During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta- and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta- and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses.
Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments.
A primary HCMV infection or virus reactivation may cause severe disease in hosts with a deficient immune system. The virus can disturb both innate and adaptive immunity by targeting dendritic cell (DC) functions. Monocytes, the precursors of DCs in vivo (MoDCs), are the primary targets of HCMV; they can also harbor latent virus. The DCs generated from infected monocytes (CMV-MoDCs) have an altered phenotype and functional defects. We have shown that CMV-MoDCs do not secrete IL-12 in response to lipopolysaccharide stimulation, cannot ingest dead cells, induce T(H)1 differentiation, or the proliferation of naive allogeneic CD4(+) T cells. We found that the GM-CSF signaling in an entire population of CMV-MoDCs was impaired, although only half of the cells were productively infected, and that IL-6 secretion and suppressors of cytokine signaling 3 induction contributed to this bystander effect. We also showed that MoDCs derived ex vivo from monocytes of viremic patients had the same altered phenotype as CMV-MoDCs, including decreased STAT5 phosphorylation, indicating defective GM-CSF signaling. We have thus described a new mechanism of HCMV-induced immunosupression, indicated how infection may disturb both GM-CSF-dependent physiologic processes and proposed GM-CSF-based therapeutic approaches.
Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions.
Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity in infected cells. We demonstrate that the PPARgamma antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARgamma, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARgamma in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARgamma human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.
Persistence of hepatitis C virus (HCV) in patients who cleared HCV is still debated. Occult HCV infection is described as the presence of detectable HCV RNA in liver or peripheral blood mononuclear cells (PBMCs) of patients with undetectable plasma HCV-RNA by conventional PCR assays. We have assessed the persistence of HCV in 26 kidney-transplant patients, followed up for 10.5 years (range 2-16), after HCV elimination while on hemodialysis. If HCV really did persist, arising out of the loss of immune control caused by institution of the regimen of immunosuppressive drugs after kidney transplantation, HCV reactivation would have taken place. Their immunosuppression relied on calcineurin inhibitors (100%), and/or steroids (62%), and/or antimetabolites (94%). An induction therapy, given to 22 patients, relied on rabbit antithymocyte globulin (59%) or anti-IL2-receptor blockers (32%). All patients had undetectable HCV RNA as ascertained by several conventional tests. At the last follow-up, no residual HCV RNA was detected in the five liver biopsies, the 26 plasma, and in the 37 nonstimulated and 24 stimulated PBMCs tested with an ultrasensitive RT-PCR assay (detection limit, 2 IU/ml). No biochemical or virologic relapse was seen during follow-up. The absence of HCV relapse in formerly HCV-infected immunocompromised patients suggests the complete eradication of HCV after its elimination while on dialysis.
That the expression of late genes is coupled to viral genome replication is well established for all herpesviruses, but the exact mechanisms of their regulation, especially by viral proteins, are poorly understood. Here, we report the identification of the Epstein-Barr virus (EBV) early protein BcRF1 as a viral factor crucial for the activation of late gene transcription following viral DNA replication during the productive cycle. In order to study the function of the BcRF1 protein, we constructed a recombinant EBV lacking this gene. In HEK293 cells, this recombinant virus underwent normal DNA replication during the productive cycle but failed to express high levels of late gene transcripts or proteins, resulting in a nonproductive infection. Interestingly, a TATT motif is present in the promoter of most EBV late genes, at the position of the TATA box. We show here that BcRF1 forms a complex with the TATT motif and that this interaction is required for activation of late viral gene expression. Moreover, our results suggest that BcRF1 acts via interaction with other viral proteins.
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