Norovirus Genome Circularization and Efficient Replication Are Facilitated by Binding of PCBP2 and hnRNP A1

Sequences and structures within the terminal genomic regions of plus-strand RNA viruses are targets for the binding of host proteins that modulate functions such as translation, RNA replication, and encapsidation. Using murine norovirus 1 (MNV-1), we describe the presence of long-range RNA-RNA interactions that were stabilized by cellular proteins. The proteins potentially responsible for the stabilization were selected based on their ability to bind the MNV-1 genome and/or having been reported to be involved in the stabilization of RNA-RNA interactions. Cell extracts were preincubated with antibodies against the selected proteins and used for coprecipitation reactions. Extracts treated with antibodies to poly(C) binding protein 2 (PCBP2) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 significantly reduced the 5′-3′ interaction. Both PCBP2 and hnRNP A1 recombinant proteins stabilized the 5′-3′ interactions and formed ribonucleoprotein complexes with the 5′ and 3′ ends of the MNV-1 genomic RNA. Mutations within the 3′ complementary sequences (CS) that disrupt the 5′-3′-end interactions resulted in a significant reduction of the viral titer, suggesting that the integrity of the 3′-end sequence and/or the lack of complementarity with the 5′ end is important for efficient virus replication. Small interfering RNA-mediated knockdown of PCBP2 or hnRNP A1 resulted in a reduction in virus yield, confirming a role for the observed interactions in efficient viral replication. PCBP2 and hnRNP A1 induced the circularization of MNV-1 RNA, as revealed by electron microscopy. This study provides evidence that PCBP2 and hnRNP A1 bind to the 5′ and 3′ ends of the MNV-1 viral RNA and contribute to RNA circularization, playing a role in the virus life cycle.     

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca²⁺-ATPase. Upon binding to the Ca₂E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca²⁺ transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser¹⁸⁶ and Asp²⁰³ interact with Glu⁴³⁹ (N-domain) and Arg⁶⁷⁸ (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp²⁰³ and Arg⁶⁷⁸ rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser¹⁸⁶ and Glu⁴³⁹. By taking advantage of the ability of wild type and mutant Ca²⁺-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca²⁺-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp²⁰³-Arg⁶⁷⁸ and Ser¹⁸⁶-Glu⁴³⁹ interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser¹⁸⁶-Glu⁴³⁹ bond are mutually exclusive in the E2P ground state.     

The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters

Eukaryotic gene expression starts off from a largely obstructive chromatin substrate that has to be rendered accessible by regulated mechanisms of chromatin remodeling. The yeast PHO5 promoter is a well known example for the contribution of positioned nucleosomes to gene repression and for extensive chromatin remodeling in the course of gene induction. Recently, the mechanism of this remodeling process was shown to lead to the disassembly of promoter nucleosomes and the eviction of the constituent histones in trans. This finding called for a histone acceptor in trans and thus made histone chaperones likely to be involved in this process. In this study we have shown that the histone chaperone Asf1 increases the rate of histone eviction at the PHO5 promoter. In the absence of Asf1 histone eviction is delayed, but the final outcome of the chromatin transition is not affected. The same is true for the coregulated PHO8 promoter where induction also leads to histone eviction and where the rate of histone loss is reduced in asf1 strains as well, although less severely. Importantly, the final extent of chromatin remodeling is not affected. We have also presented evidence that Asf1 and the SWI/SNF chromatin remodeling complex work in distinct parallel but functionally overlapping pathways, i.e. they both contribute toward the same outcome without being mutually strictly dependent.     

Requirement for C-terminal Extension to the RNA Binding Domain for Efficient RNA Binding by Ribosomal Protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

Nucleosome stability at the yeast PHO5 and PHO8 promoters correlates with differential cofactor requirements for chromatin opening

The coregulated PHO5 and PHO8 genes in Saccharomyces cerevisiae provide typical examples for the role of chromatin in promoter regulation. It has been a long-standing question why the cofactors Snf2 and Gcn5 are essential for full induction of PHO8 but dispensable for opening of the PHO5 promoter. We show that this discrepancy may result from different stabilities of the two promoter chromatin structures. To test this hypothesis, we used our recently established yeast extract in vitro chromatin assembly system, which generates the characteristic PHO5 promoter chromatin. Here we show that this system also assembles the native PHO8 promoter nucleosome pattern. Remarkably, the positioning information for both native patterns is specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract does not support proper nucleosome positioning unless supplemented with yeast extract. By competitive assemblies in the yeast extract system we show that the PHO8 promoter has greater nucleosome positioning power and that the properly positioned nucleosomes are more stable than those at the PHO5 promoter. Thus we provide evidence for the correlation of inherently more stable chromatin with stricter cofactor requirements.     

A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the ¹²⁵I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis.