Inhibition of human cytomegalovirus DNA maturation by a benzimidazole ribonucleoside is mediated through the UL89 gene product

2-Bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (BDCRB) is a member of a new class of benzimidazole ribonucleosides which inhibit human cytomegalovirus (HCMV) late in the replication cycle without inhibiting viral DNA synthesis. We show here that polygenomic concatemeric HCMV DNA does not mature to unit genome length in the presence of BDCRB. To discover the locus of action, virus resistant to BDCRB was selected by serial passage in the presence of the compound. Genetic mapping experiments with BDCRB-resistant virus demonstrated that the resistance phenotype mapped to one amino acid (Asp344Glu; low resistance) or two amino acids (Asp344Glu and Ala355Thr; high resistance) within the product of exon 2 of the HCMV U(L)89 open reading frame. The HCMV U(L)89 open reading frame and its homologs are among the most conserved open reading frames in the herpesviruses, and their products have sequence similarities to a known ATP-dependent endonuclease from the double-stranded DNA bacteriophage T4. These findings strongly suggest that BDCRB prevents viral DNA maturation by interacting with a U(L)89 gene product and that the U(L)89 open reading frame may encode an endonucleolytic subunit of the putative HCMV terminase. Further, since mammalian cell DNA replication does not involve a DNA maturation step, compounds which inhibit viral DNA maturation should be selective and safe.     

Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A

Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing apoptosis. Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser70) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kinase (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). In addition, an intact Ser70 was found to be required for Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy. We now show that phosphorylation of Bcl2 occurs rapidly after the addition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphatase (PP) 2A as the Bcl2 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bcl2 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bcl2; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bcl2 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bcl2 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bcl2. Increased association between Bcl2 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. An agonist-induced increased association of PP2A and Bcl2 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser70 site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bcl2 at Ser70 is proposed to be a dynamic process regulated by the sequential action of an agonist-activated Bcl2 kinase and PP2A.     

Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely-impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfiel.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.     

RNA recognition by the human U1A protein is mediated by a network of local cooperative interactions that create the optimal binding surface

One of the most common structural motifs in RNA-binding proteins is the RNA-binding domain (RBD). These domains share a common alpha/beta sandwich tertiary fold, and are highly conserved, though they bind diverse RNA targets with a wide range of binding affinities. The N-terminal RNA-binding domain (RBD1) of the human U1A protein binds specifically to stem/loop II of the U1 snRNA with sub-nanomolar affinity. Solvent-exposed aromatic residues on the beta-sheet surface are highly conserved among RBD domains; in RBD1, these are Tyr13 and Phe56, with a unique Gln at position 54. Effects of substitutions at these positions were examined using energetic pairwise coupling to describe the communication between these residues in both the free and RNA-bound states of the protein. 15N NMR experiments were used to determine effects of the beta-sheet substitutions on the structural and dynamic properties of this domain. The combination of thermodynamic pairwise coupling and 15N-backbone dynamics provides direct evidence for local cooperative interactions among Y13, Q54, and F56, and a non-conserved loop that directly affect RNA-binding. The results describe how conserved and non-conserved regions of an RBD can communicate with each other to mediate recognition of the RNA.     

Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains

Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes. Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits. We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences. Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices. Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins. Additionally, the N-terminal GTPase domain plays an important role in assembly. Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly.     

Virion incorporation of human immunodeficiency virus type-1 Vif is determined by intracellular expression level and may not be necessary for function

OBJECTIVE: To define the role of laparoscopic ultrasound (LUS) in the staging of pancreatic tumors. SUMMARY BACKGROUND DATA: Laparoscopy has recently been established as a valuable tool in the staging of pancreatic cancer. It has been suggested that the addition of LUS to standard laparoscopy could improve the accuracy of this procedure. METHODS: A prospective evaluation of 90 patients with pancreatic tumors undergoing laparoscopy and LUS was performed over a 27-month period. LUS equipped with an articulated curved and linear array transducer (6 to 10 MHz) was used. All patients underwent rigorous laparoscopic examination. Clinical, surgical, and pathologic data were collected. RESULTS: The median age was 65 years (range 43 to 85 years). Sixty-four patients had tumors in the head, 19 in the body, and 3 in the tail of the pancreas. Four patients had ampullary tumors. LUS was able to image the primary tumor (98%), portal vein (97%), superior mesenteric vein (94%), hepatic artery (93%), and superior mesenteric artery (93%) in these patients. LUS was particularly helpful in determining venous involvement (42%) and arterial involvement (38%) by the tumor. This resulted in a change in surgical treatment for 13 (14%) of the 90 patients in whom standard laparoscopic examination was equivocal. CONCLUSIONS: LUS is useful in evaluating the primary tumor and peripancreatic vascular anatomy. When standard laparoscopic findings are equivocal, LUS allowed accurate determination of resectability. Supplementing laparoscopy with LUS offers improved assessment and preoperative staging of pancreatic cancer.     

