The murine cytomegalovirus immediate-early 1 protein stimulates NF-κ B activity by transactivating the NF-κ B p105/p50 promoter

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Ribonucleotide reductase (RNR) is an essential enzyme for the de novo synthesis of both cellular and viral DNA and catalyzes the conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates. The enzyme consists of two nonidentical subunits, termed R1 and R2, whose expression is very low in resting cells and maximal in S-phase cells. Here we show that murine cytomegalovirus (MCMV) replication depends on ribonucleotide reduction since it is prevented by the RNR inhibitor hydroxyurea. MCMV infection of quiescent fibroblasts markedly induces both mRNA and protein corresponding to the cellular R2 subunit, whereas expression of the cellular R1 subunit does not appear to be up-regulated. The increase in R2 gene expression is due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse R2 promoter is induced following virus infection. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in R2 promoter activity. It was found that the viral gene M45, encoding a putative homologue of the R1 subunit, is expressed 24 and 48 h after infection. Meanwhile, we observed an expansion of the deoxyribonucleoside triphosphate pool between 24 and 48 h after infection; however, neither CDP reduction nor viral replication was inhibited by treatment with 10 mM thymidine. These findings indicate the induction of an RNR activity with an altered allosteric regulation compared to the mouse RNR following MCMV infection and suggest that the virus R1 homologue may complex with the induced cellular R2 protein to reconstitute a new RNR activity.The replication of both cellular and DNA virus genomes requires a balanced supply of deoxyribonucleoside triphosphates (dNTPs). In eukaryotic cells, conversion of ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates is catalyzed by ribonucleotide reductase (RNR), the rate-limiting enzyme in DNA precursor biosynthesis (56,60,61). Ribonucleotide reduction is the first of a series of metabolic reactions leading to DNA synthesis and as such is controlled at several levels. The same enzyme reduces all four ribonucleotides, and both substrate specificity and overall activity are tightly controlled by binding of NTP allosteric effectors. Substrate specificity is controlled by binding of ATP or dATP (CDP/UDP reduction), dTTP (GDP reduction), or dGTP (ADP reduction) to a specificity site in the R1 protein, while overall activity is controlled by binding ATP (activation) or dATP (inactivation) to an activity site (39). The activity of RNR is cell cycle regulated and is very low or not detectable in resting cells and maximal in S-phase cells (56,61). This is controlled both at the level of transcription and by regulation of protein stability (6,13,22,24).Three RNR classes have been characterized based on the mechanism for generation of the protein radical, metal cofactor requirement, and subunit composition (39). Human cells, like most eu...

Section: Discussionmentioning

confidence: 99%

Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts

Lembo¹,

Gribaudo²,

Hofer

et al. 2000

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“…The amplified products were digested with restriction enzyme BglII (lanes 1 and 2) or KpnI (lanes 3 and 4), and the resulting DNA fragments were separated by gel electrophoresis. (18,19), similar to that seen for HCMV, and raises the question of whether this activation is required for efficient MCMV replication. In the following set of experiments, we first sought to test whether the NF-B binding sites in the MIEP contribute to MCMV replication in fibroblasts by introducing point mutations that abolish the ability of NF-B to bind to the enhancer.…”

Section: Fig 1 Mutagenesis Of the Hcmv-miep Nf-b Binding Sites In Hmentioning

confidence: 99%

“…We further analyzed the contribution of the canonical, or classical, NF-B pathway by using dominant negative inhibitors of IB␣ and immortalized p65/RelA-deficient fibroblasts. Infection of fibroblasts by CMV has been shown to activate the translocation of transcriptionally active p50/p65 to the nucleus (10,19,27) and to induce activation of the p50 and p65 promoters by IE1 (19,54,55). Overexpression of HCMV IE1 has also recently been reported to induce transcription of the NF-B family member RelB (23).…”

Section: Discussionmentioning

confidence: 99%

Neutrality of the Canonical NF-κB-Dependent Pathway for Human and Murine Cytomegalovirus Transcription and Replication In Vitro

