Human Cytomegalovirus Manipulates Syntaxin 6 for Successful Trafficking and Subsequent Infection of Monocytes.

Human cytomegalovirus (HCMV) exhibits a complex host-pathogen interaction with peripheral blood monocytes. We have identified a unique, cell-type specific retrograde-like intracellular trafficking pattern that HCMV utilizes to gain access to the monocyte nucleus and for productive infection. We show that infection of primary human monocytes, epithelial cells, and fibroblasts leads to an increase in the amount of the trafficking protein Syntaxin 6 (Stx6). However, only knockdown (KD) of Stx6 in monocytes inhibited viral trafficking to the trans-Golgi network (TGN), a requisite step for nuclear translocation in monocytes. Conversely, KD of Stx6 in epithelial cells and fibroblasts did not change the kinetics of nuclear translocation and productive infection. Stx6 predominantly functions at the level of the TGN where it facilitates retrograde transport, a trafficking pathway used by only a few cellular proteins and seldom by pathogens. We also newly identify that in monocytes, Stx6 exhibits an irregular vesicular localization rather than being concentrated at the TGN as seen in other cell-types. Lastly, we implicate that viral particles that associate with both Stx6 and EEA1 early in infection are the viral population that successfully traffics to the TGN at later time points and undergo nuclear translocation. Additionally, we show for the first time that HCMV enters the TGN, and that lack of Stx6 prevents viral trafficking to this organelle. We argue that we have identified an essential cell-type specific regulator that controls early steps in efficient productive infection of a cell-type required for viral persistence and disease. IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe and often fatal disease in the immunocompromised. It is one of the leading infectious causes of birth defects and causes severe complications in transplant recipients. By uncovering the unique pathways used by the virus to infect key cells, such as monocytes, responsible for dissemination and persistence, we provide new potential targets for therapeutic intervention.

Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes.

Human cytomegalovirus (HCMV) infects peripheral blood monocytes and triggers biological changes that promote viral dissemination and persistence. We have shown that HCMV induces a proinflammatory state in infected monocytes, resulting in enhanced monocyte motility and transendothelial migration, prolonged monocyte survival, and differentiation toward a long-lived M1-like macrophage phenotype. Our data indicate that HCMV triggers these changes, in the absence of de novo viral gene expression and replication, through engagement and activation of epidermal growth factor receptor (EGFR) and integrins on the surface of monocytes. We previously identified that HCMV induces the upregulation of multiple proinflammatory gene ontologies, with the interferon-associated gene ontology exhibiting the highest percentage of upregulated genes. However, the function of the HCMV-induced interferon (IFN)-stimulated genes (ISGs) in infected monocytes remained unclear. We now show that HCMV induces the enhanced expression and activation of a key ISG transcriptional regulator, signal transducer and activator of transcription (STAT1), via an IFN-independent but EGFR- and integrin-dependent signaling pathway. Furthermore, we identified a biphasic activation of STAT1 that likely promotes two distinct phases of STAT1-mediated transcriptional activity. Moreover, our data show that STAT1 is required for efficient early HCMV-induced enhanced monocyte motility and later for HCMV-induced monocyte-to-macrophage differentiation and for the regulation of macrophage polarization, suggesting that STAT1 may serve as a molecular convergence point linking the biological changes that occur at early and later times postinfection. Taken together, our results suggest that HCMV reroutes the biphasic activation of a traditionally antiviral gene product through an EGFR- and integrin-dependent pathway in order to help promote the proviral activation and polarization of infected monocytes.IMPORTANCE HCMV promotes multiple functional changes in infected monocytes that are required for viral spread and persistence, including their enhanced motility and differentiation/polarization toward a proinflammatory M1 macrophage. We now show that HCMV utilizes the traditionally IFN-associated gene product, STAT1, to promote these changes. Our data suggest that HCMV utilizes EGFR- and integrin-dependent (but IFN-independent) signaling pathways to induce STAT1 activation, which may allow the virus to specifically dictate the biological activity of STAT1 during infection. Our data indicate that HCMV utilizes two phases of STAT1 activation, which we argue molecularly links the biological changes that occur following initial binding to those that continue to occur days to weeks following infection. Furthermore, our findings may highlight a unique mechanism for how HCMV avoids the antiviral response during infection by hijacking the function of a critical component of the IFN response pathway.

Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins.

UNLABELLED: Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection. IMPORTANCE: Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence in in vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence.

HCMV reprogramming of infected monocyte survival and differentiation: a Goldilocks phenomenon.

The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV) infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a "finely-tuned" macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host.