Picornavirus genome replication: assembly and organization of the VPg uridylylation ribonucleoprotein (initiation) complex

All picornaviruses have a protein, VPg, covalently linked to the 5'-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a "slide-back" mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication.     

Picornavirus RNA translation: roles for cellular proteins

Picornavirus RNA is translated within cells even when cellular cap-dependent protein synthesis is blocked. The efficiency of recognition of the viral RNA by the translational apparatus can determine viral tropism. The roles of cellular translation-initiation factors and other RNA-binding proteins in viral RNA-mediated protein synthesis are discussed.     

Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.     

Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex

Picornaviruses have a peptide termed VPg covalently linked to the 5'-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C(2)-3Dpol complex that extrapolates well to all picornaviruses.     

Identification of the amino acid residue involved in rabbit hemorrhagic disease virus VPg uridylylation

The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector. The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from [alpha-32P]UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA. The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome. These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family.     

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.     

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.     

Situational repair of replication forks: roles of RecG and RecA proteins

Replication forks often stall or collapse when they encounter a DNA lesion. Fork regression is part of several major paths to the repair of stalled forks, allowing nonmutagenic bypass of the lesion. We have shown previously that Escherichia coli RecA protein can promote extensive regression of a forked DNA substrate that mimics a possible structure of a replication fork stalled at a leading strand lesion. Using electron microscopy and gel electrophoresis, we demonstrate that another protein, E. coli RecG helicase, promotes extensive fork regression in the same system. The RecG-catalyzed fork regression is very efficient and faster than the RecA-promoted reaction (up to 240 bp s(-1)), despite very limited processivity of the RecG protein. The reaction is dependent upon ATP hydrolysis and is stimulated by single-stranded binding protein. The RecA- and RecG-promoted reactions are not synergistic. In fact, RecG functions poorly under the conditions optimal for the RecA reaction, and vice versa. When both RecA and RecG proteins are incubated with the DNA substrate, high RecG concentrations inhibit the RecA protein-promoted fork regression. The very different reaction profiles may reflect a situational application of these proteins to the rescue of stalled replication forks in vivo.     

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.     

Structure-function relationships of the RNA-dependent RNA polymerase from poliovirus (3Dpol). A surface of the primary oligomerization domain functions in capsid precursor processing and VPg uridylylation

The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication.     

DNA precursor asymmetries, replication fidelity, and variable genome evolution.

Balanced pools of deoxyribonucleoside triphosphates (dNTPs) are essential for DNA replication to occur with maximum fidelity. Conditions that create biased dNTP pools stimulate mutagenesis, as well as other phenomena, such as recombination or cell death. In this essay we consider the effective dNTP concentrations at replication sites under normal conditions, and we ask how maintenance of these levels contributes toward the natural fidelity of DNA replication. We focus upon two questions. (1) In prokaryotic systems, evidence suggests that replication is driven by small, localized, rapidly replenished dNTP pools that do not equilibrate with the bulk dNTP pools in the cell. Since these pools cannot be analyzed directly, what indirect approaches can illuminate the nature of these replication-active pools? (2) In eukaryotic cells, the normal dNTP pools are highly asymmetric, with dGTP being the least abundant nucleotide. Moreover, the composition of the dNTP pools changes as cells progress through the cell cycle. To what extent might these natural asymmetries contribute toward a recently described phenomenon, the differential rate of evolution of different genes in the same genome?     

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.     

Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.     

Polyclonal Antibodies against a Structure Mimicking the Covalent Linkage Unit between Picornavirus RNA and VPg: An Immunochemical Study

We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.     

Determination of influenza virus proteins required for genome replication.

An artificial vaccinia virus vector-driven replication system for influenza virus RNA has been developed. In this system, a synthetic NS-like gene is replicated and expressed by influenza virus proteins supplied through infection with vaccinia virus recombinant vectors. The minimum subset of influenza virus proteins needed for specific replication and expression of the viral ribonucleoprotein was found to be the three polymerase proteins (PB1, PB2, and PA) and the nucleoprotein.     

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.