Inhibition of CD4 translation mediated by human immunodeficiency virus type 1 envelope protein in a cell-free system

The human immunodeficiency virus type 1 (HIV-1) employs a number of complex strategies to interfere with the synthesis, stability, and subcellular localization of its specific cellular receptor CD4. To define better the mechanisms of inhibition of CD4 expression, we used a rabbit reticulocyte lysate in vitro system, in which cDNAs derived from HIV-1-infected cells were used to generate mRNA for the Tat, Vpu, and gp160 envelope proteins that were translated together with CD4-encoding mRNA. In the presence of microsomal membranes, we observed that cotranslation of Env mRNA resulted in a dose-dependent inhibition of CD4 translation. This effect was enhanced further when an mRNA-encoding Vpu in addition to Env mRNA was utilized. However, the activity of Vpu was mostly post-translational, since translation of Vpu alone, but not Env, was able to destabilize CD4 molecules presynthesized into microsomes. The Env-mediated inhibitory effect was specifically targeted at CD4 and did not affect the synthesis or stability of the CD8 molecule. Interestingly, mutated CD4 species, with a 20-fold lower affinity for HIV-1 Env than wild-type, were less sensitive to cotranslational inhibition. Our report identifies the envelope as the HIV-1 protein responsible for down-regulation of CD4 translation. We further propose a mechanism whereby direct interactions between gp160 and nascent CD4 molecules can cause interference with and premature termination of CD4 protein elongation.     

The interaction of arginine 106 of human prostaglandin G/H synthase-2 with inhibitors is not a universal component of inhibition mediated by nonsteroidal anti-inflammatory drugs

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.     

The interaction of eIF4E with 4E-BP1 is an induced fit to a completely disordered protein

4E binding protein 1 (4E-BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4-10, 43-46, and 56-65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady-state ?1H?-15N NOEs were measured for 45 residues in the assigned regions. Except for the two C-terminal residues, the NOEs were between -0.77 and - 1.14, indicating a high level of flexibility. Furthermore, the ?1H?-15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E-BP1 has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.     

Transactivation of the human cdc2 promoter by adenovirus E1A in cycling cells is mediated by induction of a 110-kDa CCAAT-box-binding factor

Cyclin-dependent protein kinases (Cdks) are key regulatory proteins of the eukaryotic cell cycle. Cdc2 is expressed in late G1/S phase and functions in the G2 to M phase transition. Adenovirus E1A proteins are known to induce the expression of p34cdc2 and DNA synthesis in normal quiescent cells. In this study, mutational analysis of the human cdc2 promoter revealed that transactivation of the promoter by the E1A proteins in cycling cells is mediated through the two CCAAT box binding motifs. A 110-kDa protein (CBF/cdc2) was identified in nuclear extracts from monkey kidney (CV-1) cells stably expressing E1A as well as from adenovirus-transformed human 293 cells. Further, we show that this EIA-inducible CBF/cdc2 is related to the CBF which was shown to activate the heat shock protein 70 promoter. Analyses of the functional domain(s) of E1A required for the induction of the CBF and transactivation of the cdc2 promoter in these conditions revealed that E1A mutants which were defective in binding the pRB family of proteins or the cellular p300 protein were still active in assays measuring the induction of the CBF and transactivation of the cdc2 promoter, albeit with reduced efficiencies. But the E1A mutant which lost both functional domains was inactive in these assays. These results suggest that E1A has redundant functional domains for the induction of the 110-kDa CBF and activation of human cdc2 gene expression.     

Initiation of DNA replication at palindromic telomeres is mediated by a duplex-to-hairpin transition induced by the minute virus of mice nonstructural protein NS1

The linear single-stranded DNA genome of the minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate. Amplification of this RF is initiated by the folding-back of palindromic sequences serving as primers for strand-displacement synthesis and formation of dimeric RF DNA. Using an in vitro replication assay and a cloned MVM DNA template, we observed hairpin-primed DNA replication at both MVM DNA termini, with a bias toward right-end initiation. Initiation of DNA replication is favored by nuclear components of A9 cell extract and highly stimulated by the MVM nonstructural protein NS1. Hairpin-primed DNA replication is also observed in the presence of NS1 and the Klenow fragment of the Escherichia coli DNA polymerase I. Addition of ATPgammaS (adenosine 5'-O-(thiotriphosphate)) blocks the initiation of DNA replication but not the extension of pre-existing hairpin primers formed in the presence of NS1 only. The NS1-mediated unwinding of the right-end palindrome may account for the recently reported capacity of NS1 for driving dimer RF synthesis in vitro.     

Differentiation of cultured human epidermal keratinocytes at high cell densities is mediated by endogenous activation of the protein kinase C signaling pathway

The MAGE-11 gene belongs to a family whose products were identified first in tumor tissue. The MAGE-11 gene product has not been characterized in detail. We have isolated MAGE-11 cDNA from HeLa cells and confirmed the presence of MAGE-11 protein and of at least 2 other MAGE proteins (MAGE-1 and MAGE-3) in this cell line. Monoclonal antibodies (MAbs), obtained by using a GST-MAGE-11 fusion protein, detect MAGE-11 protein in HeLa cells as a 48 kDa protein. In contrast to other known proteins of the MAGE family, MAGE-11 is found mainly in the nucleus. Immunoprecipitation out of whole-cell extracts from different species reveals that MAGE-11 protein is highly conserved among mammalian cells, suggesting a conserved and important function.     

Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism

Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive Gi-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.