Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA.

The soluble phase of the cytoplasm of poliovirus-infected cells contains an enzymatic activity able to copy RNA without an added primer. This replicase activity has been purified 60-fold; it is absent from uninfected cells. Poly(U) polymerase activity copurifies with replicase activity. Although less pure replicase fractions copy a variety of RNAs, purer fractions respond better to poliovirus RNA than to other viral RNAs. Even the less pure fractions make a specific copy of the added template, as shown by hybridization of the product to its template RNA but not to other RNAs. Among homopolymers only poly(A)-oligo(U) was copied by the replicase; other primed homopolymer templates were inactive.     

tRNA-like structures tag the 3'' ends of genomic RNA molecules for replication: implications for the origin of protein synthesis.

Single-stranded RNA viruses often have 3'-terminal tRNA-like structures that serve as substrates for the enzymes of tRNA metabolism, including the tRNA synthases and the CCA-adding enzyme. We propose that such 3'-terminal tRNA-like structures are in fact molecular fossils of the original RNA world, where they tagged genomic RNA molecules for replication and also functioned as primitive telomeres to ensure that 3'-terminal nucleotides were not lost during replication. This picture suggests that the CCA-adding activity was originally an RNA enzyme, that modern DNA telomeres with the repetitive structure CmAn are the direct descendants of the CCA terminus of tRNA, and that the precursor of the modern enzyme RNase P evolved to convert genomic into functional RNA molecules by removing this 3'-terminal tRNA-like tag. Because early RNA replicases would have been catalytic RNA molecules that used the 3'-terminal tRNA-like tag as a template for the initiation of RNA synthesis, these tRNA-like structures could have been specifically aminoacylated with an amino acid by an aberrant activity of the replicase. We show that it is mechanistically reasonable to suppose that this aminoacylation occurred by the same sequence of reactions found in protein synthesis today. The advent of such tRNA synthases would thus have provided a pathway for the evolution of modern protein synthesis.     

Structure of the Poly(G) Polymerase Component of the Bacteriophage f2 Replicase

A rifampicin-resistant poly(G) polymerase has been purified from f2 sus 11-infected cells. The poly(G) polymerase is believed to represent part of the f2 replicase on the basis of several criteria. It is present only in infected cells and shares the characteristic rifampicin resistance of crude f2 replicase activity. Partially purified poly(G) polymerase preparations exhibit replicase activity, synthesizing f2 "lus"strand RNA from denatured, partially double-stranded f2 RNA template. Highly purified poly(G) polymerase preparations, although lacking replicase activity, contain a protein which is electrophoretically identical to the protein product of the viral replicase cistron.     

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.     

Amplifiable messenger RNA.

RNA molecules were prepared that consisted of an mRNA encoding chloramphenicol acetyltransferase embedded within the sequence of midivariant RNA, which is a template for the RNA-directed RNA polymerase Q beta replicase. These recombinant RNAs were shown to be bifunctional: they are amplified exponentially by incubation with Q beta replicase, and the replicated RNA serves as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase. The availability of amplifiable mRNAs will enable relatively large amounts of protein to be synthesized in vitro.     

Minimal requirements for template recognition by bacteriophage Qβ replicase: Approach to general RNA-dependent RNA synthesis

Any oligo- or polynucleotide able to offer a C-C-C-sequence at the 3′-terminus and a second C-C-C-sequence in a defined steric position to Qβ replicase is an efficient template. Corresponding chemical modifications convert non-template RNAs to template RNAs.     

Initiation of adenovirus DNA replication in vitro requires a specific DNA sequence.

Initiation of adenovirus DNA replication in vitro occurs on a linearized plasmid DNA containing 3,327 base pairs of the adenovirus terminal sequence. Various deletions have been constructed in the plasmid DNA and their template activities examined. Deletions from an internal restriction enzyme cleavage site that retain only 20 base pairs or more of the adenovirus terminal sequence support initiation and limited chain elongation, whereas deletions that leave 14 base pairs or less of the terminal sequence do not. On the other hand, all deletions extending from the very terminus of the adenovirus DNA destroy the template activity. The terminal 20 base pairs of adenovirus DNA contain a sequence A-T-A-A-T-A-T-A-C-C, which is perfectly conserved in the DNAs from different serotypes of human adenovirus. Base changes within the conserved sequence greatly reduce the template activity. These results suggest that the terminal 20 base pairs constitute a functional origin for the initiation of adenovirus DNA replication in vitro.     

RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

Much work has been done on the isolation, purification, and characterization of the RNA-directed RNA polymerase (EC 2.7.7.48) of cucumber mosaic virus (CMV)-infected cucumbers. Uninfected plants were reported to have no such enzyme, but we recently detected low levels of the activity in cucumber. Since tobacco and cowpea contain such an enzyme that is variably increased in amount by various virus (as well as viroid) infections, we assumed that this would also be the case upon CMV infection of cucumber. However, further purification and characterization of the RNA-directed RNA polymerases from healthy and from infected cucumber suggests that these are different enzymes. The presumed CMV replicase was obtained pure and consists of a major polypeptide of Mr 100,000 and minor components of Mr 110,000 and about 10,000. The Km is 5 microM ([3H]GTP) when tobacco mosaic virus RNA is used as template.     

In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70

To gain insights into the functions of a viral RNA replicase, we have assembled in vitro and entirely from nonplant sources, a fully functional replicase complex of Tomato bushy stunt virus (TBSV). The formation of the TBSV replicase required two purified recombinant TBSV replication proteins, which were obtained from E. coli, the viral RNA replicon, rATP, rGTP, and a yeast cell-free extract. The in vitro assembly of the replicase took place in the membraneous fraction of the yeast extract, in which the viral replicase-RNA complex became RNase- and proteinase-resistant. The assembly of the replicase complex required the heat shock protein 70 (Hsp70 = yeast Ssa1/2p) present in the soluble fraction of the yeast cell-free extract. The assembled TBSV replicase performed a complete replication cycle, synthesizing RNA complementary to the provided RNA replicon and using the complementary RNA as template to synthesize new TBSV replicon RNA.     

Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.     

Reticulocyte RNA-dependent RNA polymerase

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.     

Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion

RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3′ terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at ∼60% of WT levels, and its product was found to be initiated at the WT position (−1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved.     

Isolation and Properties of RNA Replicases Induced by SP and FI Phages

New RNA replicases were isolated and purified from Escherichia coli Q13 infected with SP or FI phages showing different serological properties. These replicases showed a template specificity different from that of Qbeta replicase.     

Antibody to threonyl-transfer RNA synthetase in myositis sera

The prevalence and clinical correlations of anti-threonyl-transfer RNA synthetase (anti-PL-7), as well as the relationship of anti-PL-7 to anti-histidyl-transfer RNA synthetase (anti-Jo-1) were studied in 109 sera from patients with myositis. Inhibition of threonine aminoacylation was used to screen for anti-PL-7. Sera from 3 patients, 2 with polymyositis and 1 with polymyositis-overlap syndrome, and a fourth serum from a patient with dermatomyositis, which was previously found to contain anti-PL-7, inhibited greater than 90% of activity (3.7% of 109 sera). All 4 sera reacted strongly in an enzyme-linked immunosorbent assay with enzyme that was either affinity purified with anti-PL-7 or was biochemically purified. There was no indication of cross-reactivity by aminoacylation inhibition or, for most sera, by enzyme-linked immunosorbent assay. Anti-PL-7 is an uncommon myositis-associated antibody that is independent of anti-Jo-1, but is directed at a functionally related enzyme.     

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains.

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1-2 kcal.mol(-1) of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.