Viral Promoter Activity Research Articles

Synthesis of C-4-Substituted Steviol Derivatives and Their Inhibitory Effects against Hepatitis B Virus.

ent-13-Hydroxykaur-16-ene-19-N-butylureide (6) was one of 33 synthesized C-4-substituted steviol derivatives that were evaluated for their effects on hepatitis B virus (HBV) surface antigen (HBsAg) secretion. The IC50 (16.9 μM) and SI (57.7) values for inhibiting HBV DNA replication of compound 6 were greater than those of the reference compound, lamivudine (3-TC; IC50: 107.5 μM; SI: 22.0). Thus, the anti-HBV mechanism of 6 was investigated, and it specifically inhibited viral gene expression and reduced viral DNA levels, as well as potently attenuated all of the viral promoter activity of HBV-expressing Huh7 cells. Examination of cellular signaling pathways found that 6 inhibited the activities of the nuclear factor (NF)-κB- and activator protein (AP)-1 element-containing promoters, but had no effects on AP-2 or interferon-stimulated response element (ISRE)-containing promoters in HBV-expressing cells. Meanwhile, it significantly eliminated NF-κB and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling-related protein levels and inhibited their phosphorylation in HBV-transfected Huh7 cells. The inhibitory potency of 6 against HBV DNA replication was reversed by cotransfecting the NF-κB p65 expression plasmid. Using the MAPK-specific activator anisomycin also reversed the inhibitory effect of 6 on viral DNA replication. The present findings suggest that the anti-HBV mechanism of 6 is partly mediated through the NF-κB and MAPK signaling pathways.

Analysis of Viral Promoter Usage in EBV-Infected Cell Lines: A Comparison of qPCR Following Conventional RNA Isolation and Nuclear Run-On Assay.

To interpret the results of an epigenetic analysis in gene expression studies, it is essential to characterize the activity of the relevant promoters. According to the literature, real-time PCR assay is the most widely used method for the determination of latent EBV promoter usage. Here we describe two alternative approaches to measure the activity of viral promoters in cell lines carrying latent EBV episomes. The widespread typical approach relies on total cellular RNA isolation, whereas the nuclear run-on assay described here is based on the initial isolation of nuclei, followed by in vitro transcription in the presence of biotinylated-UTP, and purification of RNA transcripts using avidin-coated magnetic beads. Finally, both methods apply reverse transcription-based real-time PCR (i.e., quantitative polymerase chain reaction, qPCR) to quantitatively measure the amount of specific transcripts. We shall describe these methods step by step and demonstrate their use for the determination of EBER1 promoter activity in EBV-positive cell lines.

LUC7L3/CROP inhibits replication of hepatitis B virus via suppressing enhancer II/basal core promoter activity.

The core promoter of hepatitis B virus (HBV) genome is a critical region for transcriptional initiation of 3.5 kb, pregenome and precore RNAs and for the viral replication. Although a number of host-cell factors that potentially regulate the viral promoter activities have been identified, the molecular mechanisms of the viral gene expression, in particular, regulatory mechanisms of the transcriptional repression remain elusive. In this study, we identified LUC7 like 3 pre-mRNA splicing factor (LUC7L3, also known as hLuc7A or CROP) as a novel interacting partner of HBV enhancer II and basal core promoter (ENII/BCP), key elements within the core promoter, through the proteomic screening and found that LUC7L3 functions as a negative regulator of ENII/BCP. Gene silencing of LUC7L3 significantly increased expression of the viral genes and antigens as well as the activities of ENII/BCP and core promoter. In contrast, overexpression of LUC7L3 inhibited their activities and HBV replication. In addition, LUC7L3 possibly contributes to promotion of the splicing of 3.5 kb RNA, which may also be involved in negative regulation of the pregenome RNA level. This is the first to demonstrate the involvement of LUC7L3 in regulation of gene transcription and in viral replication.

Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein

The immediate early (IE) 62 protein is the major varicella-zoster virus (VZV) regulatory factor. Analysis of the VZV genome revealed 40 predicted GC-rich boxes within 36 promoters. We examined effects of ectopic expression of Sp1-Sp4 on IE62- mediated transactivation of three viral promoters. Ectopic expression of Sp3 and Sp4 enhanced IE62 activation of ORF3 and gI promoters while Sp3 reduced IE62 activation of ORF28/29 promoter and VZV DNA replication. Sp2 reduced IE62 transactivation of gI while Sp1 had no significant influence on IE62 activation with any of these viral promoters. Electrophoretic mobility shift assays (EMSA) confirmed binding of Sp1 and Sp3 but not Sp2 and Sp4 to the gI promoter. Sp1–4 bound to IE62 and amino acids 238–258 of IE62 were important for the interaction with Sp3 and Sp4 as well as Sp1. This work shows that Sp family members have differential effects on IE62-mediated transactivation in a promoter-dependent manner.

