Mutagenic analysis of the 5' arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity

Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5' arm of the influenza A virus viral RNA (vRNA) was constructed to determine the cis-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5' hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5' terminus, and (iii) the presence of base pair interactions between the 5' and 3' segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.     

Hairpin Loop Structure in the 3′ Arm of the Influenza A Virus Virion RNA Promoter Is Required for Endonuclease Activity

Previous studies have shown that the 5' arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3' arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3' arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3' hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3' arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.     

Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme

DNA polymerase III holoenzyme (holoenzyme) processively and rapidly replicates a primed single-stranded DNA circle to produce a duplex with an interruption in the synthetic strand. The precise nature of this discontinuity in the replicative form (RF II) and the influence of the 5' termini of the DNA and RNA primers were analyzed in this study. Virtually all (90%) of the RF II products primed by DNA were nicked structures sealable by Escherichia coli DNA ligase; in 10% of the products, replication proceeded one nucleotide beyond the 5' DNA terminus displacing (but not removing) the 5' terminal nucleotide. With RNA primers, replication generally went beyond the available single-stranded template. The 5' RNA terminus was displaced by 1-5 nucleotides in 85% of the products; a minority of products was nicked (9%) or had short gaps (6%). Termination of synthesis on a linear DNA template was usually (85%) one base shy of completion. Thus, replication by holoenzyme utilizes all, or nearly all, of the available template and shows no significant 5'----3' exonuclease action as observed in primer removal by the "nick-translation" activity of DNA polymerase I.     

Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs.

The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding.     

Structure of RNAs replicated by the DNA-dependent T7 RNA polymerase.

The DNA-dependent RNA polymerase of bacteriophage T7 efficiently and specifically replicates two structurally related RNAs, termed X and Y RNAs. Replication of both RNAs involves synthesis of complementary strands initiated with pppC and pppG. RNAs transcribed from DNA template containing the established sequences of X and Y RNAs were efficiently replicated by T7 RNA polymerase. Both RNAs possess palindromic sequences with a dual axis of symmetry, permitting formation of hairpin-, dumbbell-, or cloverleaf-type structures. The template must consist of RNA and not DNA sequence, and the terminal unpaired dinucleotides of the RNA are necessary for replication. Nucleotidyl transferase activity of E. coli adenylates the unpaired CCOH dinucleotide at the 3' end of a C strand of X RNA. This feature, as well as the length (64 nucleotides) and compact structure of X and Y RNAs, suggests that they may resemble tRNA molecules and tRNA-like structures at the 3' termini of many plant viral RNA genomes.     

Multiple 5' terminal cap structures in late polyoma virus RNA.

Nuclear and cytoplasmic polyoma virus-specific RNA extracted from 32P-labeled mouse embryo cells late during productive viral infection was analyzed for the presence of 5' terminal capped structures by complete digestion with RNAases T1, T2 and A, followed by two-dimensional electrophoretic fractionation. Seven major cap I structures (m7 GpppNm1pN2p) were observed in both cases. These termini were further characterized by digestion with penicillium nuclease P1, followed by product analysis in a variety of alternative separate systems. Each structure had an individual combination of N1 and N2 nucleotides, where N1 was always a purine nucleotide but N2 was any nucleotide subject to the single exception that m7GpppGmpCp is found only in low yield. Four different cap II derivatives (m7GpppNm1pNm2pN3p) of four of the cap I structures were also detected in cytoplasmic RNA. None of the termini described derived from contaminating host cell RNA. All of these cap structures mapped on the polyoma viral DNA genome between 66 and 71 map units, a region distant from the 5' end of the bodies of two of the three late polyoma mRNAs. All the polyoma virus-specific cap structures, however, were present in each of the purified 16S, 18S and 19s late mRNAs. These data suggested that families of capped leader sequences of varying sizes are attached to the main body of each late polyoma mRNA species by a splicing mechanism.     

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites.

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.     

Adrenocorticotropic hormone stimulation of adrenal RNA polymerase I and III activities. Nucleotide incorporation into internal positions and 3' chain termini.

In the presence of 50 mM (NH4)2SO4 and low concentrations of alpha-amanitin (7.7 mug/ml), adrenal nuclei synthesize predominately rRNA as characterized by size and base composition. Approximately 10% of the RNA synthesized under these conditions sediments at 4-5 S; this RNA synthesizing activity is inhibited by high concentrations of alpha-amanitin (231 mug/ml) indicating the presence of RNA polymerase III activity. ACTH administration to guinea pigs results in a twofold increase in adrenal nuclear RNA polymerase I and III activities at 14 hr of hormone treatment. Analysis of the amount of radiolabeled nucleoside triphosphate incorporated in vitro into 3' chain termini and into internal nucleotide positions has been utilized to measure the number of RNA chains and the average chain length synthesized in vitro. Incorporation into 3' chain termini is not changed by ACTH; incorporation into internal nucleotides is doubled in parallel with the increase in RNA polymerase I activity. These results are not due to an altered Km of RNA polymerase I for the four nucleoside triphosphates, nor to differential R Nase or phosphatase activity. These studies suggest that the regulation of RNA polymerase I by ACTH is accomplished in part through an increase in the rate of RNA chain elongation.     

Promoter analysis of influenza virus RNA polymerase.

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.     

Sequence homologies in mammalian 5.8S ribosomal RNA.

Oligonucleotide products of complete pancreatic or T1 RNase digestion or partial T1 RNase digestion of HeLa cell (human) and MPC-11 cell (mouse) 5.8S rRNA are identical with those obtained from Novikoff hepatoma (rat) 5.8S rRNA except for minor differences at the termini. pCp is the only major 5' terminus of both human and mouse RNAs; both pGp and pCp 5' termini were found in rat 5.8S RNA. Furthermore, HeLa cells contain C-U-U at the 3' end rather than the C-U terminus of mouse and rat. The results indicate that the nucleotide sequence has been highly conserved during the evolution of mammals and suggest that, as reported for 5S rRNA, this sequence is essentially constant throughout the Mammalia.     

Distinct RNA elements confer specificity to flavivirus RNA cap methylation events

The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.