The life cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic replication has revealed a sophisticated regulation network that controls the important events in KSHV life cycle.

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Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD+ and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.

Summary. Background: Platelet factor 4 (PF4) is an abundant protein stored in platelet a-granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of a-granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non-allelic variant PF4V1. Objectives: To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/results: Chromatin immunoprecipitation and gel-shift assays with phorbol 12-myristate 13-acetate-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at )1774/)1769 and )157/)152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions: Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in a-granule deficiency.

Sequence analysis using the Promoser program predicted two promoter-like regions for rat mtGPAT: a distal promoter approximately 30kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8- and 4.8-fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5' RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7-fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, the results suggest that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (\66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved a/b-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB -4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4-5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.

227 RUNX1/CBFA2 (Core binding factor A2) is a major transcription factor involved in hematopoiesis. RUNX1 mutations are associated with mild thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. Several patients with mutations in RUNX1 have been shown to have alpha granule deficiency characterized by decreased platelet factor 4 (PF4) content. The mechanisms leading to PF4 deficiency remain unclear in most patients with α-granule abnormalities or the Gray platelet Syndrome (GPS). GPS is a heterogeneous disorder, and defective granule formation and targeting of proteins to the granule have been postulated. Previous studies from our group have documented a patient with mild thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb-IIIa activation, which was associated with a heterozygous mutation in transcription factor RUNX1. Platelet expression profiling of this patient showed decreased expression of several genes including chemokine PF4 and its non-allelic variant PF4V1. Platelet PF4 protein was also decreased. PF4 is mainly expressed in megakaryocytes and platelets, and it serves as a lineage-specific marker of megakaryocytic differentiation. Because PF4 is downregulated in platelets from our patient with a RUNX1 mutation, we addressed the hypothesis that PF4 and PF4V1 may be direct transcriptional targets of RUNX1. Computer based TFSEARCH analysis showed six RUNX1 consensus sites on PF4 upstream (2 kb) region and eleven RUNX1 sites on PF4V1 upstream sequence (2 kb). To assess in vivo binding of RUNX1 to PF4 upstream region chromatin immunoprecipitation (ChIP) assay was performed using HEL cell genomic DNA and RUNX1 antibody. These studies were done in HEL cells treated with phorbol 12-myristate 13-acetate (PMA) to induce megakaryocytic transformation. ChIP assay revealed in vivo RUNX1 binding in two regions encompassing RUNX1 sites at −1768 (TGTGGT) and −151 (ACCGCA) on PF4 promoter. These sites were pursued with electrophoretic mobility shift assay (EMSA) using PMA treated HEL cell nuclear extract. EMSA showed specific protein binding to DNA probes encompassing each of the above sites; this was abolished by RUNX1 antibody. The ChIP and EMSA suggest that RUNX1 binds to the PF4 promoter region. To test the functional relevance of the RUNX1 binding sites wild type PF4 upstream promoter region (−1936/−27) containing both RUNX1 sites (−1768, and −151) or containing one or both sites mutated were cloned into firefly luciferase reporter gene vector pGL4 and expressed in PMA-treated HEL cells. Mutation of the −1768 site caused ∼50% reduction in luciferase activity, mutation at −151 site caused 60% reduction in activity and mutation of both sites caused 75-80% reduction in activity. These studies suggest that each RUNX1 site contributes to the transcriptional activity of PF4 promoter. Moreover, the upstream region (−1837 to +25) of the non-allelic variant PF4V1 was cloned into pGL4 plasmid; it showed negligible luciferase activity as compared to the wild PF4 promoter containing plasmid. These studies suggest that the regulation of PF4 and its variant PF4V1 is distinctly different in HEL cells. Conclusions: Our results provide the first evidence that PF4 promoter is regulated by RUNX1, and the two RUNX1 sites at −1768 and −151 are involved in its regulation. These studies provide a cogent explanation for the α-granule PF4 deficiency in our patient and others with RUNX1/CBFA2 haplodeficiency. They extend our understanding of the potential mechanisms involved in the pathogenesis of the Gray platelet syndrome. Disclosures: No relevant conflicts of interest to declare.