A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal geneexpression in plants.

A library of synthetic promoters containing the binding site of a single designer transcription activator-like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18-base long dTALE-binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty-three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.

Synthesis and anti-hepatitis B virus activity of C4 amide-substituted isosteviol derivatives

A series of novel isosteviol derivatives having C4-amide substituents were synthesized in order to test for antiviral effects against the hepatitis B virus (HBV) in vitro. Among them, IN-4 [N-(propylcarbonyl)-4α-amino-19-nor-ent-16-ketobeyeran] (5) exhibited inhibitory activity against secretion of HBsAg and HBeAg as well as inhibition of HBV DNA replication. Therefore, the mechanism of its antiviral activity was further analyzed using HBV-transfected Huh7 cells. Exposure to IN-4 produced minimal inhibitory effects on viral precore/pregenomic RNA expression. However, expression levels of the 2.4/2.1-kb preS/major S RNA of the viral surface gene significantly decreased, along with intracellular levels of HBV DNA. A promoter activity analysis demonstrated that IN-4 significantly inhibited viral X, S, and preS expression levels but not viral core promoter activities. In particular, IN-4 was observed to significantly inhibit HBV gene regulation by disrupting nuclear factor (NF)-κB-associated promoter activity. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated following IN-4 treatment, while cytoplasmic IκBα protein levels were enhanced. Meanwhile, IN-4 was observed to inhibit the binding activity of NF-κB to putative DNA elements. Furthermore, transfection of a p65 expression plasmid into Huh7 cells significantly reversed the inhibitory effect of IN-4 on HBV DNA levels, providing further evidence of the central role of NF-κB in its antiviral mechanism. It is therefore suggested that IN-4 inhibits HBV by interfering with the NF-κB signaling pathway, resulting in downregulation of viral gene expression and DNA replication.

Sorafenib overcomes the chemoresistance in HBx-expressing hepatocellular carcinoma cells through down-regulation of HBx protein stability and suppresses HBV gene expression

Previous studies have revealed that HBx expression has anti-apoptotic effects, resulting in increased drug resistance in HCC cells. Thus, we examined if sorafenib efficiently induces apoptosis in HBx-overexpressing HCC cells. Noticeably, sorafenib efficiently induced apoptosis, even in HBx-expressing HepG2 cells, indicating that the HBx protein does not attenuate sorafenib-induced apoptosis. We next investigated if sorafenib modulates autophagy, allowing HCC cells to overcome the chemoresistance conferred by the HBx protein. Although autophagy plays a cytoprotective role against sorafenib-induced lethality, sorafenib was effective irrespective of HBx protein overexpression. We next examined if sorafenib exerts its cytotoxic effect via direct effects on the HBx protein. Importantly, sorafenib decreased HBx protein stability through a proteasome-dependent degradation pathway. Moreover, sorafenib decreased HBV gene expression and viral promoter activity. Taken together, sorafenib efficiently induces apoptotic cell death in HBx-expressing HCC cells via the downregulation of the HBx protein, a key factor in the anti-cancer drug resistance observed in HBV-induced HCC.

Recombinant adeno-associated virus utilizes cell-specific infectious entry mechanisms.

Understanding the entry and trafficking mechanism(s) of recombinant adeno-associated virus (rAAV) into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin D blocked rAAV transduction of HeLa cells (>2-fold) but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was due to neither viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced single-stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement) but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery and could help unify the divergent accounts of rAAV cellular entry mechanisms. There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early trafficking through the host cellular machinery, the mechanisms of which are under continued study. However, should the early entry and trafficking mechanisms of rAAV differ across virus serotype or be dependent on host cell environment, this could expand our ability to target particular cells and tissue for selective transduction. Thus, the observation that inhibiting macropinocytosis leads to cell-specific enhancement or inhibition of rAAV transduction that extends to the organismic level exposes a new means of modulating vector targeting.

Antiviral activity of p-hydroxyacetophenone isolated from Artemisia morrisonensis against hepatitis B virus in vitro

p-Hydroxyacetophenone (PHAP) isolated from Artemisia morrisonensis was found to have potential anti-HBV effects in HepG2 2.2.15 cells. The 2.4-kb preS RNA of viral surface gene increased significantly relative to the 2.1-kb S RNA with PHAP. Promoter activity analysis demonstrated that PHAP had a potent effect on augmenting the viral preS promoter activity. Interestingly, PHAP specifically reduced ER stress related GRP78 RNA/protein levels, but not those of GRP94 in treated Huh7 cells while PHAP also led to the significant intracellular accumulation of virus. In conclusion, this study suggests that the mechanism of HBV inhibition by PHAP might involve the regulation of viral surface gene expression and block virion secretion by interference with the ER stress signaling pathway.

