Biology subjects

Crepin, T.

Publications and source records attributed to Crepin, T..

3 recordsLinked to original sources

bioRxiv2024

Inhibition of influenza virus replication by artificial proteins (αReps) targeting its RNA-polymerase

Seasonal epidemics and pandemics caused by influenza A viruses still represent a main public health burden in the world. Influenza viruses replicate and transcribe their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol) through extensive structural rearrangements and differential interactions with host cell factors. To get insights into its functioning, we screened a phage-display library of biosynthetic proteins (named Reps and build on a rigid alpha-helicoidal HEAT-like scaffold) against the structurally invariant FluPol core and several flexibly-linked domains of the FluPol PB2 subunit. Several Reps specific of the cap binding domain [CBD], the 627-domain and the NLS domain of the PB2 FluPol subunit displayed FluPol inhibitory and virus neutralization activities when transiently expressed in the cytosol. Furthermore, intracellular ectopic inducible expression of the Reps C3 and F3 (specific of the CBD and the 627-domain, respectively) in influenza virus permissive cells blocked transcription and multiplication of viruses representative of the H1N1, H3N2 and H7N1 subtypes, even when induced at late times post-infection. A synergic inhibitory effect on FluPol activity and virus multiplication was evidenced when the two Reps were covalently linked. These results suggest that i) interfering with FluPol structural rearrangements that are concomitant to its various activities may represent a promising strategy to block virus multiplication and to design new types of antivirals such as dual binders targeting distant sites on FluPol and ii) the 627-domain could be efficiently targeted to design influenza antivirals. Author SummaryThe influenza virus RNA-polymerase (FluPol) ensures genome transcription and replication in the nucleus of the infected cells. To select ligands able to interfere with FluPol functions, we screened a library of phages encoding biosynthetic proteins (named Reps) for binding to FluPol subunits and domains. When expressed intracellularly, several of them display efficient FluPol blocking and virus neutralizing activities. Reps C3 and F3 assembled through covalent linkages blocked FluPol activity more efficiently than their precursors. These Reps impaired multiplication of H1N1, H3N2 and H7N1 viruses, showing that their binding sites may constitute effective targets for new antiviral development.

microbiology↗

bioRxiv2023

Antiviral activity of intracellular nanobodies targeting the influenza virus RNA-polymerase core

Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, several of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) targets PA, but preferentially bind the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to impair the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin {beta} RanBP5 and subsequent steps in FluPol assembly. These data suggest that the VHH16 neutralization activity is likely due to an alteration of the import of the PA-PB1 dimer into the nucleus, resulting to an inhibition of FluPol functioning. VHH16 binding site represent a potential target for antiviral development. Author SummaryThe influenza virus RNA-polymerase (FluPol) ensures genome transcription and replication in the nucleus of the infected cells. To select ligands able to block FluPol activities, we screened a library of phages encoding nanobodies and resulting from the immunization of a llama with FluPol subunits. When expressed intracellularly, one of the nanobodies displays highly efficient FluPol blocking and virus neutralizing activities. This nanobody binds FluPol with high affinity and recognizes preferentially the PA-PB1 assembled subunits. Furthermore, it was found to interfere with the transport of the PA-PB1 dimer into the nucleus, suggesting that targeting FluPol trafficking between the cytoplasm and the nucleus may constitute a powerful strategy to develop new antivirals.

microbiology↗

bioRxiv2023

Multivalent dynamic colocalization of avian influenza polymerase and nucleoprotein by intrinsically disordered ANP32A reveals the molecular basis of human adaptation

Adaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remain poorly understood. Here we characterise ternary complexes formed between ANP32A, FluPol and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted Pol, containing the E627K mutation, simultaneously bind the identical extended linear motif on human ANP32A, in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.

biochemistry↗