Genomic RNAs of Borna disease virus are elongated on internal template motifs after realignment of the 3' termini.

The terminal structures of the Borna disease virus (BDV) genome (vRNA) and antigenome (cRNA) differ from those of other negative strand RNA viruses, as both molecules possess four nucleotides at the 3' terminus without an apparent template at the 5' end of the opposite strand. Consequently, the v- and cRNA molecules are not perfect mirror images, a situation that is not compatible with conventional strategies to maintain genetic information. We show here that recombinant viruses recovered from cDNA lacking the nontemplated nucleotides efficiently reconstitute the 3' overhangs. Analyses of recombinant viruses encoding genetic markers in potential alternative template sequences demonstrated that the BDV v- and cRNA molecules are extended by a realign-and-elongation process on internal template motifs located in close proximity to the 3' ends of v- and cRNA, respectively. The data further suggest that cRNA elongation is restricted to a single template motif of the nascent strand, whereas elongation of vRNA might use multiple template motifs. We propose that the elongation of the 3' termini supports the terminal integrity of the genomic RNA molecules during BDV persistence, and furthermore provides an elegant strategy to eliminate the triphosphate groups from the 5' termini of the BDV v- and cRNA without compromising the genetic information of the virus.

Identification of high-affinity PB1-derived peptides with enhanced affinity to the PA protein of influenza A virus polymerase.

The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.

Limited compatibility of polymerase subunit interactions in influenza A and B viruses.

Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A(1-25)) with that of FluB (PB1-B(1-25)) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B(1-25) peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A(1-25) peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.

Identification of a PA-binding peptide with inhibitory activity against influenza A and B virus replication.

There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.

Processing of genome 5' termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction.

Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5' triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5' triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5' monophosphate-specific exonuclease indicates that the RIG-I-activating 5' triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5' end as a strategy of viruses to evade the innate immune response.

RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication.

De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5'-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5' termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.

Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome.

The essential Saccharomyces cerevisiae PRP43 gene encodes a 767-amino acid protein of the DEXH-box family. Prp43 has been implicated in spliceosome disassembly (Arenas, J. E., and Abelson, J. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11798-11802). Here we show that purified recombinant Prp43 is an RNA-dependent ATPase. Alanine mutations at conserved residues within motifs I ((119)GSGKT(123)), II ((215)DEAH(218)) and VI ((423)QRAGRAGR(430)) that diminished ATPase activity in vitro were lethal in vivo, indicating that ATP hydrolysis is necessary for the biological function of Prp43. Overexpression of lethal, ATPase-defective mutants in a wild-type strain resulted in dominant-negative growth inhibition. The ATPase-defective mutant T123A interfered in trans with the in vitro splicing function of wild-type Prp43. T123A did not affect the chemical steps of splicing or the release of mature mRNA from the spliceosome, but it blocked the release of the excised lariat-intron from the spliceosome. We show that the lariat-intron is not accessible to debranching by purified Dbr1 when it is held in the T123A-arrested splicing complex. Our results define a new ATP-dependent step of splicing that is catalyzed by Prp43.