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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 β 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. Author summary: The 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 phages library 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 activity. This nanobody recognizes the PA subunit and the PA-PB1 dimer with high affinity. The VHH binds an epitope overlapping or close to the binding sites of ANP32A and RNA-polymerase II, two key cell proteins involved in FluPol replication and transcription activities, respectively. Furthermore, the VHH was found to interfere with the transport of the PA-PB1 dimer into the nucleus. Thus, the high inhibitory activity of the VHH may result from diverse properties, altering FluPol subunits trafficking inside the cell and FluPol transcription and replication. Targeting the VHH binding site may constitute a powerful strategy to develop new antivirals. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions. [ABSTRACT FROM AUTHOR]

Bacteriophages are also challenging objects for crystallization with their large icosahedral-capsid-harboring tails of varying length and flexibility. Genome packaging is highly studied for bacteriophages and herpesviruses and plays a pivotal role in protecting, transporting and facilitating genetic exchange. Sci. 2017; 33: 1111-1117 3 Durieux Trouilleton Q., Barata-Garcia S., Arragain B., Reguera J., Malet H. Structures of active Hantaan virus polymerase uncover the mechanisms of Hantaviridae genome replication. [Extracted from the article]

This paper focuses on the nucleoprotein (NP) of the newly identified member of the Orthomyxoviridae family, Influenza D virus. To date several X-ray structures of NP of Influenza A (A/NP) and B (B/NP) viruses and of infectious salmon anemia (ISA/NP) virus have been solved. Here we purified, characterized and solved the X-ray structure of the tetrameric D/NP at 2.4 Å resolution. The crystal structure of its core is similar to NP of other Influenza viruses. However, unlike A/NP and B/NP which possess a flexible amino-terminal tail containing nuclear localization signals (NLS) for their nuclear import, D/NP possesses a carboxy-terminal tail (D/NPTAIL). We show that D/NPTAIL harbors a bipartite NLS and designed C-terminal truncated mutants to demonstrate the role of D/NPTAIL for nuclear transport. [ABSTRACT FROM AUTHOR]

Influenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility. This tail contains the Nuclear Location Signal (NLS). The nuclear trafficking of the viral components mobilizes cellular import factors at different stages, making these host-pathogen interactions promising targets for new therapeutics. NP is imported into the nucleus by the importin-α/β pathway, through a direct interaction with importin-α isoforms. Here we provide a combined nuclear magnetic resonance and small-angle X-ray scattering (NMR/SAXS) analysis to describe the dynamics of the interaction between influenza B NP and the human importin-α. The NP of influenza B does not have a single NLS nor a bipartite NLS but our results suggest that the tail harbors several adjacent NLS sequences, located between residues 30 and 71.

tRNAs occupy a central role in the cellular life, and they are involved in a broad range of biological processes that relies on their interaction with proteins and RNA. Crystallization and structure resolution of tRNA or/and tRNA/partner complexes can yield in valuable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA, is often challenging. Here we review two methods to produce and purify tRNA in quantity and quality to perform X-ray studies.

Transcription and replication by influenza virus are carried out by protein-RNA complexes named RNPs. There are eight of these complexes, each containing one of the eight segments of viral RNA, multiple copies of the viral nucleoprotein and each complex carries a copy of the viral RNA-dependent RNA polymerase. The polymerase itself is a complex of three subunits: PB1, PB2 and PA. Through an effort by laboratories from all over the world, atomic structures have been determined of nucleoproteins of several viral strains and of protein domains of PA and PB2. For PB1, only the structures of the small interfaces with PA and PB2 have been determined. Even though a full understanding of the fundamental processes in the viral life cycle is still lacking, the structures have revealed how nucleoprotein can oligomerize and binds to RNA, how PB1 binds to PA and how the polymerase binds to capped cellular pre-messenger RNA (mRNA) and cleaves this RNA in order to make a capped primer for its own mRNAs (cap-snatching mechanism). The structures also stimulated structure-aided drug design efforts and first generation inhibitors against nucleoprotein oligomerization, binding of PB1 to PA and the cap-snatching activity have been published. Such inhibitors may be developed into new anti-influenza drugs.