A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

Circadian and Ultradian Rhythms in Blood Glucose and Plasma Insulin of Healthy Adults

The circadian and ultradian variations of blood glucose and plasma insulin have been characterized individually and as a group phenomenon in five healthy young adults studied while adhering as closely as possible to their usual routine of sleep, activity, meal content and timing. Three complementary methods were used to analyze the data: displaying raw data as a function of time; cosinor method according to Nelson and Halberg; and time series analyses as proposed by De Prins and Malbecq. The subjects were studied in the laboratory and their life routine were controlled, but very close to that of their habitual routine. They had mainly ultradian rhythms of blood glucose (mainly about 6 hr) and circadian rhythms of immunoreactive insulin (I.R.I.). Blood glucose ultradian rhythms seem to be mainly but not exclusively mealtime dependent, while I.R.I, circadian rhythms appear to be primarily endogenous in origin. Therefore, the role played by insulin in the control of blood glucose levels seems to be programmed on a circadian basis rather than by a time independent feedback phenomenon as postulated by the conventional homeostatic hypothesis. The advantage of this chronophysiologic approach is to consider circadian rhythms of both I.R.I. and insulin effectiveness as an adaptive phenomenon able to maintain blood sugar changes in the ultradian domain of rhythms.     

Circadian Rhythms in Competitive Sabre Fencers: Internal Desynchronization and Performance

During a 7-10 day span, circadian rhythms of sleep-wake, self-rated fatigue and mood, oral temperature, eye-hand skill and right and left hand grip strength were investigated in eight subjects: five males (21-28 years of age), members of the French sabre fencing team selected for the 1984 Olympic Games in Los Angeles, and three females (19-26 years of age) practicing fleuret (foil) fencing as a sports activity. On the average six measurements/day/variable/subject were performed. The single cosinor method showed that a circadian rhythm was detectable for only 26 out of the 56 time series (46.4%). Power spectrum analysis gave almost the same figure (19 out of 48: 39.5%) with regard to rhythms with τ=24hr indicating that with one exception (subject JFL) rhythms were internally desynchronized including differences τ between right and left hand grip strength rhythms for three subjects. Results suggest: (a) a physiologic synchronization of circadian rhythms may be a predictor of good performance; (b) however, internal desynchronization as shown previously may be a trivial phenomenon and thus does not imply in itself alterations of either health or performance; (c) chronobiologic methods should be recommended for a better understanding of changes in performance by those participating in competitive and other sports.     

Effect of trophic conditions on aspartate transamination in wheat plants

The enzymatic transamination reactions between aspartic and α-ketoglutaric acid and between aspartic and pyruvic acid were studied in fresh dialysed extracts of young wheat plants cultivated under various trophical conditions, in mineral solution (Knop), in the solution of an soil organic substance (potassium humate) and without nutrients (H₂O). Simultaneously, the level of endogenic aspartic acid, glutamic acid and the growth values were determined. The enzymatic reactions were characterized by determining the optimum pH, the time course, and the effect of coenzyme and of inhibitors. The activity of the aspartate-glutamate transaminase from the root system of plants was considerably higher than the activity of the overground organs. The enzymatic activity from both parts of the plant was inversely proportional to the growth rate: intensive growth of the plants from the Knop variant was connected with their low enzymatic activity; the level of endogenic glutamic acid was high. The slow growth of the plants without nutrients was connected with a higher enzymatic activity; the level of endogenic glutamic acid was low. The plants from the potassium humate variant had an intermediate position between these two variants from the point of view of growth as well as from that of enzymatic activity. The plants with insufficient nutrition (slow growth, low level of endogenic glutamic acid) apparently have a low capacity for supplementing the glutamic acid deficit, which is essential for the metabolic processes, by increasing the activity of the reactions leading to glutamic acid synthesis (Asp-Glu) and, on the other hand, by decreasing the reactions utilizing it (Glu-Ala). For wheat plants the active aspartate-glutamate reaction is obviously physiologically more important than the direct reaction glutamate-aspartate and the reaction aspartate-alanine which in all cases had a very low activity.     

Influence of the soya meal fractions on gibberellic acid and gibberellin A production in submerse cultivation ofGibberella fujikuroi

The relationship of different soya meal components to gibberellin (GA) production was studied. Fluorometric assay confirmed that under the given fermentation conditions, only gibberellic acid (GA₃) was synthesized on medium containing corn steep. On substituting soya flour for corn steep, the same amount of GA₃ was produced and in addition gibberellin A (GA₁) was formed. The GA₃: GA₁ ratio was 1∶1. The course of fermentation in media containing the soya meal protein fraction (fraction I), the soya meal amino acid complex, the corn steep amino acid complex and individual amino acids (γ-aminobutyric acid or tryptophane) was the same as in the control medium containing soya meal. The soya meal fraction II, which is characterized by a high cellulose and carbohydrate content, raised GA production by 25% as compared with production in medium containling soya meal; it simultaneously stimulated GA₃ production, so that the final GA₃: GA₁ ratio was 4∶1.