Alik Honigman

Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus

Human cytomegalovirus, a chief pathogen in immunocompromised people, can persist in a healthy immunocompetent host throughout life without being eliminated by the immune system. Here we show that pp65, the main tegument protein of human cytomegalovirus, inhibited natural killer cell cytotoxicity by an interaction with the activating receptor NKp30. This interaction was direct and specific, leading to dissociation of the linked CD3ζ from NKp30 and, consequently, to reduced killing. Thus, pp65 is a ligand for the NKp30 receptor and demonstrates a unique mechanism by which an intracellular viral protein causes general suppression of natural killer cell cytotoxicity by specific interaction with an activating receptor.

A Pivotal Role of Cyclic AMP-Responsive Element Binding Protein in Tumor Progression

Tumor microenvironment controls the selection of malignant cells capable of surviving in stressful and hypoxic conditions. The transcription factor, cyclic AMP-responsive element binding (CREB) protein, activated by multiple extracellular signals, modulates cellular response by regulating the expression of a multitude of genes. Previously, we have demonstrated that two cystein residues, at the DNA binding domain of CREB, mediate activation of CREB-dependent gene expression at normoxia and hypoxia. The construction of a dominant-positive CREB mutant, insensitive to hypoxia cue (substitution of two cystein residues at position 300 and 310 with serine in the DNA binding domain) and of a dominant negative CREB mutant (addition of a mutation in serine133), enabled a direct assessment, in vitro and in vivo, of the role of CREB in tumor progression. In this work, we demonstrate both in vitro and in vivo that CREB controls hepatocellular carcinoma growth, supports angiogenesis, and renders resistance to apoptosis. Along with the identification, by DNA microarray, of the CREB-regulated genes in normoxia and hypoxia, this work demonstrates for the first time that in parallel to other hypoxia responsive mechanisms, CREB plays an important role in hepatocellular carcinoma tumor progression.

Early Emergence of Ganciclovir-Resistant Human Cytomegalovirus Strains in Children with Primary Combined Immunodeficiency

Children with primary combined immunodeficiency (CID) and human cytomegalovirus (HCMV) infection often deteriorate despite antiviral therapy. In this study, the emergence of ganciclovir-resistant strains was examined in 6 children with CID and HCMV infection, using sequence analysis of the HCMV UL97 gene and virus susceptibility assays. Mutations in the proposed ATP binding site associated with ganciclovir resistance were found in 4 of the 6 children. In 1 patient with B severe CID, an unusual multiplicity of mutations was found in the UL97 substrate binding domain between aa 590-606. All mutations were detected within 10 days to 3 weeks from initiation of therapy. The emergence of resistant strains in children with CID appears earlier than in other groups of HCMV-infected patients. These findings may have relevance to the cellular pathways involved in viral DNA repair and mutagenesis, and they indicate the need for early and frequent genotypic monitoring and prompt therapeutic modification in this patient population.

cis Acting RNA sequences control the gag-pol translation readthrough in murine leukemia virus.

Abstract The pol gene of the Moloney murine leukemia virus (M-MuLV) is expressed as a Gag-Pol fusion protein through an in-frame suppression of the UAG termination codon located between the two genes. The role of nucleotide context in suppression was investigated, in a rabbit reticulocyte lysate translation system, using site-directed mutagenesis. The results indicate that the translational readthrough is mediated by at least 50 bases long RNA sequence located 3′ to the gag UAG termination codon. Within this sequence a short purine-rich sequence adjacent to the amber codon, highly conserved among different retroviruses, appears essential for M-MuLV suppression. Two alternative putative stem and loop like RNA structures can be drawn at the gag-pol junction, one abutting the gag UAG codon, and the second downstream to it. None of these structures appears to be important to the suppression process.

Transcriptional regulation of the human sterol 27-hydroxylase gene (CYP27) and promoter mapping.

