Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses

Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.     

The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function

Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme.     

c-Cbl is not required for ERK1/2-dependent degradation of BimEL

Bim_EL the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim_EL. It has been proposed that the RING finger protein Cbl binds to Bim_EL and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim_EL is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim_EL turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim_EL is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim_EL does not involve its tyrosine phosphorylation; indeed, Bim_EL is not a tyrosine phosphoprotein. Furthermore, Bim_EL fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim_EL turnover proceeds normally in Cbl-containing or Cbl−/− fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim_EL turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.     

Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.     

Calpain activation is not required for AIF translocation in PARP-1-dependent cell death (parthanatos)

Apoptosis-inducing factor (AIF) is critical for poly(ADP-ribose) polymerase-1 (PARP-1)-dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell-free system in intact cells under conditions where endogenous calpain was activated by either NMDA or N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62-kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA- or MNNG-induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP-1 inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-iso-quinolinone. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.     

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.     

Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.     

RNA helicase A is not required for RISC activity

It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects.     

Preannealing of poly(A) template and oligo(dT) primer is not required for hepatitis C virus RNA-dependent RNA polymerase activity

Preannealed homopolymeric DNAs or RNAs are often used as templates and/or primers to characterize activities of DNA or RNA-dependent RNA polymerases. Based on the calculated melting temperatures (T_m values), however, poly(A)/oligo(dT_12–18) is not expected to form stable duplexes. To determine this, we compared the enzymatic activity of hepatitis C virus polymerase using poly(A)/oligo(dT₁₂) that were or were not preannealed. No significant differences were observed. These results suggest that it is not necessary to perform preannealing reactions for poly(A) and oligo(dT₁₂), making it possible to characterize mechanism of inhibition of NS5B inhibitors against either template RNA poly(A) or primer oligo(dT₁₂) independently.     

The 24,000 Da subunit is not required for the RNA synthesis activity of chicken leukemia RNA polymerase II

The partially purified RNA polymerase II from chicken leukemia cells (Chuang R. Y., Chuang L. F. & Israel M. (1986) Biochem. Pharmacol. 35, 1293-1297) contained multiple subunits with molecular masses (in Da) ranging from 220,000 to 24,000, as shown by SDS-polyacrylamide gel electrophoresis. The enzyme was further purified through phosphocellulose column and fractions containing the enzyme activity were collected and concentrated 400-fold through a microconcentrator. The microconcentrator contained a membrane with a molecular weight cutoff around 30,000 and, hence, removed the 24,000 Da polypeptide from the enzyme. It was found that the resulting enzyme retained all the catalytic activity as compared to the enzyme preparation before the concentration step, suggesting that the stoichiometric amount of the 24,000 Da polypeptide is not required for RNA synthesis activity with a denatured DNA template.     

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.     

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.     

Protein synthesis is not required for the inhibitory effect of selenite on cell colony formation and RNA synthesis

Selenite has been shown to undergo intracellular metabolism that results in its conversion to other low molecular weight Secontaining species and also to its incorporation into a selenocysteine residue in selenoprotein. In order to investigate whether the incorporation into protein is required for the cytotoxic effects of selenite, we have examined whether inhibition of protein synthesis prevents the inhibitory effect of selenite on the ability of cells to form colonies or to synthesize RNA. We have found that treatment of HeLa cells with cycloheximide inhibited protein synthesis by >90% but had no effect on the inhibitory effect of selenite on cell colony formation or RNA synthesis. Since protein synthesis is not necessary for these cytotoxic effects of selenite they are unlikely to result from an increase in the synthesis of selenoproteins.     

Preproenkephalin is a Th2 cytokine but is not required for Th2 differentiation in vitro.

Preproenkephalin (PPNK) mRNA expression has been detected in many cells of the immune system, including monocytes and lymphocytes. In the present paper, the expression of PPNK mRNA in purified CD4+ Th1 and Th2 lymphocyte subpopulations is investigated and correlated with the presence of the opioid neuropeptides leu- and met-enkephalin. We found that PPNK mRNA and met-enkephalin were present at higher levels in the Th2 cultures compared with the Th1 cultures. Lymphocytes from PPNK-deficient mice were then used to look at the role of PPNK in Th2 lymphocyte differentiation. Lymphocytes from these mice could be driven into a Th2 phenotype, suggesting that cultures containing IL-4 do not require PPNK for Th2 differentiation.