The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

Abstract

Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies.

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity

“…In contrast to conventional antibodies, many VHHs do not require intrachain disulfide bonds to fold and maintain their function in the reducing environment of the cytosol [16][17][18] . To test whether the VHHs we identified could be expressed and bind their target in the mammalian cytosol, we expressed the indicated VHHs, equipped with HA tags, in mouse embryonic fibroblasts (MEFs) and performed immunoprecipitations with anti-HA conjugated beads.…”

“…VHHs were amplified from a cDNA library generated from alpaca lymphocytes and subcloned into pD vector. VHHs enriched after 2 rounds of panning were subcloned to the pHEN6 vector for expression in E. coli and to pINDUCER20 vector for expression in mammalian cells as described [16][17][18] .…”

“…Many VHHs do not require glycosylation or disulfide bond formation to retain their proper fold and can therefore bind to their targets when expressed in the cytosol of mammalian cells. We have used cytoplasmically expressed VHHs to block nuclear import of nuclear protein (NP) during influenza infection 16 , to protect cells from VSV infection 17 , as well as to track the formation of Asc filaments during inflammasome activation 18 . We generated VHHs that target endogenous UBC6e as a new tool to explore its activity.…”

Protein Vaccination Directs the CD4 + T Cell Response toward Shared Protective Epitopes That Can Be Recalled after Influenza Virus Infection

“…Beyond HA, our studies also evaluated epitopes derived from NP, based on several considerations. NP is currently a human vaccine candidate (35)(36)(37)(38) to elicit broadly protective immunity. Also, NP resides in the cytoplasm and nucleus of infected cells (39) and thus might have preferential access to the cytosolic proteasome, be processed after infection by a nonendosomal route, and generate a pattern of peptides distinct from that generated by exogenous protein-based vaccines.…”

“…Independently of the mechanisms involved in the antigen handling, processing, and presentation in vivo that lead to MHC class II peptide display and the recruitment of CD4 ϩ T cells, the results presented here support the use of protein-based vaccines for eliciting protective immunity from influenza virus. Many new protein vaccines, particularly those involving chimeric HA proteins designed to target antibodies reactive to the highly genetically conserved stalk domain (56)(57)(58)(59)(60), as well as other highly conserved viral antigens, such as NP and M1 (35)(36)(37)(38), are being developed. Based on the results presented here, the CD4 ϩ T cells elicited by these protein-based vaccines should help produce neutralizing antibody responses to HA and should also be able to be recruited and deliver an effector function after natural infection.…”