Synthesis and antiviral effects of isosteviol-derived analogues against the hepatitis B virus

Among several isosteviol-derived analogues, NC-8 (ent-16-oxobeyeran-19-N-methylureido) showed inhibitory potency against the hepatitis B virus (HBV) in HepG2 2.2.15 cells. Its anti-HBV mechanism was then next investigated in a human hepatoma cell culture system. Results showed that it specifically inhibited viral gene expression and reduced the level of encapsidated viral DNA intermediates in Huh7 cells that expressed replicating HBV. It also potently attenuated all viral promoter activity in HBV-expressing Huh7 cells, but not in cells lacking HBV expression. By examining its antiviral mechanism in cellular signaling pathways, NC-8 was found to inhibit the activity of the nuclear factor (NF)-κB element-containing promoter, but only slightly enhanced activities of activator protein (AP)-1- and interferon-sensitive response element (ISRE)-containing promoters in HBV-expressing cells. NC-8 also significantly eliminated NF-κB (p65/p50) and Toll-like receptor (TLR)2 proteins, but increased the IκBα protein level in a dose-dependent manner in HBV-transfected Huh7 cells, while these protein levels were apparently unchanged in non-transfected cells. Meanwhile, NC-8-treated nuclear extracts that co-expressed HBV inhibited the binding of NF-κB to the CS1 site of HBV major surface gene and specifically attenuated CS1-containing promoter activity. Taken together, this study suggests that the antiviral mechanism of NC-8 appears to be mediated by disturbing replication and gene expression of HBV and by inhibiting the host TLR2/NF-κB signaling pathway.

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.

Antiviral activity of chemical compound isolated from Artemisia morrisonensis against hepatitis B virus in vitro

The compound p-hydroxyacetophenone (PHAP) isolated from Artemisia morrisonensis was found to have potential anti-HBV effects in HepG2 2.2.15 cells. We clarified its antiviral mode further and HBV-transfected Huh7 cells were used as the platform. During viral gene expression, treatment with PHAP had no apparent effects on the viral precore/pregenomic RNA. However, the 2.4-kb preS RNA of viral surface gene increased significantly relative to the 2.1-kb S RNA with PHAP. Promoter activity analysis demonstrated that PHAP had a potent effect on augmenting the viral preS promoter activity. The subsequent increase in the large surface protein and induce endoplasmic reticular (ER) stress has been reported previously. Interestingly, PHAP specifically reduced ER stress related GRP78 RNA/protein levels, but not those of GRP94, in treated Huh7 cells while PHAP also led to the significant intracellular accumulation of virus. Moreover, treatment with the ER chaperone inducer thapsigargin relieved the inhibitory effect of PHAP based on the supernatant HBV DNA levels of HBV-expressed cells. In conclusion, this study suggests that the mechanism of HBV inhibition by PHAP might involve the regulation of viral surface gene expression and block virion secretion by interference with the ER stress signaling pathway.

Genetic variability and in vitro transcriptional permissibility of primary ovine beta-retrovirus promoter isolates

To assess genomic sequence conservation and variation in the proviral promoter of enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) in tissue samples from 3 sheep with nasal adenocarcinoma associated with ENTV and 3 sheep with pulmonary adenocarcinoma associated with JSRV and to identify a cell culture system that supports transcriptional activity of the ENTV and JSRV viral promoters. 6 adult sheep. Standard PCR procedures for detection of the ENTV and JSRV long terminal repeat (LTR) promoter region were performed on samples from the 3 nasal adenocarcinomas and 3 pulmonary adenocarcinomas, respectively. The LTRs were cloned into shuttle vectors, amplified, sequenced, and analyzed. The cloned LTR regions were transferred into reporter plasmids and multiple human and ruminant cell lines, and primary cells were transfected with the promoter-reporter plasmids. The viral promoter activity was evaluated by use of an in vitro β-galactosidase reporter assay. Each isolate had a unique nucleotide sequence. Single nucleotide polymorphisms were the most common LTR mutation and rarely occurred at transcription factor binding sites. Relative to ENTV, the JSRV promoter isolates had a conserved 66-bp U3 insertion, including the lung-specific transcription factor HNF-3β binding site. Among the cell lines used, human embryonic kidney (293T) and goat synovial membrane cells supported promoter transcription. The LTRs of ENTV and JSRV have extensive blocks of sequence conservation. Human 293T and goat synovial membrane cell lines may be suitable in vitro cell culture systems for further research of viral promoter functions.

