Nucleozin targets cytoplasmic trafficking of viral ribonucleoprotein-Rab11 complexes in influenza A virus infection

Abstract

Novel 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.

Fig 1

Time-of-addition experiments examining the effect of nucleozin on IAV replication and macromolecular synthesis. (A) A549 cells were infected with either the WT or the NP Y289H mutant virus, and 1 μM nucleozin (NCZ) was added at the indicated time points. Supernatants were collected and plaque titrated in MDCK cells. Virus titers (white bars) are expressed as the mean percentage ± the standard deviation (n = 3). (B) Cell lysates were analyzed by Western blotting for the indicated proteins. (C) Total cellular RNA was extracted and analyzed by radioactive primer extension, urea-PAGE and autoradiography for mRNA and vRNA from segments 3 and 5, as well as 5S rRNA. (D) vRNA accumulation was quantified by densitometry of primer extension autoradiographs with ImageJ and plotted as the mean percentage of the amount from untreated cells at 8 hpi ± the standard deviation (n = 3).

Fig 2

Nucleozin induces cytoplasmic aggregates containing RNP and Rab11. A549 cells were infected with the WT virus or mock infected and either left untreated or treated with 1 μM nucleozin as shown. Samples were fixed at the times shown and stained for Rab11 and NP (A) or processed for FISH for the indicated vRNAs (B) before imaging by confocal microscopy. Merged images include a 4′,6-diamidino-2-phenylindole (DAPI) channel shown in blue. Scale bars, 10 μm. Images are representative of three independent experiments.

Fig 3

Nucleozin treatment does not disrupt RNPs. 293T cells were transfected with plasmids expressing GFP alone or GFP-NP, incubated for 24 h, and infected with the WT virus or mock infected. At 6 hpi, one set of cells was treated with 1 μM nucleozin (NCZ). Cell lysates prepared at 8 hpi were analyzed by Western blotting for the indicated polypeptides after GFP-Trap affinity selection into supernatant and bound fractions. Sample loading is such that the supernatants are equivalent to 1/10 of the bound fractions. RNA was analyzed by primer extension for segment 3 vRNA and 5S rRNA before or after GFP-Trap selection. The experimental procedure was performed twice. Cont., control.

Fig 4

Live cell trafficking of GFP-NP and RFP-Rab11. A549 cells were transfected with GFP-NP and RFP-Rab11 and 12 h later infected with WT PR8 or mock infected before imaging under time-lapse conditions (approximately every 4 s) at 8 hpi. Selected still images are shown. Arrows indicate the time of drug addition (1 μM). (A) Mock-infected cell. (B) Infected cell without drug treatment. (C) Infected cell with nucleozin treatment. Scale bars, 10 μm. Images were acquired with an SPE confocal microscope, and images were processed with LAS AF Lite. See Movies S1 to S3 in the supplemental material.

Fig 5

Nucleozin does not disrupt the main exocytic pathway. (A to C, E, F) A549 cells were infected with the WT virus, and at 6 hpi, 1 μM nucleozin (NCZ) was added to one set. Samples were fixed at 8 hpi and stained for NP and calnexin (A); NP and GM130 (B); NP and clathrin, EEA1, or LAMP1 in the presence of nucleozin (C); M2 (E); HA (PR8) and NA (F); or NP and α-tubulin (α-tub) (G). Merged images include a DAPI channel shown in blue. Scale bars, 10 μm unless indicated otherwise. In panel D, lysates from cells treated and harvested as indicated were analyzed by Western blotting for HA, M2, and (as a loading control) α-tubulin (α-tub). Images are representative of three independent experiments.

Fig 6

TEM visualization of budding virions in nucleozin-treated cells. A549 cells were infected with WT or NP Y289H mutant virus, treated with the drug, and fixed at the time points shown before imaging by TEM. Arrowheads indicate defective budding events (A) Representative images. Scale bars, 100 nm. (B) Numbers of WT budding virions were calculated over the whole surface of one side of the cell. (C) Sizes of budding virions from cells infected with WT PR8 were determined (as shown) for at least 30 particles from three independent experiments and plotted as the average ± the standard error of the mean. Nonparametric statistical analysis values obtained by Kruskal-Wallis analysis of variance with a significance of 95% were calculated with GraphPad Prism. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no statistically significant difference. (D) Gallery of representative budding events with or without nucleozin (NCZ) treatment (1 μM, added at the indicted times postinfection). Arrows indicate defective particles. Images were compiled from three independent experiments.