Significance: Human cytomegalovirus (HCMV) pathogenesis relies upon viral infection of monocytes, as this is the key cell type mediating the hematogenous dissemination of HCMV throughout the host. Here we report that HCMV glycoprotein B surprisingly remains bound to the epidermal growth factor receptor (EGFR) throughout the duration of viral postentry events to induce chronic signaling through EGFR. This chronic EGFR signaling is required to facilitate viral localization to correct trafficking vesicles, as well as promoting nuclear translocation of viral DNA into the monocyte nucleus, both of which are necessary for infection of monocytes. Our data highlight how HCMV regulates the EGFR kinase externally and, uniquely, internally to promote separate and distinct events required for productive infection, spread, and persistence.

Human cytomegalovirus (HCMV) infection is endemic throughout the world regardless of socioeconomic conditions and geographic locations with a seroprevalence reaching up to 100% in some developing countries. Although asymptomatic in healthy individuals, HCMV can cause severe multiorgan disease in immunocompromised or immunonaive patients. HCMV disease is a direct consequence of monocyte-mediated systematic spread of the virus following infection. Because monocytes are short-lived cells, HCMV must subvert the natural short life-span of these blood cells by inducing a distinct activation of Akt, a serine/theonine protein kinase. In this work, we demonstrate that HCMV glycoproteins gB and gH work in tandem to reroute classical host cellular receptor signaling to aberrantly activate Akt and drive survival of infected monocytes. Deciphering how HCMV modulates the cellular pathway to induce monocyte survival is important to develop a new class of anti-HCMV drugs that could target and prevent spread of the virus by eliminating infected monocytes.

Blood monocytes mediate the hematogenous dissemination of human cytomegalovirus (HCMV) in the host. However, monocytes have a short 48-hour (h) lifespan and are not permissive for viral replication. We previously established that HCMV infection drives differentiation of monocytes into long-lived macrophages to mediate viral dissemination, though the mechanism was unclear. Here, we found that HCMV infection promoted monocyte polarization into distinct macrophages by inducing select M1 and M2 differentiation markers and that Akt played a central role in driving differentiation. Akt’s upstream positive regulators, PI3K and SHIP1, facilitated the expression of the M1/M2 differentiation markers with p110δ being the predominant PI3K isoform inducing differentiation. Downstream of Akt, M1/M2 differentiation was mediated by caspase 3, whose activity was tightly regulated by Akt in a temporal manner. Overall, this study highlights that HCMV employs the PI3K/SHIP1/Akt pathway to regulate caspase 3 activity and drive monocyte differentiation into unique macrophages, which is critical for viral dissemination.

Establishing a nonproductive, quiescent infection within monocytes is essential for the spread of human cytomegalovirus (HCMV). We investigated the mechanisms through which HCMV establishes a quiescent infection in monocytes. US28 is a virally encoded G protein–coupled receptor (GPCR) that is essential for silent infections within cells of the myeloid lineage. We found that preformed US28 was rapidly delivered to monocytes by HCMV viral particles, whereas the de novo synthesis of US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28Δ) was unable to establish a quiescent infection, resulting in a fully productive lytic infection able to produce progeny virus. Infection with US28Δ HCMV resulted in the phosphorylation of the serine and threonine kinase Akt at Ser ⁴⁷³ and Thr ³⁰⁸ , in contrast with the phosphorylation of Akt only at Ser ⁴⁷³ after WT viral infection. Inhibiting the dual phosphorylation of Akt prevented the lytic replication of US28Δ, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of wild-type HCMV. Mechanistically, we found that US28 was necessary and sufficient to attenuate epidermal growth factor receptor (EGFR) signaling induced during the entry of WT virus, which led to the site-specific phosphorylation of Akt at Ser ⁴⁷³ . Thus, particle-delivered US28 fine-tunes Akt activity by limiting HCMV-induced EGFR activation during viral entry, enabling quiescent infection in monocytes.

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