'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs

Abstract

Many viruses exploit cellular polarity to constrain the assembly and release of progeny virions to a desired surface. Influenza virus particles are released only from the apical surface of epithelial cells and this polarization is partly owing to specific targeting of the viral membrane proteins to the apical plasma membrane. The RNA genome of the virus is transcribed and replicated in the nucleus, necessitating nuclear export of the individual ribonucleoprotein (RNP) segments before they can be incorporated into budding virus particles. We show that the process of polarized virus assembly begins in the nucleus with the RNPs adopting a novel asymmetric distribution at the inner nuclear membrane prior to their export to the cytoplasm. The viral nucleoprotein, the major protein component of RNPs, displays the same polarized intranuclear distribution in the absence of other influenza virus components, suggesting the existence of a hitherto unrecognized polarity within the mammalian cell nucleus.

Figure 1

Polarized intranuclear localization of NP. (a–c) BHK cells were (a) infected and (i) fixed at 3 h p.i. or (ii) treated with 11 nM LMB and fixed at 9 h p.i. before immunofluorescent staining for NP (green) and Nup62 (red). (b,c) 5×10⁴ cells were transfected with (b) 10 or (c) 100 ng of NP plasmid and stained 16 h later for NP (green) and Rch1 (red). (d) NP localization patterns were scored (nuclear (N) diffuse, N-polar or cytoplasmic (C)) through a time-course of infection in 293-T cells and plotted as the percentage of total cells per time-point. A minimum of 100 cells were counted per datum from two independent experiments and the mean±range plotted. (e) Gallery of images taken from 8 μm frozen sections cut through infected 293-T cells at 4.5 h p.i. stained for NP (green) and LAP-2 (red). (f) Infected 293-T cells were stained for NP (red) and the indicated cellular antigen (green) at 4.5 h p.i. Single optical sections through the midline of the nucleus (xy) and z-axis reconstructions (z) are shown. Arrowheads indicate the plane of matching z-sections. Scale bar, 5 μm.

Figure 2

Intranuclear localization of NP in epithelial cells. (a) Infected 293-T cells on cover-slips or (b) Caco-2 cells on filters were stained at (a) the indicated times p.i. or (b) at 4.5 h p.i. for NP (green) and LAP-2 (red) or NP (red) and Nup 62 or β-actin (green) as labelled. Scale bars: (a) 10 μm; (b) 5 μm.