CD14 + monocytes are a reservoir for latent human cytomegalovirus, and virus replication is reactivated during their differentiation to macrophages or dendritic cells. It has not been clear whether the virus can establish latency upon direct infection of monocytes or whether it must first become quiescent in a progenitor cell that subsequently differentiates to generate a monocyte. We report that infection of primary human monocytes with a clinical strain of human cytomegalovirus exhibits the hallmarks of latency. We established conditions for culturing monocytes that prevent differentiation for at least 25 d, as evidenced by cell surface marker expression. Infection of these monocytes with the FIX clinical strain resulted in transient accumulation of many viral lytic RNAs and sustained expression of four previously described latency-associated transcripts. The amount of viral DNA remained constant after infection, and cell surface and total HLA-DR proteins were substantially reduced on a continuing basis after infection. When treated with cytokine mixtures that stimulate differentiation to a macrophage or dendritic cell phenotype, infected monocytes reactivated virus replication and produced infectious progeny. Treatment of infected monocytes with IL-6 alone also was sufficient for reactivation, and the particles produced after exposure to this cytokine were about fivefold more infectious than virions produced by other treatments. We propose that in vivo microenvironments influence not only the efficiency of reactivation but also the infectivity of the virions produced from latently infected monocytes.herpesvirus | myeloid biology | cell culture | antigen presentation | immunology H uman cytomegalovirus (HCMV) is a dangerous opportunistic pathogen (1) that replicates in many cell types but enters latency in others, allowing persistence of the viral genome without production of progeny. Viral DNA and a small subset of viral RNAs have been found in naturally infected CD34 + hematopoietic stem cells (HSCs) and CD33 + progenitor cells (2). Experimental infections of CD34 + and CD33 + cells also display the hallmarks of latency. HCMV DNA and a subset of viral transcripts are present in these cells after infection in culture, and the virus can be reactivated to produce progeny if the cells differentiate (2). The choice of HCMV strain can influence the outcome of infections (3). Two clinical isolates entered and exited latency, whereas the AD169 laboratory strain failed to become latent in CD34 + cells. AD169 lacks the UL138 gene, which is important for entry into latency (3, 4).Like HSCs, naturally infected CD14 + monocytes harbor HCMV DNA (5-7), and viral replication is activated by differentiation (8, 9). Monocytes from peripheral blood are nonpermissive for HCMV replication (10), but when differentiated to a macrophage phenotype, they support the production of infectious progeny (11, 12). Thus, natural and experimental infections argue that differentiation from monocyte to macrophage or dendritic cell marks a divi...
Cells activate the transcription factor NFB in a wide variety of situations, including responses to stress-inducing insults such as UV irradiation and virus infection or in response to cytokines such as tumor necrosis factor alpha (TNF-␣). NFB has an important role in suppression of apoptosis and regulates the expression of many important antiapoptotic functions (5,31,48,50). NFB, as a p65/p50 heterodimer, is normally sequestered in the cytoplasm in a complex with inhibitor of B (IB). IB␣ is targeted for phosphorylation at serine residues 32 and 36, and IB is targeted for phosphorylation at serine residues 19 and 23 (42, 46, 51), by the multisubunit IB kinase (IKK) (11,23,36,52,58). This phosphorylation triggers its polyubiquitylation and destruction by the 26S proteosome (6,7,12), and, as a result, NFB is translocated to the nucleus (14, 57). Key roles for IKK and IB in NFB signaling were demonstrated in studies in which overexpression of a kinase-dead, trans-dominant form of IKK prevents IB phosphorylation and inhibits NFB activation (36, 52). IKK is activated by phosphorylation mediated by mitogen-activated protein (MAP) kinase kinase kinases (MAP3Ks) MEKK1, -2, or -3 or NFBinducing kinase (NIK) (23,30,37,59). Under stress conditions, the double-stranded RNA-activated protein kinase (PKR) has been shown to activate NFB through a pathway dependent on NIK and IKK (57). Distinct roles for the catalytic components of the IKK have been recognized. IKK␣ appears to play a major role in transducing signals for NFB activation during embryonic development (18, 45), while IKK is essential for cytokine and other stress-induced signaling pathways (10,27,29). Besides cytoplasmic roles in the activation of NFB, recent studies have identified IKK␣ and the IKK scaffold protein IKK␥/NEMO in direct regulation of NFB-dependent transcription in the nucleus (2, 49, 53). NFB activation is also dependent on distinct signaling pathways which target p65 for phosphorylation (32).The ability of herpes simplex virus type 1 (HSV-1) to activate NFB has been well documented (1,15,40). Beginning at 3 to 5 h postinfection (hpi), HSV-1 induced a strong and persistent nuclear translocation, increased p50/p65-dependent DNA binding activity as measured by electrophoretic mobility shift assay (EMSA), and induced activation of a 3XNFB-luciferase reporter. Persistent NFB activation required virus binding and entry as well as de novo infected cell protein synthesis, including expression of functional viral immediateearly (IE) protein ICP27. Activation was also accompanied by increased IKK activity and loss of both IB␣ and IB. Interference with NFB activation occurred following expression of a dominant-negative IB␣ (DNIB) containing alanine substitutions for serine residues 32 and 36 normally targeted by IKK. The resulting substantial reduction in NFB EMSA activity correlated with a reduction in virus yield. The latter may be related to the reported role of NFB in preventing HSV-1-induced apoptosis (15). The foregoing results argue that the observe...