Characterization of simian T-cell leukemia virus type 1 in naturally infected Japanese macaques as a model of HTLV-1 infection

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed.ResultsWe identified spliced transcripts of STLV-1 corresponding to HTLV-1 tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-β signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1+ cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration.ConclusionsSTLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals.

CMV promoter is repressed by p53 and activated by JNK pathway

Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.

Functional Cooperation between Human Adenovirus Type 5 Early Region 4, Open Reading Frame 6 Protein, and Cellular Homeobox Protein HoxB7

Human adenovirus type 5 (HAdV5) E4orf6 (early region 4 open reading frame 6 protein) is a multifunctional early viral protein promoting efficient replication and progeny production. E4orf6 complexes with E1B-55K to assemble cellular proteins into a functional E3 ubiquitin ligase complex that not only mediates proteasomal degradation of host cell substrates but also facilitates export of viral late mRNA to promote efficient viral protein expression and host cell shutoff. Recent findings defined the role of E4orf6 in RNA splicing independent of E1B-55K binding. To reveal further functions of the early viral protein in infected cells, we used a yeast two-hybrid system and identified the homeobox transcription factor HoxB7 as a novel E4orf6-associated protein. Using a HoxB7 knockdown cell line, we observed a positive role of HoxB7 in adenoviral replication. Our experiments demonstrate that the absence of HoxB7 leads to inefficient viral progeny production, as HAdV5 gene expression is highly regulated by HoxB7-mediated activation of various adenoviral promoters. We have thus identified a novel role of E4orf6 in HAdV5 gene transcription via regulation of homeobox protein-dependent modulation of viral promoter activity.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.

The Shrimp NF-κB Pathway Is Activated by White Spot Syndrome Virus (WSSV) 449 to Facilitate the Expression of WSSV069 (ie1), WSSV303 and WSSV371

The Toll-like receptor (TLR)-mediated NF-κB pathway is essential for defending against viruses in insects and mammals. Viruses also develop strategies to utilize this pathway to benefit their infection and replication in mammal hosts. In invertebrates, the TLR-mediated NF-κB pathway has only been well-studied in insects and has been demonstrated to be important in antiviral responses. However, there are few reports of interactions between viruses and the TLR-mediated NF-κB pathway in invertebrate hosts. Here, we studied Litopenaeus vannamei Pelle, which is the central regulator of the Toll pathway, and proposed that a similar TLR/MyD88/Tube/Pelle/TRAF6/NF-κB cascade may exist in shrimp for immune gene regulation. After performing genome-wild analysis of white spot syndrome virus (WSSV) encoded proteins, we found that WSSV449 shows 15.7-19.4% identity to Tube, which is an important component of the insect Toll pathway. We further found that WSSV449 activated promoters of Toll pathway-controlled antimicrobial peptide genes, indicating WSSV449 has a similar function to host Tube in activating the NF-κB pathway. We suspected that WSSV449 activated the Toll-mediated NF-κB pathway for regulating viral gene expression. To test this hypothesis, we analyzed the promoters of viral genes and found 40 promoters that possess NF-κB binding sites. A promoter screen showed that the promoter activities of WSSV069 (ie1), WSSV303 and WSSV371 can be highly induced by the shrimp NF-κB family protein LvDorsal. WSSV449 also induced these three viral promoter activities by activating the NF-κB pathway. To our knowledge, this is the first report of a virus that encodes a protein similar to the Toll pathway component Tube to upregulate gene expression in the invertebrate host.

Epigenetic Regulation of HIV-1 Transcription

After entry into the target cell and reverse transcription, HIV-1 genes are integrated into the host genome. It is now well established that the viral promoter activity is directly governed by its chromatin environment. Nuc-1, a nucleosome located immediately downstream of the HIV-1 transcriptional initiation site directly impedes long-terminal repeat (LTR) activity. Epigenetic modifications and disruption of Nuc-1 are a prerequisite to the activation of LTR-driven transcription and viral expression. The compaction of chromatin and its permissiveness for transcription are directly dependent on the post-translational modifications of histones such as acetylation, methylation, phosphorylation and ubiquitination. Understanding the molecular mechanisms underlying HIV-1 transcriptional silencing and activation is thus a major challenge in the fight against AIDS and will certainly lead to new therapeutic tools.

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state.

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.