A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation.

The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication.

Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

Breast cancer-associated gene 3 interacts with Rac1 and augments NF-κB signaling in vitro, but has no effect on RANKL-induced bone resorption in vivo.

Breast cancer-associated gene 3 (BCA3) is a recently identified adaptor protein whose functions are still being defined. BCA3 has been reported to be an important regulator of nuclear factor-κB (NF-κB) signaling. It has also been reported to interact with the small GTPase, Rac1. Consistent with that observation, in the present study, BCA3 was found to interact with nuclear Rac1 in 293 cells and influence NF-κB signaling. Additional experiments revealed that depending on cell type, BCA3 augmented, attenuated or had no effect on NF-κB signaling in vitro. Since canonical NF-κB signaling is a critical downstream target from activated receptor activator of nuclear factor κB (RANK) that is required for mature osteoclast formation and function, BCA3 was selectively overexpressed in osteoclasts in vivo using the cathepsin K promoter and the response to exogenous receptor activator of nuclear factor κB ligand (RANKL) administration was examined. Despite its ability to augment NF-κB signaling in other cells, transgenic animals injected with high-dose RANKL had the same hypercalcemic response as their wild­type littermates. Furthermore, the degree of bone loss induced by a 2-week infusion of low-dose RANKL was the same in both groups. Combined with earlier studies, the data from our study data indicate that BCA3 can affect NF-κB signaling and that BCA3 plays a cell-type dependent role in this process. The significance of the BCA3/NF-κB interaction in vivo in bone remains to be determined.

Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

Latency of Epstein-Barr virus is disrupted by gain-of-function mutant cellular AP-1 proteins that preferentially bind methylated DNA.

ZEBReplication Activator (ZEBRA), a viral basic zipper protein that initiates the Epstein-Barr viral lytic cycle, binds to DNA and activates transcription through heptamer ZEBRA response elements (ZREs) related to AP-1 sites. A component of the biologic action of ZEBRA is attributable to binding methylated CpGs in ZREs present in the promoters of viral lytic cycle genes. Residue S186 of ZEBRA, Z(S186), which is absolutely required for disruption of latency, participates in the recognition of methylated DNA. We find that mutant cellular AP-1 proteins, Jun(A266S) and Fos(A151S), with alanine-to-serine substitutions homologous to Z(S186), exhibit altered DNA-binding affinity and preferentially bind methylated ZREs. These mutant AP-1 proteins acquire functions of ZEBRA; they activate expression of many viral early lytic cycle gene transcripts in cells harboring latent EBV but are selectively defective in activating expression of some viral proteins and are unable to promote viral DNA replication. Transcriptional activation by mutant c-Jun and c-Fos that have acquired the capacity to bind methylated CpG challenges the paradigm that DNA methylation represses gene expression.

Breast cancer-associated gene 3 (BCA3) is a novel Rac1-interacting protein.

UNLABELLED: BCA3 was identified in a yeast two-hybrid screen as a novel Rac1-interacting partner in osteoclasts. BCA3 binds directly to Rac and, in vivo, binds GTP-Rac but not GDP-Rac. Perinuclear co-localization of BCA3 and Rac1 is observed in CSF-1-treated osteoclasts. Overexpression of BCA3 attenuates CSF-1-induced cell spreading. We conclude that BCA3 regulates CSF-1-dependent Rac activation. INTRODUCTION: Rac1, a ubiquitously expressed GTPase, is a mediator of colony-stimulating factor 1 (CSF-1)-dependent actin remodeling in osteoclasts. Because the role of Rac in osteoclasts has not been fully defined, we undertook a yeast two-hybrid screen to identify Rac-interacting partners in these cells. MATERIALS AND METHODS: A yeast two-hybrid screen was undertaken using a cDNA library prepared from osteoclast-like cells as prey and either native Rac1 or constitutively active Rac1 (Q61L) as bait. Radiolabeled breast cancer-associated gene 3 (BCA3) protein constructs were generated in vitro using rabbit reticulate lysates and used in vitro binding assays with Rac1. In vivo binding was assessed using myc-tagged Rac1(Q61L) and HA-tagged BCA3. PBD pull-down assays were used to determine if GTP-loaded Rac1 preferentially bound BCA3. Co-localization of Rac1 and BCA3 in osteoclasts was assessed using confocal immunofluorescence. The functional significance of the BCA3-Rac1 interaction was assessed by examining the effect of overexpressing BCA3 in RAW 264.7 cells on the subsequent spreading response to CSF-1. RESULTS: One of three positive clones from the wildtype Rac1 screen and all three positive clones from the Rac1(Q61L) screen encoded the same protein, BCA3. BCA3 expression in osteoclasts was confirmed by RT-PCR and immunocytochemistry. BCA3 bound directly to Rac1 in vitro. Deletional analysis indicated that amino acids 76-125 in BCA3 are important for its ability to bind Rac. In vivo association of the two proteins was shown by co-immunoprecipitation of BCA3 and Rac1. Only GTP-bound-Rac but not GDP-bound Rac could interact with BCA3 in vivo. Confocal immunocytochemistry showed perinuclear co-localization of BCA3 and Rac1 in CSF-1-treated neonatal rat osteoclasts but not in resting osteoclasts. Overexpression of BCA3 markedly attenuated the spreading response to CSF-1 in RAW 264.7 cells. CONCLUSIONS: These data establish that BCA3 is a novel Rac1-interacting protein and suggest that it may influence the ability of Rac1 to remodel the actin cytoskeleton.

