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, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and 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 interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin ß RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development.

Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation.

Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo-electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.

Advances in Structural Virology via Cryo-EM in 2022.

In recent years, cryo-electron microscopy (cryo-EM) has emerged as an important standalone technique within structural biology [...].

Borna Disease Virus 1 Phosphoprotein Forms a Tetramer and Interacts with Host Factors Involved in DNA Double-Strand Break Repair and mRNA Processing.

Determining the structural organisation of viral replication complexes and unravelling the impact of infection on cellular homeostasis represent important challenges in virology. This may prove particularly useful when confronted with viruses that pose a significant threat to human health, that appear unique within their family, or for which knowledge is scarce. Among Mononegavirales, bornaviruses (family Bornaviridae) stand out due to their compact genomes and their nuclear localisation for replication. The recent recognition of the zoonotic potential of several orthobornaviruses has sparked a surge of interest in improving our knowledge on this viral family. In this work, we provide a complete analysis of the structural organisation of Borna disease virus 1 (BoDV-1) phosphoprotein (P), an important cofactor for polymerase activity. Using X-ray diffusion and diffraction experiments, we revealed that BoDV-1 P adopts a long coiled-coil α-helical structure split into two parts by an original ß-strand twist motif, which is highly conserved across the members of whole Orthobornavirus genus and may regulate viral replication. In parallel, we used BioID to determine the proximal interactome of P in living cells. We confirmed previously known interactors and identified novel proteins linked to several biological processes such as DNA repair or mRNA metabolism. Altogether, our study provides important structure/function cues, which may improve our understanding of BoDV-1 pathogenesis.