Mutations in the 5' trailer region of a respiratory syncytial virus minigenome which limit RNA replication to one step

The 3' termini of the genomic and antigenomic RNAs of human respiratory syncytial virus (RSV) are identical at 10 of the first 11 nucleotide positions and 21 of the first 26 positions. These conserved 3'-terminal sequences are thought to contain the genomic and antigenomic promoters. Furthermore, the complement of each conserved sequence (i.e., the 5' end of the RNA it encodes) might contain an encapsidation signal. Using an RSV minigenome system, we individually mutated each of the last seven nucleotides in the 5' trailer region of the genome. We analyzed effects of these mutations on encapsidation of the T7 polymerase-transcribed negative-sense genome, its ability to function as a template for RSV-driven synthesis of positive-sense antigenome and mRNA, and the ability of this antigenome to be encapsidated and to function as template for the synthesis of more genome. As a technical complication, mutations in the last five nucleotides of the trailer region were found to affect the efficiency of the adjoining T7 promoter over more than a 10-fold range, even though three nonviral G residues had been included between the core promoter and the trailer to maximize the efficiency of promoter activity. This was controlled in all experiments by monitoring the levels of total and encapsidated genome. The efficiency of encapsidation of the T7 polymerase-transcribed genome was not affected by any of the trailer mutations. Furthermore, neither the efficiency of positive-sense RNA synthesis from the genome nor the efficiency of encapsidation of the encoded antigenome was affected by the mutations. However, nucleotide substitution at positions 2, 3, 6, or 7 relative to the 5' end of the trailer blocked the production of progeny genome, whereas substitution at positions 1 and 5 allowed a low level of genome production and substitutions at position 4 were tolerated. Position 4 is the only one of the seven positions examined that is not conserved between the 3' ends of genomic and antigenomic RNA. The mutations that blocked the synthesis of progeny genome thus limited RNA replication to one step, namely, the synthesis and encapsidation of antigenome. Restoration of terminal complementarity for one of the trailer mutants by making a compensatory mutation in the leader region did not restore synthesis of genomic RNA, confirming that its loss was not due to reduced terminal complementarity. Interestingly, this leader mutation appeared to prevent antigenome synthesis with only a slight effect on mRNA synthesis, apparently providing a dissociation between these two synthetic activities. Genomes in which the terminal 24 or 325 nucleotides of the trailer have been deleted were competent for encapsidation and the synthesis of mRNA and antigenomic RNA, further confirming that terminal complementarity was not required for these functions.     

A single amino acid substitution in the phosphoprotein of respiratory syncytial virus confers thermosensitivity in a reconstituted RNA polymerase system

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.     

A novel mechanism for the initiation of Tacaribe arenavirus genome replication.

The ends of arenavirus genome and antigenome RNAs are highly conserved and where determined directly, always contain a 3' G (referred to as position +1). However, primers extended to the 5' ends of Tacaribe virus genomes and antigenomes extend to position -1. When genomes and antigenomes are annealed either inter or intramolecularly and treated with RNase A or T1, there appears to be a single unpaired G at the 5' ends of the hybrids. A single extra G is also found by cloning the 5' ends of S antigenomes, and studies with capping enzyme detect (p)ppG at the 5' ends of genome and antigenome chains. A model is proposed in which genome replication initiates with pppGpC to create the nontemplated extra G. In contrast, the nontemplated bases at the 5' ends of the N mRNAs, which extend to positions -1 to -5, were found to be capped and also heterogeneous in sequence.     

Molecular genetics of bacteriophage P22 scaffolding protein's functional domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.     

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (~0.4–0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (~0.4–0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4–0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.     

Intracellular vesicular stomatitis virus leader RNAs are found in nucleocapsid structures.

Previous studies demonstrated that cytoplasmic extracts of cells infected with vesicular stomatitis virus contain plus-strand leader RNAs which sediment at 18S on sucrose gradients as a complex with viral N protein. The work presented in this paper demonstrated that these 18S complexes were stable on CsCl density gradients, banding at a buoyant density near that of genome nucleocapsids, and exhibited a morphology in an electron microscope similar to the disk structures found in virus genome nucleocapsids. Minus-strand leader RNAs were also found in 18S complexes on sucrose gradients. Quantitation of intracellular leader RNA suggested that, late in infection, approximately three-quarters of total intracellular leader RNA was encapsidated.     

5'-proximal hot spot for an inducible positive-to-negative-strand template switch by coronavirus RNA-dependent RNA polymerase

Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3' nested set of subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3' end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5'-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3'-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5'-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.     

Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.     

Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.

T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.     

The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication

The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.     

A high-throughput genome-walking method and its use for cloning unknown flanking sequences

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5′ ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5′ end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5′ flanking regions/promoters of selected plant genes.