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.

The establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34 ϩ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/ trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34 ϩ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34 ϩ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mockinfected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34 ϩ HPCs by HCMV.IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV from its latent reservoir in the bone marrow causes significant morbidity and mortality in immunologically compromised individuals, such as bone marrow and solid organ transplant patients. Lifelong persistent infection has also been linked with the development of various cardiovascular diseases in otherwise healthy individuals. Current HCMV therapeutics target lytic replication, but not the latent viral reservoir; thus, an understanding of the molecular basis for viral latency and persistence is paramount to controlling or eliminating HCMV infection. Here, we show that the viral signalosome activated by HCMV binding to its entry re-

The ability of human cytomegalovirus (HCMV) to reactivate from latent infection of hematopoietic progenitor cells (HPCs) is intimately linked to cellular differentiation. HCMV encodes UL7 that our group has shown is secreted from infected cells and induces angiogenesis. In this study, we show that UL7 is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R), a well-known critical factor in HPC differentiation. We observed that UL7 directly binds Flt-3R and induces downstream signaling cascades, including phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. Importantly, we show that UL7 protein induces differentiation of both CD34+ HPCs and CD14+ monocytes. Last, we show that an HCMV mutant lacking UL7 fails to reactivate in CD34+ HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.

Japanese encephalitis (JE) cases have been increasingly reported recently especially in Seoul and its vicinity. Pigs are known as amplifying host of JE virus (JEV), but do not play an important role in these recent events because pig-breeding is not common in Seoul. The distribution and the density of migratory birds are correlated with JE cases in cities and they might be highly potential hosts contributing to transmit JEV in metropolitan areas. JE genotype and sero-prevalence in birds should be determined for the verification of the transmission route of JEV in the recent sporadic occurrence of JE cases in Seoul.

We initiated experiments to examine the infection of monocytes postentry. New data show that human cytomegalovirus (HCMV) DNA is detected in the nucleus beginning only at 3 d postinfection in monocytes, compared with 30 min postinfection in fibroblasts and endothelial cells, suggesting that HCMV nuclear translocation in monocytes is distinct from that seen in other cell types. We now show that HCMV is initially retained in early endosomes and then moves sequentially to the trans-Golgi network (TGN) and recycling endosomes before nuclear translocation. HCMV is retained initially as a mature particle before deenvelopment in recycling endosomes. Disruption of the TGN significantly reduced nuclear translocation of viral DNA, and HCMV nuclear translocation in infected monocytes was observed only when correct gH/gL/UL128-131/integrin/c-Src signaling occurred. Taken together, our findings show that viral binding of the gH/gL/UL128-131 complex to integrins and the ensuing c-Src signaling drive a unique nuclear translocation pattern that promotes productive infection and avoids viral degradation, suggesting that it represents an additional viral evasion/survival strategy.monocytes | viral trafficking | gH/gL/UL128-131 complex | integrins | nuclear translocation pathway H uman cytomegalovirus (HCMV) infection usually results in an asymptomatic lifelong persistent infection in healthy individuals after primary infection (1, 2). Although HCMV infection rarely causes disease in immunocompetent individuals, it can cause severe/ fatal disease in immunocompromised patients, such as congenitally infected neonates, patients with AIDS, and transplant recipients (3-6). HCMV infection is also associated with the development of cardiovascular diseases, including atherosclerosis, restenosis, and transplant vascular sclerosis, as well as some cancers (7-10). This wide range of pathological complications results from viral spread to multiple organs and the broad cellular tropism displayed by HCMV following infection (11).Monocytes are a primary site of persistent HCMV infection, and HCMV-infected monocytes are believed to play a key role in the hematogenous dissemination of the virus to target organs following primary infection (12). For HCMV to infect primary monocytes, it must overcome several biological barriers to successfully mediate viral spread and persistence. For example, monocytes do not initially support de novo HCMV gene expression and viral replication (13), and they have a lifespan of only 1-3 d in circulation under normal homeostatic conditions (14, 15). Understanding how HCMV overcomes these biological barriers following infection may provide clues to the underlying causes of HCMV pathogenesis and persistence. Our laboratory has provided molecular evidence for how HCMV evolved to deal with these biological barriers (16)(17)(18)(19)(20)(21)(22).We have shown that the biological changes in HCMV-infected monocytes are triggered by the binding of gB to EGFR (19) and binding of the gH/gL/UL128-131 complex to β1 and β3 integri...

Genes enhancing lycopene production in Escherichia coli were identified through colorimetric screening of shot-gun library clones constructed with E. coli chromosomal DNA. These E. coli cells had been engineered to produce lycopene, a red-colored carotenoid, which enabled screening for genes that enhance lycopene production. Six clones with enhanced lycopene production were isolated. Among 13 genes in these clones, dxs, appY, crl, and rpoS were found to be involved in enhanced lycopene production. While dxs and rpoS have been already reported to enhance lycopene production, appY and crl have not. DXP (1-deoxy-D-xylulose-5-phosphate) synthase is encoded by dxs and participates in the rate-limiting step in the synthesis of isopentenyl pyrophosphate (IPP), a building block of lycopene. Sigma S factor, encoded by rpoS, regulates transcription of genes induced at the stationary phase. The appY and crl genes encode transcriptional regulators related to anaerobic energy metabolism and the formation of curli surface fibers, respectively. E. coli harboring appY plasmids produced 2.8 mg lycopene/g dry cell weight (DCW), the same amount obtained with dxs despite the fact that appY is not directly involved in the lycopene synthesis pathway. The co-expression of appY, crl, and rpoS with dxs synergistically enhanced lycopene production. The co-expression of appY with dxs produced eight times the amount of lycopene (4.7 mg/g DCW) that was produced without expression of both genes (0.6 mg/g DCW).

Monocytes IMPORTANCEHematogenous 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. H uman cytomegalovirus (HCMV) is a ubiquitous host-restricted betaherpesvirus that infects 60 to 90% of the population and persists for the lifetime of the infected individual (1). HCMV infection results in a wide range of pathogenic outcomes dependent upon the age and immune status of the host (2). Infection of immunocompetent hosts is usually asymptomatic or only mildly symptomatic (3, 4); however, it can cause infectious mononucleosis, is a risk factor for the development of cardiovascular disease (5-9), and has been linked to certain types of cancers in otherwise healthy individuals (10-15). In contrast, HCMV infection leads to significant morbidity and mortality in the immunocompromised. HCMV is an important opportunistic pathogen in AIDS patients (16)(17)(18)(19)(20), is a leading infectious cause of complications in transplant recipients (21-28), and causes severe neurological disease in congenitally infected neonates (29)(30)(31)(32)(33)(34).HCMV pathogenesis and disease result from viral spread to multiple organ sites following primary HCMV infection, a process which appears to be a critical step in the viral persistence strategy, as it allows for the establishment of lifelong persistence within the host, as well as for viral shedding and spread to additional hosts (1,35,36). Monocytes are the primary blood-borne targets for HCMV infection and are thought to be centrally involved in the hematogenous dissemination of the virus to target organ systems (37-41). We propose that HCMV reprograms the biology of infected monocytes, creating the ideal cell type to serve as "Trojan horses" to carry HCMV to target host organ sites and then to

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 HCMVinduced 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 EGFRand integrin-dependent pathway in order to help promote the proviral activation and polarization of infected monocytes.