Targeting of viral capsids to nuclear pores in a cell-free reconstitution system

Abstract

Many viruses deliver their genomes into the nucleoplasm for viral transcription and replication. Here, we describe a novel cell-free system to elucidate specific interactions between viruses and nuclear pore complexes (NPCs). Nuclei reconstituted in vitro from egg extracts of Xenopus laevis, an established biochemical system to decipher nuclear functions, were incubated with GFP-tagged capsids of herpes simplex virus, an alphaherpesvirus replicating in the nucleus. Capsid binding to NPCs was analyzed using fluorescence and field emission scanning electron microscopy. Tegument-free capsids or viral capsids exposing inner tegument proteins on their surface bound to nuclei, while capsids inactivated by a high-salt treatment or covered by inner and outer tegument showed less binding. There was little binding of the four different capsid types to nuclei lacking functional NPCs. This novel approach provides a powerful system to elucidate the molecular mechanisms that enable viral structures to engage with NPCs. Furthermore, this assay could be expanded to identify molecular cues triggering viral genome uncoating and nuclear import of viral genomes.

Keywords: Xenopus nuclei; herpes simplex virus; herpesviruses; nuclear pore; reconstitution.

Figure 1. An in vitro assay to analyze the binding of viral capsids to NPCs.

Nuclear capsids (dark green) were isolated from the nuclei of HSV1 infected cells by gradient sedimentation (top left). Viral capsids were generated from mature extracellular particles released from HSV1 infected cells by lysis with 1% TX-100 in the presence of 1 M, 0.5 M or 0.1 M KCl (light and dark green), and purified through sucrose cushions (top middle). The four different capsid types were re-suspended in BRB80 buffer before tip sonication and DNase/RNase treatment. Nuclei harboring functional NPCs were generated in vitro from Xenopus egg extracts around a chromatin template. The formation of NPCs was inhibited in some reactions by the addition of Imp β^45-462 or BAPTA to the assembly reaction to create nuclei enclosed by continuous membranes lacking functional NPCs (top right). After nuclear assembly and re-sedimentation the nuclear or viral 1 M, 0.5 M or 0.1 M capsids were incubated with different nuclei for 20 to 90 min at room temperature. Nuclear assembly and capsid binding were analyzed by fluorescence light microscopy or field emission scanning electron microscopy (FESEM). Modified from Figure 1 of (19).

Figure 2. Protein composition of different HSV1 capsids types.

Nuclear capsids were isolated from BHK cells infected with HSV1-GFPVP26 using TNE- or MKT-buffer for cell suspension, and their protein composition was compared to that of viral capsids treated with TX-100 and 1.0 M, 0.5 or 0.1 M KCl by immunoblotting with antibodies raised against the capsid proteins VP5 (mAb H1.4), pUL25 (mAb #166), VP19c (pAb NC-2), VP21/VP22a (pAb NC3/4), or VP23 (pAb5) or the tegument proteins pUL36 (pAb #147), pUL37 (pAb anti-pUL37), or VP13/14 (pAb R220).

Figure 3. Electron cryo tomography reveals surface features of different HSV1 capsids types.

Results from sub-volume averaging are presented for capsids isolated from intact virions (HSV1 strain F) and extracted with TX-100 and 1 M (A), 0.5 M (B) or 0.1 M (C) KCl. Row (i): cross sections of the averages from 183 (1 M), 246 (0.5 M) or 217 (0.1 M) capsid sub-volumes. Row (ii): close-up view of the top vertex in row (i). Row (iii): iso-surface representation of the averages. Row (iv): close-up view of a salt treated capsid vertex (left), compared to nuclear C-capsids (66). Row (v): difference map between the respective salt treated capsid group and the nuclear C-capsids average, superimposed onto the nuclear C-capsids average. Row (vi): close-up view of a vertex from row (v). The capsids have a diameter of 125 nm.

Figure 4. Targeting of HSV1 capsids to NPCs on Xenopus nuclei in vitro.

A to C. Functional (A), Imp β^45-462 (B) or BAPTA (C) nuclei were reconstituted in vitro. Completion of nuclear assembly was monitored by epi-fluorescence microscopy by DNA staining with Hoechst 33258 (i and iii), NPC staining with fluorescently labeled mAb414 (ii) and the transport capacity of the assembled NPCs was confirmed by the nuclear import of TRITC-NLS-BSA (iv). Scale bar: 10 µm. D, E. Binding of viral 0.5 M KCl capsids extracted from extracellular particles of HSV1-GFPVP26 (ii and iii, green, white arrowheads) to Xenopus nuclei was analyzed using confocal fluorescence laser scanning microscopy and a serial z-slicing of 0.37 μm optical sections. A surface view (D) and one midsection (E) of a representative nucleus are shown. NPCs were labeled with Imp β^45-462-TRITC (i and iii, red). Scale bar: 10 µm. F, G. HSV1-GFPVP26 viral 0.5 M capsids were incubated with Xenopus nuclei harboring functional NPCs for 45 min, labeled with a polyclonal rabbit antiserum raised against intact HSV1 capsids (Remus, bleed V) and colloidal gold with a diameter of 12 nm coated with anti-rabbit antibodies. The specimens were analyzed by field emission scanning electron microscopy. (F) 3D surface topography of reconstituted nuclei from different views in the in-lens images (Fig. 4Fi-iii). The same areas were imaged through a backscatter electron detector revealing the positions of gold-conjugated antibodies (Fig. 4Gi-iii; inverted color mode). HSV1 capsids (white arrowheads) are bound to the cytoplasmic face of the NPCs (white arrows). Scale bar: 100 nm.

Figure 5. HSV1 capsid binding to nuclei requires specific capsid surface features and NPCs.

Viral capsids isolated from viral particles with TX-100 and 0.1, 0.5 or 1.0 M KCl or nuclear C capsids were incubated with Xenopus nuclei reconstituted in vitro. The number of HSV1 capsids bound to nuclei harboring functional NPCs (control), or to nuclei assembled in the presence of Imp β^45-462 or BAPTA was determined by epi-fluorescence microscopy. Images were acquired at different focal planes, merged, bound capsids were counted, and the data normalized to 100% for 0.5 M viral capsids binding to functional nuclei. The error bars indicate the standard error of the mean. Asterisks indicate p<0.0015 and “ns” p>0.05 as determined in two-tailed student’s t-test.