Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for replication

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5' and 3' ends and (ii) base pairing in the stems of both the 5' and 3' hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3' hairpin loop were crucial for promoter activity in vivo. However, the 3' hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3' hairpin loop remains unknown.     

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.     

Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.     

Promoter elements in the influenza vRNA terminal structure.

The role of the partially double-stranded influenza vRNA terminal structure and its constitutive elements as a promoter signal was studied in vivo in a series of nucleotide substitution and insertion derivatives. A series of single and complementary double exchanges restoring intrastrand base pairing shows that a distal promoter element consists of a six-base pair double-stranded RNA rod in long-range complementary interaction. Within the distal element, all base pair positions are freely exchangeable, and hence no nucleotide-specific recognition could be identified. The proximal promoter element consists of nine partially complementary nucleotides at the vRNA 5' and 3' end. The nine plus six base pair panhandle rod of protein-free vRNA is interrupted by a central third element, a single unpaired nucleotide: adenosine 10 or various substitute residues, which appears to cause a bulged conformation in the overall structure. Mutagenization studies in the promoter proximal element indicate that, upon binding to polymerase, nucleotides at positions 2 and 3 interact with positions 9 and 8 within each branch (5' or 3') in short-range base pairing. In this conformation, the intermediate positions 4-7 are exposed as a single-stranded tetra-loop, which includes invariant guanosine residue 5 in the top conformational position of the 5' segment loop. Altogether, the three base paired segments in angular conjunction to each other adopt a conformation that is described in a "corkscrew model" for an activated stage of vRNA/polymerase interaction.     

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.     

Solubilization and promoter analysis of RNA polymerase from rice stripe virus.

The RNA-dependent RNA polymerase associated with rice stripe virus was dissociated from viral RNA (vRNA) by CsCl centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template 50 nucleotides in length carrying the 5'- and 3'-terminal conserved sequences of all four genome RNA segments. A 3'-terminal half molecule of the model template was also active as a template. Hence, we propose that the 3'-terminal conserved sequence serves as a promoter for the rice stripe virus-associated RNA polymerase. The solubilized enzyme, however, was unable to transcribe vRNA. The failure of the solubilized enzyme to transcribe vRNA is discussed in relation to the apparent loss of RNA polymerase activity after treatment of virions with high concentrations of salt.     

The packaging signal of influenza viral RNA molecules.

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5'-bulged promoter structure, as caused by the central unpaired residue A10 in its 5' branch. Upon insertion of two uridine residues in the 3' branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3'-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5'-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 5'A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 5' bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP- or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.     

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.     

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.     

本氏烟Ⅰ型启动子的克隆及其转录起始位点分析

启动子是转录水平上一个重要的调控元件,其决定着基因的表达模式和表达强度。Ⅰ型启动子具有高转录活性和种属间特异性等特点。如将其应用于植物RNA病毒载体表达系统,有利于提高表达系统的表达效率和生物安全性。本氏烟(Nicotiana benthaminana)是一种被广泛地应用于植物生物反应器和植物病理学的模式生物,但是现有核酸数据库中尚没有其Ⅰ型启动子的相关信息。因此,克隆本氏烟Ⅰ型启动子并分析其转录起始位点就具有重要的应用价值。通过半巢式PCR获得了514 bp的本氏烟Ⅰ型启动子序列(KC352713);生物信息学分析初步预测其转录起始位点位于其核心序列TATA(G)TA(N)GGGGG中的第3位A处;通过植物RNA病毒表达载体和5'RACE技术在体内验证本氏烟Ⅰ型启动子转录起始位点与生物信息学预测结果一致。研究结果为深入研究Ⅰ型启动子和构建Ⅰ型启动子介导转录的植物RNA病毒载体表达系统奠定了基础。