The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

Abstract

We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as “vehicles” for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.

Author Summary

We previously demonstrated that HCMV, by engaging cellular receptors, changes the biology of blood monocytes, allowing for efficient viral entry into these cells and their use as virus-carriers in HCMV systemic spread. However, it was unclear how HCMV induces receptor-mediated signaling in infected cells. Here we report that HCMV by expressing a specific complex of five glycoproteins, present on HCMV clinical isolates, engages cellular integrin receptors and subsequently triggers integrin-mediated signaling leading to efficient viral entry into monocytes and productive infection of monocyte-derived macrophages. We also demonstrate that the HCMV pentameric complex has an inhibitory effect on integrin-mediated signaling in fibroblasts, an in vitro model system of HCMV infection, suggesting that the presence of the pentameric complex is not advantageous for HCMV infection of fibroblasts. Together, our results argue that HCMV uses distinct mechanisms to enter monocytes and fibroblasts. In support, our findings indicate that HCMV utilizes an endocytic-like route of entry into monocytes that is in contrast to viral fusion at the cell surface seen in fibroblasts. Our studies provide a molecular explanation for a previously observed critical role of the HCMV pentameric complex during infection of clinically relevant cell types, which in the future may lead to the development of better targets for antiviral therapy.