We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK The stress-activated protein kinases (SAPKs) p38 and JNK are part of a larger family of serine/threonine terminal kinases termed mitogen-activated protein kinases (MAPK), which includes ERK1 and -2. SAPK pathways are normally activated by UV irradiation, anoxia, and engagement of proinflammatory cytokines or Fas ligand by their cognate receptors. Following ligand binding to a cognate receptor, signaling is initiated through receptor-associated kinases to a MAPKKK (MAPK kinase kinase). MAPKKKs capable of initiating the p38 or JNK pathway include MEKK1, -2, -3, and -4, ASK-1, TAK-1, and TAO. These in turn activate dual-specificity MAPK kinase kinases (MAPKKs) MKK3/6 and MKK4/7, which directly bind and phosphorylate p38 and JNK, respectively, on both tyrosine and threonine, resulting in their activation (for a review, see reference 78).All three subfamilies of human herpesviruses activate one or more of the MAPKs during infection. The betaherpesvirus cytomegalovirus activates both p38 and ERK by a mechanism dependent on viral gene expression (14,39,76) and the upstream kinases MKK3/6 and MKK1/2, respectively, (39, 40). ERK activates the early gene UL112-113 promoter (76) and phosphorylates immediate-early 86 and 72 proteins to alter their transactivation activity (40), while p38 phosphorylates retinoblastoma protein and HSP27 (39). ERK and p38 activation are required for cytomegalovirus DNA replication (16). The gammaherpesvirus Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor activates ERK and p38, causing increased vascular endothelial growth factor expression via phosphorylation of hypoxia-inducible factor 1␣ (86). The gammaherpesvirus Epstein-Barr virus activates p38 and JNK (1, 21). The immediate-early proteins BZLF1 and BRLF1 induce increased phosphorylation of p38 and JNK and activation of the ATF-2 transcription factor (1). BZLF1 expression during BRLF1-driven virus reactivation requires p38 activity (1). Among the alphaherpesviruses, HSV-2 has been reported to activate ERK through ICP10-PK binding to RAS-GAP, leading to further expression of ICP10 and prevention of apoptosis (68).Two previous reports documented the activation of the p38 and JNK (SAPK) pathways by HSV-1. Increased JNK and p38 kinase activity was observed as early as 3 hours postinfection and remained active throughout the course of infection (56,97). SAPKs were activated downstream of Ras, as infection with a dominant-negative form of Ras did not affect activation of the transcription factors ATF-2 and c-Jun, downstream targets of p38 and JNK, respectively (56). Furthermore, activation was specific for SAPKs, as ERK was not activated during infect...
The ability of herpes simplex virus type 1 (HSV-1) to activate NF-B has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-B-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IB␣ and IB. In this study, we identified loss of IB␣ as a marker of NF-B activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IB␣ loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IB␣ loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-B activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-B activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IB␣ and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-B activation during HSV infection, highlighting the important role that NF-B may play in infection.NF-B is a cellular integrator of diverse signaling pathways leading to programs of immune, inflammatory, and antiapoptotic gene expression. These pathways are initiated through the engagement of cell surface receptors by a variety of chemical ligands, such as cytokines, phorbol esters, lipopolysaccharide, and virion glycoproteins, or by stresses such as UV irradiation or changes in osmolarity. NF-B is normally sequestered in the cytoplasm through interactions with its inhibitory binding partner IB. While a variety of NF-B activation pathways have been characterized, many ultimately converge on and activate the IB kinase (IKK) complex, resulting in phosphorylation of IB. Once IB is phosphorylated, polyubiquitin-dependent proteolysis of IB occurs, releasing NF-B and allowing its translocation to the nucleus, where it participates in transcription activation in conjunction with other transcription factors and coactivators.Transcription of the herpes simplex virus (HSV) genome during lytic infection is temporally regulated (see reference 62 for a review). Three immediate-early (IE) proteins have important roles in regulating the temporal pattern of gene expression. ICP4 is essential for E and L gene expression and colocalizes with viral DNA (22, 23, 85), RNA polymerase II holoenzyme, and general transcription factors (10, 79). ICP0 can trans-activate viral IE, E, and L promoters alone or in combination with ICP4 and/or ICP27 in transient reporte...
Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors in vivo. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14+ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14+ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14+ cells.
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