Identification of a PA-Binding Peptide with Inhibitory Activity against Influenza A and B Virus Replication

Wunderlich, Kerstin; Mayer, Daniel; Ranadheera, Charlene; Holler, Anne-Sophie; Mänz, Benjamin; Martin, Aaron; Chase, Geoffrey; Tegge, Werner; Frank, Ronald; Keßler, Ulrich; Schwemmle, Martin

doi:10.1371/journal.pone.0007517

Cited by 80 publications

(102 citation statements)

References 53 publications

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“…The interaction between L protein and these NP sites may be a target for antiviral intervention (42). Among the Old World arenaviruses, the main components of the replication complex have the potential to functionally and physically interact across species boundaries.…”

Section: Discussionmentioning

confidence: 99%

Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Kerber

Rieger

Busch

et al. 2011

J Virol

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Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa, Mopeia virus (MOPV) from EastAfrica, and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Little is known about how the components of the arenavirus replication machinery, i.e., the genome, nucleoprotein (NP), and L protein, interact. In addition, it is unknown whether these components can function across species boundaries. We established minireplicon systems for MOPV and LCMV in analogy to the existing LASV system and exchanged the components among the three systems. The functional and physical integrity of the resulting complexes was tested by reporter gene assay, Northern blotting, and coimmunoprecipitation studies. The family Arenaviridae comprises more than 20 virus species and is divided into the Old World and the New World complexes (9). Main representatives of the Old World complex are the prototype arenavirus lymphocytic choriomeningitis virus (LCMV), Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa (14), and Mopeia virus (MOPV) from East Africa. MOPV is closely related to LASV but, in contrast to the latter, is neither associated with human disease nor pathogenic in animal models (39).The genomes of arenaviruses consist of two negative-strand RNA segments. The large (L) segment and the small (S) segment each contain two genes in opposite orientation separated by an intergenic region (IGR). During genome replication, full-length copies of S and L RNA are synthesized. Viral transcripts contain a cap structure and terminate within the IGR (1). The IGR contains a stable secondary structure and plays a role in both transcription termination and virus assembly (31).The L RNA encodes the small Z protein and the 220-kDa L protein (26). The latter is a multidomain protein (5, 20) that harbors the viral RNA-dependent RNA polymerase (RdRp) (13, 17) and a cap-snatching endonuclease (22,29). It oligomerizes (34) and interacts with the viral promoter sequences (16,20). Z protein functions as a matrix protein (30, 37) and interacts with L protein (19,40). The S RNA encodes the glycoprotein precursor (GPC) and the nucleoprotein (NP). The virus genome, NP, and L protein represent the minimal cis-and trans-acting components required for replication and transcription (15,21,24). NP is the major component of the viral ribonucleoprotein (RNP) complex. It physically associates with itself as well as with L and Z proteins (7,19,23,35). Association with Z protein and with the ALIX/AIP1, an ESCRTassociated host protein, is important for incorporation of NP into virus-like particles (7,23,35,36). In addition, NP acts as an interferon antagonist (27,28). Recently published X-ray crystallographic data revealed a nucleotide binding site in the N terminus and a 3Ј-5Ј exoribonuclease in the C terminus of NP; the latter is critical for NP to function as an interferon antagonist (18,32).While a substantial amount of information has been accumulated on the individual components of the replication mac...

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Section: Discussionmentioning

confidence: 99%

Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Kerber

Rieger

Busch

et al. 2011

J Virol

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“…The NS1-and NEP-coding plasmids were cloned using the gene-specific forward and reverse primers for NS1 and NEP and cloned into pCAGGS vectors using NotI and XhoI. The pCAGGS expression plasmids encoding the respective C-terminally HA-tagged polymerase subunits (PA, PB1, PB2) or PB2 truncation mutants were generated by PCR and ligated into pCAGGS PA SC35M -HA vector 29 linearized with XmaI and NotI. The N-terminal Strep-tag 20 was cloned in pCAGGS-HA-NEP KAN − 1 expression plasmid linearized with EcoRI and NotI.…”

Section: Methodsmentioning

confidence: 99%

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

et al. 2012

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Infection of mammals by avian influenza viruses requires adaptive mutations to achieve high-level replication in the new host. However, the basic mechanism underlying this adaptation process is still unknown. Here we show that avian polymerases, lacking the human signature PB2-E627K, are incapable of generating usable complementary RnA templates in cultured human cells and therefore require adaptation. Characterization of the highly pathogenic human H5n1 isolate A/Thailand/1(KAn-1)/2004 that retained the avian PB2-E627 reveals that the defect in RnA replication is only partially compensated by mutations in the polymerase. Instead, mutations in the nuclear export protein are required for efficient polymerase activity. We demonstrate that adaptive mutations in nuclear export proteins of several human isolates enhance the polymerase activity of avian polymerases in human cultured cells. In conclusion, when crossing the species barrier, avian influenza viruses acquire adaptive mutations in nuclear export protein to escape restricted viral genome replication in mammalian cells.

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“…The holoenzyme further assembles with genomic RNA and NP to form higher-order vRNPs. Defects or the intentional perturbation of any of these assembly steps can impair polymerase activity (9,11,(38)(39)(40). Polymerase activity was reconstituted with the P[P/F]AAAPP or basic face mutants, and immunoprecipitation assays were used to test whether impaired polymerase activity resulted from defects in the assembly process.…”

Section: G T D T Q I I K L L P a A A P P Q S R M Q S S L T V N V R V mentioning

confidence: 99%

Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

Kirui

Bucci

Poole

et al. 2014

J Virol

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Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/ P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals.The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

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Identification of a PA-Binding Peptide with Inhibitory Activity against Influenza A and B Virus Replication

Cited by 80 publications

References 53 publications

Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

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