Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo

The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.     

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

Interaction of scavenger receptor class B type I with peroxisomal targeting receptor Pex5p

Scavenger receptor class B type I (SR-BI) is an HDL receptor that mediates selective HDL lipid uptake. Peroxisomes play an important role in lipid metabolism and peroxisomal targeting signal type 1 (PTS1)-containing proteins are translocated to peroxisomes by the peroxisomal targeting import receptor, Pex5p. We have previously identified a PTS1 motif in the intracellular domain of rat SR-BI. Here, we examine the possible interaction between Pex5p and SR-BI. Expression of a Flag-tagged intracellular domain of SR-BI resulted in translocation to the peroxisome as demonstrated by double labeling with anti-Flag IgG and anti-catalase IgG analyzed by confocal microscopy. Immunoprecipitation experiments with anti-SR-BI antibody showed that Pex5p co-precipitated with SR-BI. However, when an antibody against Pex5p was used for immunoprecipitation, only the 57kDa, non-glycosylated form, of SR-BI co-precipitated. We conclude that the PTS1 domain of SR-BI is functional and can mediate peroxisomal interaction via Pex5p, in vitro.     

Distinct effects on heparan sulfate structure by different active site mutations in NDST-1

Heparan sulfate polymerization and modification take place in the Golgi compartment. The modification reactions are initiated by glucosaminyl N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme that removes N-acetyl groups from selected N-acetyl-d-glucosamine units followed by N-sulfation of the generated free amino groups. Four isoforms of NDST have been identified. NDST-1 and -2 have a wide and largely overlapping tissue distribution, but it is not known if they can act on the same heparan sulfate chain. We have introduced point mutations into NDST-1 cDNA, which selectively destroy the N-deacetylase or N-sulfotransferase activity of the enzyme [Wei, Z., and Swiedler, S. J. (1999) J. Biol. Chem. 274, 1966-70 and Sueyoshi, T., et al. (1998) FEBS Lett. 433, 211-4]. Stable 293 cell lines expressing the NDST-1 mutants were then generated. Structural analyses of heparan sulfate synthesized by these cells and by cells overexpressing wild-type NDST-1 demonstrate that the N-deacetylation step is not only prerequisite but also rate-limiting, determining the degree of N-sulfation. Transfection of mutant NDST-1 lacking N-deacetylase activity had no effect on heparan sulfate sulfation, while cells expressing wild-type enzyme or NDST-1 lacking N-sulfotransferase activity both resulted in the production of oversulfated heparan sulfate. Since no increase in the amount of N-unsubstituted glucosamine residues was seen after transfection of the mutant lacking N-sulfotransferase activity, the results also suggest that two different enzyme molecules can act on the same glucosamine unit. In addition, we show that oversulfation of heparan sulfate produced by cells tranfected with wild-type NDST-1 or the mutant lacking N-sulfotranferase activity results in decreased sulfation of chondroitin sulfate.     

Roles of individual amino acids in helix 14 of the membrane domain of proton-translocating transhydrogenase from Escherichia coli as deduced from cysteine mutagenesis

Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane alpha helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues betaLeu240 to betaSer260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of betaGly245, betaGly249, and betaGly252, also to leucines. In addition to the residues forming the helix, residues betaAsn238 and betaAsp239 were also mutated. Except for betaI242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines betaGly245, betaGly249, and betaGly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of betaGly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines betaSer250, betaSer251, and betaSer256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.     

Development of a large-scale biocalorimeter to monitor and control bioprocesses

Calorimetry has shown real potential at bench-scale for chemical and biochemical processes. The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor. To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation implemented to enable on-line monitoring and dynamic heat balance estimation. Providing that the signals are sufficiently precise, such a heat balance would enable calculation of the heat released or taken up during an operational (bio)process. Two electrical Wattmeters were developed, the first for determination of the power consumption by the stirrer motor and the second for determination of the power released by an internal calibration heater. Experiments were designed to optimize the temperature controller of the bioreactor such that it was sufficiently rapid so as to enable the heat accumulation terms to be neglected. Further calibration experiments were designed to correlate the measured stirring power to frictional heat losses of the stirrer into the reaction mass. This allows the quantitative measurement of all background heat flows and the on-line quantitative calculation of the (bio)process power. Three test fermentations were then performed with B. sphaericus 1593M, a spore-forming bacterium pathogenic to mosquitoes. A first batch culture was performed on a complex medium, to enable optimization of the calorimeter system. A second batch culture, on defined medium containing three carbon sources, was used to show the fast, accurate response of the heat signal and the ability to perfectly monitor the different growth phases associated with growth on mixed substrates, in particular when carbon sources became depleted. A maximum heat output of 1100 W was measured at the end of the log-phase. A fed-batch culture on the same defined medium was then carried out with the feed rate controlled as a function of the calorimeter signal. A maximum heat output of 2250 W was measured at the end of the first log-phase. This work demonstrates that real-time quantitative calorimetry is not only possible at pilot-scale, but could be readily applied at even larger scales. The technique requires simple, readily available devices for determination of the few necessary heat flows, making it a robust, cost-effective technique for process development and routine monitoring and control of production processes.     

Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.     

Site-specific loss of acetylation upon phosphorylation of histone H3

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser(10)) facilitates acetylation of lysine 14 (Lys(14)) by Gcn5 in vitro (, ). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser(10) in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser(10) to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys(9)) in vivo. Acetylation of Lys(9) is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser(10) in H3 and the concomitant loss of Lys(9) acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser(10) mutations that are reminiscent of delays in G(2)/M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys(9) and Lys(14), and they highlight the importance of these histone modifications to normal cell functions.     

Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e. the cis isomer of amiclenomycin is a potent inhibitor, whereas the trans isomer is much less reactive. The crystal structure of the complex of the holoenzyme and amiclenomycin at 1.8 A resolution reveals that the internal aldimine linkage between the cofactor and the side chain of the catalytic residue Lys-274 is broken. Instead, a covalent bond is formed between the 4-amino nitrogen of amiclenomycin and the C4' carbon atom of pyridoxal-phosphate. The electron density for the bound inhibitor suggests that aromatization of the cyclohexadiene ring has occurred upon formation of the covalent adduct. This process could be initiated by proton abstraction at the C4 carbon atom of the cyclohexadiene ring, possibly by the proximal side chain of Lys-274, leading to the tautomer Schiff base followed by the removal of the second allylic hydrogen. The carboxyl tail of the amiclenomycin moiety forms a salt link to the conserved residue Arg-391 in the substrate-binding site. Modeling suggests steric hindrance at the active site as the determinant of the weak inhibiting potency of the trans isomer.