Gα q Directly Activates p63RhoGEF and Trio via a Conserved Extension of the Dbl Homology-associated Pleckstrin Homology Domain

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Gα12/13 and Gαq/11 families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Gα12/13-mediated RhoA activation, proteins that functionally couple Gαq/11 to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Gαq/11 signaling to RhoA based on its ability to synergize with Gαq/11 resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Gαq directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Gαq relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Gαq

The Epstein-Barr Virus (EBV)-Encoded Protein Kinase, EBV-PK, but Not the Thymidine Kinase (EBV-TK), Is Required for Ganciclovir and Acyclovir Inhibition of Lytic Viral Production

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC50] = 1.5 μM) and ACV (IC50 = 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC50 = 19.6 μM) and ACV (IC50 = 36.4 μM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC50 = 1.2 μM) and ACV (IC50 = 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities

Auto-inhibition of the Dbl Family Protein Tim by an N-terminal Helical Motif

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process

Different linkages in the long and short regions of the genomes of duck enteritis virus Clone-03 and VAC Strains

<p>Abstract</p> <p>Background</p> <p>Duck enteritis virus (DEV) is an unassigned member in the family <it>Herpesviridae</it>. To demonstrate further the evolutionary position of DEV in the family <it>Herpesviridae</it>, we have described a 42,897-bp fragment. We demonstrated novel genomic organization at one end of the long (L) region and in the entire short (S) region in the Clone-03 strain of DEV.</p> <p>Results</p> <p>A 42,897-bp fragment located downstream of the <it>LOFR11 </it>gene was amplified from the Clone-03 strain of DEV by using 'targeted gene walking PCR'. Twenty-two open reading frames (ORFs) were predicted and determined in the following order: 5'<it>-LORF11-RLORF1</it>-<it>ORF1</it>-<it>ICP4</it>-<it>S1-S2-US1-US10-SORF3-US2-MDV091.5-like-US3-US4-US5-US6-US7-US8-ORFx-US1-S2-S1-ICP4 </it>-3'. This was different from that of the published VAC strain, both in the linkage of the L region and S region, and in the length of the US10 and US7 proteins. The <it>MDV091.5-like </it>gene, <it>ORFx </it>gene, <it>S1 </it>gene and <it>S2 </it>gene were first observed in the DEV genome. The lengths of DEV US10 and US7 were determined to be 311 and 371 amino acids, respectively, in the Clone-03 strain of DEV, and these were different from those of other strains. The comparison of genomic organization in the fragment studied herein with those of other herpesviruses showed that DEV possesses some unique characteristics, such as the duplicated US1 at each end of the US region, and the US5, which showed no homology with those of other herpesviruses. In addition, the results of phylogenetic analysis of ORFs in the represented fragment indicated that DEV is closest to its counterparts VZV (<it>Varicellovirus</it>) and other avian herpesviruses.</p> <p>Conclusion</p> <p>The molecular characteristics of the 42,897-bp fragment of Clone-03 have been found to be different from those of the VAC strain. The phylogenetic analysis of genes in this region showed that DEV should be a separate member of the subfamily <it>Alphaherpesvirinae</it>.</p

A Live-Attenuated HSV-2 ICP0− Virus Elicits 10 to 100 Times Greater Protection against Genital Herpes than a Glycoprotein D Subunit Vaccine

Glycoprotein D (gD-2) is the entry receptor of herpes simplex virus 2 (HSV-2), and is the immunogen in the pharmaceutical industry's lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0− virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A) produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain). In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0− virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein

Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial–mesenchymal transition in metastatic nasopharyngeal carcinoma

Background:Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial–mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT.Methods:We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells.Results:In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells.Conclusions:This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC

Mutual Inhibition between Kaposi's Sarcoma-Associated Herpesvirus and Epstein-Barr Virus Lytic Replication Initiators in Dually-Infected Primary Effusion Lymphoma

Background: Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are members of the human gamma herpesvirus family: each is associated with various human cancers. The majority of AIDS-associated primary effusion lymphoma (PEL) are co-infected with both KSHV and EBV. Dually-infected PELs selectively switch from latency to lytic replication of either KSHV or EBV in response to chemical stimuli. KSHV replication and transcription activator (K-RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication, while EBV BZLF1 gene product (EBV-Z) is a critical initiator for induction of EBV lytic replication. Methodology/Principal Findings: We show K-RTA and EBV-Z are co-localized and physically interact with each other in dually-infected PELs. K-RTA inhibits the EBV lytic replication by nullifying EBV-Z-mediated EBV lytic gene activation. EBV-Z inhibits KSHV lytic gene expression by blocking K-RTA-mediated transactivations. The physical interaction between K-RTA and EBV-Z are required for the mutual inhibition of the two molecules. The leucine heptapeptide repeat (LR) region in K-RTA and leucine zipper region in EBV-Z are involved in the physical interactions of the two molecules. Finally, initiation of KSHV lytic gene expression is correlated with the reduction of EBV lytic gene expression in the same PEL cells. Conclusions/Significance: In this report, how the two viruses interact with each other in dually infected PELs is addressed. Our data may provide a possible mechanism for maintaining viral latency and for selective lytic replication in dually infected PELs, i.e., through mutual inhibition of two critical lytic replication initiators. Our data about putative interactions between EBV and KSHV would be applicable to the majority of AIDS-associated PELs and may be relevant to the pathogenesis of PELs

Epstein-Barr Virus BGLF4 Kinase Retards Cellular S-Phase Progression and Induces Chromosomal Abnormality

Epstein-Barr virus (EBV) induces an uncoordinated S-phase-like cellular environment coupled with multiple prophase-like events in cells replicating the virus. The EBV encoded Ser/Thr kinase BGLF4 has been shown to induce premature chromosome condensation through activation of condensin and topoisomerase II and reorganization of the nuclear lamina to facilitate the nuclear egress of nucleocapsids in a pathway mimicking Cdk1. However, the observation that RB is hyperphosphorylated in the presence of BGLF4 raised the possibility that BGLF4 may have a Cdk2-like activity to promote S-phase progression. Here, we investigated the regulatory effects of BGLF4 on cell cycle progression and found that S-phase progression and DNA synthesis were interrupted by BGLF4 in mammalian cells. Expression of BGLF4 did not compensate Cdk1 defects for DNA replication in S. cerevisiae. Using time-lapse microscopy, we found the fate of individual HeLa cells was determined by the expression level of BGLF4. In addition to slight cell growth retardation, BGLF4 elicits abnormal chromosomal structure and micronucleus formation in 293 and NCP-TW01 cells. In Saos-2 cells, BGLF4 induced the hyperphosphorylation of co-transfected RB, while E2F1 was not released from RB-E2F1 complexes. The E2F1 regulated activities of the cyclin D1 and ZBRK1 promoters were suppressed by BGLF4 in a dose dependent manner. Detection with phosphoamino acid specific antibodies revealed that, in addition to Ser780, phosphorylation of the DNA damage-responsive Ser612 on RB was enhanced by BGLF4. Taken together, our study indicates that BGLF4 may directly or indirectly induce a DNA damage signal that eventually interferes with host DNA synthesis and delays S-phase progression