We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation.

Human cytomegalovirus (HCMV) pathogenesis is characterized by multiple organ system involvement due to viral spread to host organs after a cell-associated viremia. The cell type responsible for HCMV dissemination is unknown. Monocytes are the most likely candidate since they are the predominant cell type infected in the blood. However, monocytes are not productive for viral replication and are abortively infected. The results presented here provide a potential answer to this conundrum. We report that primary HCMV infection of monocytes induces transendothelial migration and monocyte-to-macrophage differentiation and that these HCMV-differentiated macrophages are productive for viral replication. Together, our data suggest a novel mechanism for HCMV pathogenesis; HCMV induces cellular changes in monocytes to promote viral replication and spread to host organs.Human cytomegalovirus (HCMV) is a leading cause of morbidity and mortality in immunocompromised hosts. It is the leading cause of congenital central nervous system damage and deafness in neonates (58), one of the most common viral opportunistic infections in AIDS patients (38), and an important infectious agent affecting organ transplant recipients (23). In immunocompetent hosts, HCMV causes some cases of infectious mononucleosis and is associated with cardiovascular disease (1,17,31,33,39,46,57,60). A hallmark of HCMV infection is a broad range of pathological complications attributed to viral spread to virtually every organ in the host, including the gastrointestinal tract, liver, salivary glands, brain, central nervous system, kidney, retina, and lung (44,53,63).Systemic distribution of HCMV occurs during symptomatic and asymptomatic infections (63) and is believed to be essential for HCMV survival through the establishment of persistent infection in host organs, with subsequent viral shedding and spread to additional hosts (53). Therefore, the ability of HCMV to persist in a host and in the general population is dependent on efficient viral spread to multiple organ systems, which in immunocompromised hosts leads to overt organ disease. During primary infection, HCMV spreads from the initial site of infection to the peripheral blood, from which dissemination to host organ tissue occurs (53). It is known that HCMV viremia is cell associated, suggesting that peripheral blood leukocytes are involved (51, 53). However, the mechanism by which viral spread to host organ tissue occurs is unresolved, and the cell type responsible is unknown (53).Because HCMV infection of host organs causes severe disease in immunocompromised patients (24), the investigation of the mechanism(s) for hematogenous spread is of key importance for understanding HCMV pathogenesis. Monocytes have been proposed to be the cell type responsible for dissemination and disease for several reasons. First, monocytes are the primary cell type infected in the blood during acute HCMV infection, as determined by the detection of HCMV DNA and antigens in monocytes (51,53,61). Second, monocytes are th...

Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.T he wide range of pathological complications associated with human cytomegalovirus (HCMV) infection is a direct consequence of viral spread to peripheral organ sites and the broad cellular tropism of the virus (1). Monocytes are primary in vivo targets for HCMV and are believed to be responsible for hematogenous dissemination of HCMV to multiple organ systems (2). We previously showed that HCMV infection of monocytes polarized the infected cell toward a distinct proinflammatory phenotype that possessed the distinct biological changes necessary to promote viral spread (3-5). Specifically, HCMV infection induced polarization of infected monocytes toward an M1 proinflammatory cell type that simultaneously exhibited characteristics associated with an M2 antiinflammatory macrophage (6). The infected monocytes also displayed a high level of chemokinesis when compared with monocytes activated by LPS or phorbol 12-myristate 13-acetate (PMA) (4, 5). Moreover, an accelerated rate of differentiation from short-lived monocytes (nonpermissive for HCMV replication) into long-lived macrophages (permissive for HCMV replication) was observed following infection with HCMV (4). Based on our studies, we suggest that during primary infection, newly infected peripheral monocytes acquire a motile phenotype that promotes exit of the infected cell from the circulating blood into multiple organ tissue despite the absence of a chemotactic gradient. Once in the surrounding tissue, differentiation into HCMV replication-competent macrophages occurs, resulting in viral spread...

Monocytes are primary targets for human CMV (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Monocytes acquire different functional traits during polarization to the classical proinflammatory M1 macrophage or the alternative antiinflammatory M2 macrophage. We hypothesized that HCMV induced a proinflammatory M1 macrophage following infection to promote viral dissemination because, biologically, a proinflammatory state provides the tools to drive infected monocytes from the blood into the tissue. To test this hypothesis of monocyte conversion from a normal quiescent phenotype to an inflammatory phenotype, we used Affymetrix Microarray to acquire a transcriptional profile of infected monocytes at a time point our data emphasized is a key temporal regulatory point following infection. We found that HCMV significantly up-regulated 583 (5.2%) of the total genes and down-regulated 621 (5.5%) of the total genes ≥1.5-fold at 4 h postinfection. Further ontology analysis revealed that genes implicated in classical M1 macrophage activation were stimulated by HCMV infection. We found that 65% of genes strictly associated with M1 polarization were up-regulated, while only 4% of genes solely associated with M2 polarization were up-regulated. Analysis of the monocyte chemokinome at the transcriptional level showed that 44% of M1 and 33% of M2 macrophage chemokines were up-regulated. Proteomic analysis using chemokine Ab arrays confirmed the secretion of these chemotactic proteins from HCMV-infected monocytes. Overall, the results identify that the HCMV-infected monocyte transcriptome displayed a unique M1/M2 polarization signature that was skewed toward the classical M1 activation phenotype.