We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary peripheral blood monocytes, allowing HCMV to use monocytes as "vehicles" for its systemic spread. HCMV infection of monocytes results in rapid induction of phosphatidylinositol-3-kinase [PI(3)K] and NF-B activities. Integrins, which are upstream of the PI(3)K and NF-B pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src signaling pathway are essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected-monocyte transcriptome and found that the integrin/Src signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry into monocytes. New results confirmed that HCMV entry into target monocytes was significantly reduced in cells deficient in paxillin expression or the integrin/Src/ paxillin signaling pathway. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination.Human cytomegalovirus (HCMV), a betaherpesvirus, is a prevalent infectious agent, with seropositivity reaching 50 to 80% among adults in the United States (15). In immunocompromised individuals, viral infection can lead to significant morbidity and mortality (19,35). HCMV is the leading cause of congenital central nervous system damage and a leading opportunistic pathogen in AIDS and transplant patients (19,35). In immunocompetent individuals, HCMV infection is usually mild or asymptomatic, although results now show that HCMV is a strong risk factor in the development of cardiovascular diseases (CVDs) (25,(42)(43)(44)47).After primary infection, HCMV establishes a lifelong persistent infection, with frequent reactivations and active infection of new target cells. The virus can be shed in nearly all body fluids, illustrating HCMV's broad cellular tropism and capacity to spread to and infect most organ systems (5, 27, 54). It is postulated that effective multiorgan viral spread is critical for HCMV survival and persistence within infected hosts (35). HCMV has been shown to infect circulating cells in the blood (30), such as monocytes, and to use these cells as viral carriers, allowing dissemination to target tissues (37). In support, HCMV infection is c...