Activated c-Fms recruits Vav and Rac during CSF-1-induced cytoskeletal remodeling and spreading in osteoclasts.

Colony-stimulating factor-1 (CSF-1) induces osteoclast spreading that requires activation of c-Src and phosphatidyl inositol 3-kinase (PI3-K), both of which are recruited to activated c-Fms, the CSF-1 receptor. The present report provides evidence that the hemopoietic guanine nucleotide exchange factor (GEF), Vav, and its target GTPase, Rac, lie downstream from this initial signaling complex. CSF-1 treatment of osteoclast-like cells induced translocation of Vav to the plasma membrane, an increase in its phosphotyrosine content, and a concomitant decline in the amount of phosphoinositol 4,5-bisphosphate bound to Vav, changes known to induce Vav's GEF activity. CSF-1 induced the association of Vav and Rac and increased Rac's GTPase activity. CSF-1 also induced rapid translocation of Rac to the periphery of spreading neonatal rat osteoclasts where it co-localized primarily with Vav3 and to a lesser extent with Vav1. Wortmannin, an inhibitor of PI3-K, blocked CSF-1-induced Rac translocation and prevented CSF-1-induced spreading and actin reorganization in osteoclasts. CSF-1-induced osteoclast spreading was not significantly reduced in osteoclasts isolated from Vav1 knock-out mice and Vav1 knock-out mice had normal bone density. Microinjection of constitutively active Rac, but not constitutively active Cdc42 or RhoA, induced lamellipodia formation and osteoclast spreading, mimicking the effects of CSF-1. Dominant-negative Rac blocked CSF-1-induced osteoclast spreading, whereas neither dominant-negative Cdc42 nor C3, an inhibitor of RhoA, affected the response to CSF-1. These data demonstrate that Vav and Rac lie downstream from activated PI3-K in CSF-1-treated osteoclasts and that Rac is required for CSF-1-induced cytoskeletal remodeling in these cells.

LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1.

LATS (large tumour suppressor) is a family of conserved tumour suppressors identified in Drosophila and mammals. Here we show that human LATS1 binds to LIMK1 in vitro and in vivo and colocalizes with LIMK1 at the actomyosin contractile ring during cytokinesis. LATS1 inhibits both the phosphorylation of cofilin by LIMK1 and LIMK1-induced cytokinesis defects. Inactivation of LATS1 by antibody microinjection or RNA-mediated interference in cells, or gene knockout in mice, abrogates cytokinesis and increases the percentage of multinucleate cells. Our findings indicate that LATS1 is a novel cytoskeleton regulator that affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1.

Cloning of Human Myelin Protein Zero-like Genes by Bioinformatics Strategy.

To clone novel myelin protein related genes, two human ESTs, which shared significant similarity with the human myelin protein zero gene, were found by the comparison of homologue between the cDNA coding region sequences of MPZ gene and the EST database of NCBI. An 801 bp EST contig was assembled, which was 100% identical with a 128 kb genomic sequence, mapped to 1q24. A 435 bp open reading frame (ORF) within the 801 bp contig was shown by computer analysis. Two primers designed according to the sequence of the contig, were coupled with the primers(lambdagt10-5 and gt10-5) on the sequences flanking cloning site of the cDNA library vector to amplify the cDNA library sequences by nested PCR. New primers, designed based on novel cDNA sequences, were used for the PCR amplification with lambdagt10-5 and gt10-5 in the same way as above. Finally, the human myelin protein zero like gene isoform I and II (MPZL1a, MPZL1b GenBank AF095727, AF092424) were cloned. Comparison of gene and protein structures between MPZL1 and MPZ revealed that MPZL1 is the second member of MPZ family. Mutation analysis of MPZL1 gene was performed in 24 Charcot-Marie-Tooth disease (CMT) families and 26 nonsyndrome deafness families, but no mutation was found.