tetano
Editor, Senior Moderator
J Virol. 2014 Dec 24. pii: JVI.02693-14. [Epub ahead of print]
[h=1]Intracellular expression of camelid single domain antibodies specific for influenza virus nucleoprotein (NP) uncovers distinct features of its nuclear localization.[/h] Ashour J[SUP]1[/SUP], Schmidt F[SUP]1[/SUP], Hanke L[SUP]1[/SUP], Cragnolini J[SUP]1[/SUP], Cavallari M[SUP]1[/SUP], Altenburg A[SUP]1[/SUP], Brewer R[SUP]1[/SUP], Ingram J[SUP]1[/SUP], Shoemaker C[SUP]2[/SUP], Ploegh H[SUP]3[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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 flu-encoded proteins. We generated a panel of camelid-derived single domain antibody fragments (VHHs) against influenza nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza 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 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.
[h=4]IMPORTANCE:[/h] Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function, and how to disrupt said function through pharmaceutical intervention, is difficult. We present a novel method based on single domain antibody technology that permits specific targeting and disruption of an essential flu protein in the absence of genetic manipulation of the flu 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.
Copyright ? 2014, American Society for Microbiology. All Rights Reserved.
PMID: 25540369 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/25540369
[h=1]Intracellular expression of camelid single domain antibodies specific for influenza virus nucleoprotein (NP) uncovers distinct features of its nuclear localization.[/h] Ashour J[SUP]1[/SUP], Schmidt F[SUP]1[/SUP], Hanke L[SUP]1[/SUP], Cragnolini J[SUP]1[/SUP], Cavallari M[SUP]1[/SUP], Altenburg A[SUP]1[/SUP], Brewer R[SUP]1[/SUP], Ingram J[SUP]1[/SUP], Shoemaker C[SUP]2[/SUP], Ploegh H[SUP]3[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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 flu-encoded proteins. We generated a panel of camelid-derived single domain antibody fragments (VHHs) against influenza nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza 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 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.
[h=4]IMPORTANCE:[/h] Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function, and how to disrupt said function through pharmaceutical intervention, is difficult. We present a novel method based on single domain antibody technology that permits specific targeting and disruption of an essential flu protein in the absence of genetic manipulation of the flu 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.
Copyright ? 2014, American Society for Microbiology. All Rights Reserved.
PMID: 25540369 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/25540369