Benedict

Angulo

Patterson

et al. 2004

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor B (NF-B) after infectionCytomegalovirus (CMV), the prototypic betaherpesvirus, shows sequential regulation of gene expression, as is the case for all the members of family Herpesviridae (35). CMV replication begins with expression of the immediate-early (IE/␣) proteins shortly after infection and is dependent upon the host RNA polymerase. Expression of the CMV ie genes leads to expression of viral transactivators that promote induction of CMV early (␤) genes and eventually late (␥) genes, which are expressed after DNA replication has been initiated. Transcription of the major ie genes is controlled by the major immediate-early promoter (MIEP). The enhancer of the MIEP is a highly complex region, and its activity depends on a diverse number of positive and negative cis-acting elements. Although the MIEP enhancers of mouse and human CMV (MCMV and HCMV, respectively) show limited homology at the nucleotide level, they share many common functional regulatory elements (6, 13). The MIE proteins in HCMV and MCMV (IE1/IE2 and IE1/IE3, respectively) have been shown to transactivate viral and host gene expression as well as to regulate their own expression through binding the MIEP (24,30,35,(46)(47)(48). The MIEP enhancer of MCMV has been shown to play an important yet dispensable role in initiation and potentiation of viral replication in tissue culture (2) and more recently has been found to be absolutely essential for in vivo growth (14). Replacement of the MIEP enhancer of MCMV with the paralogous region from HCMV does not restrict replication of this MCMV "enhancer swap" virus in tissue culture (2) or in vivo (20). Directed mutagenesis of the HCMV MIEP has identified the distal region of the enhancer as important for optimal expression of IE genes and viral replication at low multiplicity of infection (32,33). Accordingly, expression of the MIE proteins is required for efficient CMV growth (1,17,32,34,36), and significant work has been done to delineate what specific host factors are required for the initiation of ie gene expression.The nuclear factor B (NF-B) family of transcription factors (including c-Rel, p65/RelA, RelB, p50/NF-B1, and p52/ NF-B2) is critical for mounting both innate and adaptive host immune responses to pathogen infection through induction of various "inflammatory" genes (15). Stimulation of cells with agonists (including microbial products, viruses, or host cytokines) can trigger various signal transduction pathways, ultimately leading to activation of the inhibitor of NF-B kinase

“…Of these, the NF-B pathway plays a crucial role by transactivating the major IE enhancer-promoter that regulates the expression of the major IE gene products during both replication and reactivation from latency (14, 18). NF-B translocations into the nucleus and DNA binding, in fact, are hallmarks of CMV infection (10,11,15,24,28,29).Several stimuli leading to NF-B activation converge onto a multiprotein kinase complex designated IKK (IB kinase) or signalosome (9). This consists of two catalytic subunits, IKK1 (IKK␣) and IKK2 (IKK␤), and the regulatory subunit IKK␥ (NF-B essential modulator).…”

mentioning

confidence: 99%

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confidence: 99%

Human Cytomegalovirus Stimulates Cellular IKK2 Activity and Requires the Enzyme for Productive Replication

Caposio

Dréano

Garotta

et al. 2004

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Human cytomegalovirus (HCMV) exploits the host transcription factor NF-B to enhance its own replication, dissemination, and reactivation from latency. Here we report that HCMV infection activates the upstream IB kinase (IKK) complex and that its catalytic IKK2 subunit is required for HCMV-induced NF-B activation, as well as the replication of different HCMV strains. These results indicate that IKK2 is essential for HCMV replication and emphasize the feasibility of blocking NF-B activation as a way of inhibiting infection.Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that generally causes benign or asymptomatic infections. However, it is the leading cause of congenital viral infection in humans and a primary cause of morbidity in immunocompromised hosts (5,12,16,18,20).During HCMV infection, a coordinated cascade of events must occur. The activity of viral immediate-early (IE) proteins is essential for HCMV replication, since they regulate the subsequent expression of early (E) and late (L) genes. Expression of IE genes is closely associated with cellular activation pathways and involves several transcription factors whose activities are stimulated by infection (5,6,16,18). Of these, the NF-B pathway plays a crucial role by transactivating the major IE enhancer-promoter that regulates the expression of the major IE gene products during both replication and reactivation from latency (14, 18). NF-B translocations into the nucleus and DNA binding, in fact, are hallmarks of CMV infection (10,11,15,24,28,29).Several stimuli leading to NF-B activation converge onto a multiprotein kinase complex designated IKK (IB kinase) or signalosome (9). This consists of two catalytic subunits, IKK1 (IKK␣) and IKK2 (IKK␤), and the regulatory subunit IKK␥ (NF-B essential modulator). Activated IKK phosphorylates the cytoplasmic NF-B inhibitors, IBs, and tags them for proteasomal degradation. It thus allows NF-B proteins to translocate into the nucleus and activate the transcription of cellular and viral responsive genes.Viral protein products that activate NF-B appear to act through several distinct mechanisms involving either alteration of the normal cellular signal transduction pathways acting upstream from the IKK complex (as in the case of Epstein-Barr virus LMP1 protein), or promoting a persistent degradation of IBs (as for hepatitis B virus), or by direct association with the IKK complex (as for the HTLV-1 tax protein) (13, 26). However, it is not yet known which mechanism is exploited by HCMV.Since elucidation of the molecular mechanisms of virusmediated regulation of the host biochemical pathway may identify new targets suitable for the design of molecules with antiviral activity, we investigated the effects of HCMV infection on IKK activity. The results showed that HCMV infection indeed stimulates IKK2 activity and that it is required for HCMV replication.HCMV infection stimulates cellular IKK activity. To determine whether HCMV regulates the activity of the upstream IKK complex, quiescent HELF cells were infec...