title: Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization.
abstract: Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of influenza virus-encoded proteins. We generated a panel of camelid-derived single-domain antibody fragments (VHHs) against influenza virus nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza virus genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (αNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the viral ribonucleoprotein (vRNP) complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle.
  
  
  OBJECTIVE
  Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function and determining how to disrupt said function through pharmaceutical intervention are difficult. We present a novel method based on single-domain-antibody technology that permits specific targeting and disruption of an essential influenza virus protein in the absence of genetic manipulation of influenza virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Ashour
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Florian I
authors.initials: FI
authors.lastname: Schmidt
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Leo
authors.initials: L
authors.lastname: Hanke
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Juanjo
authors.initials: J
authors.lastname: Cragnolini
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Cavallari
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Arwen
authors.initials: A
authors.lastname: Altenburg
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Rebeccah
authors.initials: R
authors.lastname: Brewer
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ingram
authors.affiliation: Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Shoemaker
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA ploegh@wi.mit.edu.
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry* / Q000821:virology*; D004285:Dogs; D015870:Gene Expression; D009980:Influenza A virus / Q000502:physiology*; D008967:Molecular Biology / Q000379:methods; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins / Q000032:analysis*; D061905:Single-Domain Antibodies / Q000235:genetics / Q000378:metabolism*; D014758:Viral Core Proteins / Q000032:analysis*; D014773:Virology / Q000379:methods*; D014779:Virus Replication
chemical_list: C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D061905:Single-Domain Antibodies; D014758:Viral Core Proteins