While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX3C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis.

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to proteintyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK⅐Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK⅐Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2⅐FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH 2 -terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration. Human cytomegalovirus (HCMV)1 is a ubiquitous herpesvirus that establishes a life-long latent infection after the primary infection has been cleared. Although anti-viral therapy has appreciably reduced disease in transplant and AIDS patients, HCMV is still a significant problem in congenital disease and bone marrow transplant patients. In addition, HCMV has also been associated with long term diseases such as atherosclerosis, restenosis after angioplasty, chronic rejection after solid organ transplantation, and malignancies (1-4). The development of vascular disease involves a chronic inflammatory process with many contributing factors, and of these, chemokines and their receptors have been identified as key mediators. Interestingly, HCMV encodes a CXC chemokine (UL146), a potential CC chemokine (UL128), and four potential chemokine receptors (US27, US28, UL33, and UL78) with the most characterized being US28 (5-8). We have previously reported that US28 mediates arterial smooth muscle cell (SMC) migration and that this activity may contribute to viral dissemination and/or acceleration of vascular disease development (9).US28 contains homology to the CC-chemokine receptors (10) and binds to a broad spectrum of chemokines including the CC chemokines RANTES, MCP-1, MCP-3, and MIP-1␤ and the CX 3 C chemokine Fractalkine/CX3CL1 (11, 12). That CC chemokines fail to compete out Fractalkine binding suggests that Fractalkine binds to additional unique regions of US28 compared with the CC chemokines (11). In 293 cells, RANTES binding to US28 activates ERK1/2 pathways through the Gproteins G␣ i1 and G␣ 16 (13). We have...

Coupling of G proteins to ligand-engaged chemokine receptors is the paramount event in G-protein-coupled receptor signal transduction. Previously, we have demonstrated that the human cytomegalovirus-encoded chemokine receptor US28 mediates human vascular smooth muscle cell (SMC) migration in response to either RANTES or monocyte chemoattractant protein 1. In this report, we identify the G proteins that couple with US28 to promote vascular SMC migration and identify other signaling molecules that play critical roles in this process. US28-mediated cellular migration was enhanced with the expression of the G-protein subunits G␣12 and G␣13, suggesting that US28 may functionally couple to these G proteins. In correlation with this observation, US28 was able to activate RhoA, a downstream effector of G␣12 and G␣13 in cell types with these G proteins but not in those without them and activation of RhoA was dependent on US28 stimulation with RANTES. In addition, inactivation of RhoA or the RhoA-associated kinase p160ROCK with a dominantnegative mutant of RhoA or the small molecule inhibitor Y27632, respectively, abrogated US28-induced SMC migration. The data presented here suggest that US28 functionally signals through G␣12 family G proteins and RhoA in a ligand-dependent manner and these signaling molecules are important for the ability of US28 to induce cellular migration.

No abstract

Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration.