. Our results demonstrate that HCMV exerts fine control over the CXCL12/CXCR4 pathway, which could lead to enhanced virus dissemination, altered immune cell trafficking, and serious health implications for HCMV patients.

Modulation of the Host Environment by Human Cytomegalovirus with Viral Interleukin 10 in Peripheral Blood.

Background: Human cytomegalovirus (HCMV) is a herpesvirus with both lytic and latent life cycles. Human cytomegalovirus encodes 2 viral cytokines that are orthologs of human cellular interleukin 10 (cIL-10). Both cytomegalovirus interleukin 10 (cmvIL-10) and Latency-associated cytomegalovirus interleukin 10 (LAcmvIL-10) (collectively vIL-10) are expressed during lytic infection and cause immunosuppressive effects that impede virus clearance. LAcmvIL-10 is also expressed during latent infection of myeloid progenitor cells and monocytes and facilitates persistence. Here, we investigated whether vIL-10 could be detected during natural infection. Methods: Plasma from healthy blood donors was tested by enzyme-linked immunosorbent assay for anti-HCMV immunoglobulin G and immunoglobulin M and for cIL-10 and vIL-10 levels using a novel vIL-10 assay that detects cmvIL-10 and LAcmvIL-10, with no cross-reactivity to cIL-10. Results: vIL-10 was evident in HCMV+ donors (n = 19 of 26), at levels ranging 31-547 pg/mL. By comparison, cIL-10 was detected at lower levels ranging 3-69 pg/mL. There was a strong correlation between vIL-10 and cIL-10 levels (P = .01). Antibodies against vIL-10 were also detected and neutralized vIL-10 activity. Conclusions: vIL-10 was detected in peripheral blood of healthy blood donors. These findings suggest that vIL-10 may play a key role in sensing or modifying the host environment during latency and, therefore, may be a potential target for intervention strategies.

The DRY box and C-terminal domain of the human cytomegalovirus US27 gene product play a role in promoting cell growth and survival.

Human cytomegalovirus (HCMV) is a widespread pathogen that can lay dormant in healthy individuals and establish lifelong latent infection. This successful co-existence is facilitated by a number of viral gene products that manipulate host cellular functions and immune responses. Among these immunomodulatory genes are four G-protein coupled receptors (GPCRs) encoded by HCMV, designated US27, US28, UL33, and UL78. Studies have shown the US28 gene product to be a functional chemokine receptor that signals both constitutively and in a ligand-dependent manner, resulting in a wide range of cellular effects. In previous work, we have found that US27 expression results in at least two biological effects: enhanced CXCR4 signaling and increased in cellular proliferation in HEK293 cells. Here, we examined the involvement of two protein domains, the DRY box and the C-terminal intracellular domain (CTD) of US27, in mediating both cell proliferation and survival. While both domains were required for a proliferative effect, loss of either domain only moderately impacted cell survival, suggesting that US27 may interact with cell survival pathways through protein regions other than the DRY box and CTD. Quantitative RT-PCR arrays were used to profile changes in cellular gene expression in the HEK293-US27 cell line, and down-regulation of cell cycle regulators CDKN1A/p21/CIP1 (cyclin dependent kinase inhibitor 1A) and SESN (Sestrin2 or Hi95) was observed. These results indicate that increased cell proliferation due to US27 may be linked to suppression of negative growth regulators, and that these interactions require the DRY box and CTD.

The human cytomegalovirus US27 gene product enhances cell proliferation and alters cellular gene expression.

Human cytomegalovirus (HCMV) is a prevalent pathogen worldwide. Although generally harmless in healthy individuals, HCMV can pose a serious threat to immune compromised individuals and developing fetuses in utero. HCMV encodes four genes predicted to give rise to G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. The US28 gene product is a functional chemokine receptor that enhances cell growth in some cell types but induces apoptosis in others. In contrast, the US27 gene product has not been demonstrated to signal either constitutively or in a ligand-induced manner. In this study, US27 was expressed in transfected cells, and both cell proliferation and DNA synthesis were significantly increased compared to control cells. PCR array analysis revealed that expression of US27 led to changes in a limited number of cellular genes, but genes that were up-regulated included the pro-survival factor Bcl-x, AP-1 transcription factor components jun and fos, and the IL-6 family cytokine oncostatin M. These results demonstrate that US27 can impact host cell physiology and may shed light on the function of this orphan viral GPCR.