47 research outputs found
DNA Polymerase Processivity Factor of Human Cytomegalovirus May Be a Key Molecule for Molecular Coupling of Viral DNA Replication to Transcription
Unexpected Instability of Family of Repeats (FR), the Critical cis-Acting Sequence Required for EBV Latent Infection, in EBV-BAC Systems
A group of repetitive sequences, known as the Family of Repeats (FR), is a critical cis-acting sequence required for EBV latent infection. The FR sequences are heterogeneous among EBV strains, and they are sometimes subject to partial deletion when subcloned in E. coli-based cloning vectors. However, the FR stability in EBV-BAC (bacterial artificial chromosome) system has never been investigated. We found that the full length FR of the Akata strain EBV was not stably maintained in a BAC vector. By contrast, newly obtained BAC clones of the B95-8 strain of EBV stably maintained the full length FR during recombinant virus production and B-cell transformation. Investigation of primary DNA sequences of Akataβderived EBV-BAC clones indicates that the FR instability is most likely due to a putative secondary structure of the FR region. We conclude that the FR instability in EBV-BAC clones can be a pitfall in E. coli-mediated EBV genetics
Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase
AbstractThe Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial
Degradation of Phosphorylated p53 by Viral Protein-ECS E3 Ligase Complex
p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection
The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene
Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5' mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells
Role of the Proximal Enhancer of the Major Immediate-Early Promoter in Human Cytomegalovirus Replication
The human cytomegalovirus (CMV) enhancer has a distal component (positions β550 to β300) and a proximal component (β300 to β39) relative to the transcription start site (+1) of the major immediate-early (MIE) promoter. Without the distal enhancer, human CMV replicates slower and has a small-plaque phenotype. We determined the sequence requirements of the proximal enhancer by making 5β²-end deletions to positions β223, β173, β116, β67, and β39. Even though recombinant virus with the proximal enhancer deleted to β39 has the minimal TATA box-containing MIE promoter element, it cannot replicate independently in human fibroblast cells. Recombinant virus with a deletion to β67 has an Sp-1 transcription factor binding site which may represent a minimal enhancer element for recombinant virus replication in human fibroblast cells. Although recombinant virus with a deletion to β223 replicates to titers at least 100-fold less than that of the wild-type virus, it replicates to titers 8-fold higher than that of recombinant virus with a deletion to β173 and 20-fold higher than that of virus with a deletion to β67. Recombinant virus with a deletion to β173 replicates more efficiently than that with a deletion to β116. There was a direct correlation between the level of infectious virus replication and time after infection, amount of MIE gene transcription, MIE and early viral protein synthesis, and viral DNA synthesis. The extent of the proximal enhancer determines the efficiency of viral replication
Dynamic load balancing for distributed network management
Abstract: The scalability limitations of centralized management models have motivated distributed management models, in which management programs describing some of management tasks are distributed and executed on managed systems. In the models, management program distribution that considers dynamic network resource utilization is one of the most important challenges, in striking a load balance between management and managed systems for an entire managed network. Some methods for load balancing have been studied; however, they cannot adequately be achieved throughout an entire managed network. This arises from criteria for load balancing that lacks dynamic network resource utilization, or from a localized subnetwork in which the performance is limited, although it does include processing loads for dynamic network resource utilization. To solve this, a new dynamic load balancing method is proposed for distributed network management. Thus, systems that execute management programs are decided dynamically on the basis of CPU utilization for each system and the bandwidth required for executing all management programs. Two typical algorithms derived from the proposed method, each having different criteria in the form of mean deviation and range types with respect to CPU utilization, are introduced. They were evaluated analytically according to capability, i.e., how well they perform as close to load balancing as possible, as well as time complexity. The results show that the mean deviation type algorithm performs better at almost the same computational cost. A prototype system is also implemented based on the proposed method, and evaluated empirically by applying it to an operational LAN. The proposed method performs well in trials with a trivial overhead
Sharing sensor networks
Many industrial applications rely on sensors and sensor networks residing on machinery, transport containers or in the environment. For distributed processes in such domains the sharing of those sensor networks is crucial. This paper introduces a peer-topeer (P2P) architecture for sharing services provided by existing sensor networks with any Internet based application. The differences between the sensor networks are abstracted using a service-oriented approach. The proposed technology is able to build up a global sensor Internet across multiple domain boundaries. Our implementation is evaluated with 300+ sensor nodes organized in a P2P network across continents. 1
Optimum Capacity and Placement of Storage Batteries Considering Photovoltaics
In recent years, due to the enforcement of the Feed-in tariff (FIT) scheme for renewable energy, a large number of photovoltaic (PV) has been introduced, which causes fluctuations in the supply-demand balance of a power system. As measures against this, the introduction of large capacity storage batteries and demand response has been carried out, and the balance between supply and demand has been adjusted. However, since the increase in capacity of the storage battery is expensive, it is necessary to optimize the capacity of the storage battery from an economic point of view. Therefore, in the power system to which a large amount of photovoltaic power generation has been introduced, the optimal capacity and optimal arrangement of storage batteries are examined. In this paper, the determination of storage battery placement and capacity considering one year is performed by three-step simulation based on probability density function. Simulations show the effectiveness of storage batteries by considering the introduction of demand response and comparing with multiple cases
The CREB Site in the Proximal Enhancer Is Critical for Cooperative Interaction with the Other Transcription Factor Binding Sites To Enhance Transcription of the Major Intermediate-Early Genes in Human Cytomegalovirus-Infected Cells βΏ
One of the two SP1 sites in the proximal enhancer of the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter is essential for transcription in human fibroblast cells (H. Isomura, M. F. Stinski, A. Kudoh, T. Daikoku, N. Shirata, and T. Tsurumi, J. Virol. 79:9597-9607, 2005). Upstream of the two SP1 sites to β223 relative to the +1 transcription start site, there are an additional five DNA binding sites for eukaryotic transcription factors. We determined the effects of the various transcription factor DNA binding sites on viral MIE RNA transcription, viral gene expression, viral DNA synthesis, or infectious virus production. We prepared recombinant HCMV bacterial artificial chromosome (BAC) DNAs with either one site missing or one site present upstream of the two SP1 sites. Infectious recombinant HCMV BAC DNAs were transfected into various cell types to avoid the effect of the virion-associated transactivators. Regardless of the cell type, which included human fibroblast, endothelial, and epithelial cells, the CREB site had the most significant and independent effect on the MIE promoter. The other sites had a minor independent effect. However, the combination of the different transcription factor DNA binding sites was significantly stronger than multiple duplications of the CREB site. These findings indicate that the CREB site in the presence of the other sites has a major role for the replication of HCMV