Recent evidence suggests that sterol 27-hydroxylase may play a role in cholesterol homeostasis and affect atherogenesis. The major objective of the study was to map and characterize the sterol 27-hydroxylase (CYP27) promoter region. Here we show that CYP27 gene has a TATA-less promoter and transcription initiates at a cluster of sites. The basic promoter is located between -166 and -187 bp from the translation initiation site. Possible positive transcription regulation sites are located at position -187 to -320 and -857 to -1087 bp. A negative transcription regulator site is located in position -320 to -413 bp. An enhancer sequence is located upstream to position -1087. CYP27 is upregulated by dexamethasone and downregulated by cyclosporin A and cholic acid. The dexamethasone responsive element is located between 1087 and 678 bp upstream to the putative ATG. Cyclosporin A affects bile acid metabolism by repressing CYP27 at the transcriptional level. The cyclosporin A- responsive element is mapped to between 1087 and 4000 bp upstream of the ATG. Cholic acid represses sterol 27-hydroxylase mRNA level by affecting the stability of its mRNA. The results obtained here imply that CYP27 has a potentially important role in cholesterol homeostasis in human cells, and is regulated by several substances that were previously shown to affect bile acid metabolism.

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase

SummaryThe cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy.

A Key role for cyclic AMP-responsive element binding protein in hypoxia-mediated activation of the angiogenesis factor CCN1 (CYR61) in Tumor cells.

Hypoxia is a prominent feature of solid tumors known to contribute to malignant progression and therapeutic resistance. Cancer cells adapt to hypoxia using various pathways, allowing tumors to thrive in a low oxygen state. Induction of new blood vessel formation via the secretion of proangiogenic factors is one of the main adaptive responses engaged by tumor cells under hypoxic conditions. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays a pivotal role in mediating such responses. In addition, several other transcription factors have also been implicated in hypoxic gene regulation, either independently or in cooperation with HIF-1. In this work, we show that the expression of the angiogenesis-related, immediate early gene CCN1 (formerly known as CYR61), considered to be involved in tumor growth and invasiveness, is enhanced upon hypoxia stress primarily in a protein kinase A and cyclic AMP-responsive element binding protein (CREB) and CRE–dependent manner in various cell lines. The hypoxia-mediated activation of the CCN1 promoter is independent of HIF-1 and HIF-2, as shown by small interfering RNA knockdown. We identify the cis element in the mouse CCN1 promoter responsible for CREB binding to be one of two partial CRE sites present in the promoter. Moreover, we report for the first time that CREB-mediated CCN1 transcription is enhanced in hypoxic regions of tumors in vivo. Identifying and characterizing the molecular mechanisms that govern the response of tumors to hypoxia may be instrumental to identify the tumors that will respond favorably to inhibition of angiogenesis and thus lead to the development of treatments that could complement hypoxia-inducing treatment modalities. (Mol Cancer Res 2008;6(9):1397–409)

Plasmid vectors for positive selection of DNA inserts controlled by the λ pl promoter, repressor and antitermination function

Hybrid plasmids consisting of pBR322 or pOP203-3 and the EcoRI-D fragment of lambda DNA kill their bacterial host upon expression of a lambda gene (probably the kil function) located either between or across the SalI sites. The plasmids from surviving hosts are acquired deletions that remove the lambda kil gene or insertions that block the transcription of the kil gene. Some plasmids probably carry point mutations. Based on these findings, we constructed two vector plasmids, pKL1 and pHA10, which can be used for a direct positive selection of cloned fragments. These plasmids are particularly useful for the cloning and selection of N-unresponsive termination signals using BamHI and its isoschizomers. The DNA fragments cloned into these plasmids are under control of the strong pL promoter, which can be regulated by the lambda repressor, and the antitermination activity of the N gene product.

Emergence of Multiple Human Cytomegalovirus Ganciclovir-Resistant Mutants with Deletions and Substitutions within the UL97 Gene in a Patient with Severe Combined Immunodeficiency

ABSTRACT Infection with multiple ganciclovir-resistant human cytomegalovirus mutants, containing different substitutions and deletions in the UL97 gene, was found in a patient with severe combined immunodeficiency (SCID) within 3 weeks of ganciclovir therapy. A novel 11-codon deletion at positions 590 to 600 was identified. These unique findings may be related to the nature of the immunodeficiency in the SCID patient.

Human Cytomegalovirus UL97 Kinase and Nonkinase Functions Mediate Viral Cytoplasmic Secondary Envelopment

ABSTRACT Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.