Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

Abstract

dNovel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as "molecular staples" that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early-and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

“…In the samples that had been selectively precipitated with the Rab11a-specific antibody, PB2 (as proxy of vRNPs) was only detected in infected cells, and the levels increased during infection (Fig. 4A), corroborating findings that Rab11 is able to attach to vRNPs (Amorim et al, 2011(Amorim et al, , 2013Eisfeld et al, 2011;Momose et al, 2011). Conversely, FIP2 was present in non-infected cells, and its intensity decreased as infection progressed.…”

“…By this method, individual vRNPs are 'stuck' by nucleozin, causing their aggregation in the cytoplasm (Fig. 5C, and for detailed description on nucleozin readers are referred to Amorim et al, 2013). Furthermore, because nucleozin has affinity for nucleoprotein and not Rab11, its action is specific to infected cells.…”

“…Furthermore, because nucleozin has affinity for nucleoprotein and not Rab11, its action is specific to infected cells. Interestingly, the 3D forms created included Rab11 and vRNPs but excluded markers from several organelles, including GM130 (Golgi) and calnexin (endoplasmic reticulum) (Amorim et al, 2013). By performing confocal inspection, infected cells exhibited 3D forms of Rab11-vRNP vesicles (Amorim et al, 2013) upon nucleozin incubation (Fig.…”

“…HeLa cells were concurrently transfected with GFP-nucleoprotein and infected with PR8. This method has been established and characterized previously (Amorim et al, 2013), but for clarity will be briefly explained here. In uninfected cells, GFPnucleoprotein, which contains several nuclear localization signals, is found exclusively in the nucleus.…”

“…To demonstrate the accuracy of the CLEM alignments, we thought of using a strategy that altered the distribution of Rab11 vesicles, far more than normal infection, therefore artificially creating dramatic 3D forms that span the entire volume of the cell (Amorim et al, 2013). If the forms captured by confocal microscopy matched in shape and volume the ultrastructural features found in electron micrographs, our system would be validated.…”