Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro

Abstract

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.

FIG. 1

Incoming HSV-1 capsids bind to NPCs in vivo. Ultrathin Epon sections of Vero cells infected with HSV-1 at an MOI of 500 PFU/cell in the presence of cycloheximide are shown. At later times of infection, both DNA-containing, filled capsids (black arrow in panel A) and uncoated, empty capsids (black arrow in panel B) are located in close proximity to the NPC. Occasionally, capsids can be seen associated with fibers emanating from the pores (arrowheads in panels A and B).

FIG. 2

Uncoating of HSV-1 in Vero cells. Vero cells were infected with [³H]thymidine- or [³⁵S]methionine-labeled virus at an MOI of 10 PFU/cell. At various time points, the extracellular viruses were removed by proteinase K and the cells were lysed. The postnuclear supernatants were loaded onto a linear sucrose gradient for ultracentrifugation analysis of the internalized capsids. The sedimentation profiles of internalized [³H]thymidine-labeled and [³⁵S]methionine-labeled capsids at 30 min (A) and 4 h (B) p.i. are shown. Sedimentation profiles from one experiment are shown. However, the analysis was repeated three times, and mean values (with standard deviations) of two independent experiments with [³H]thymidine-labeled virus presented as percentages of total counts are shown in Table 1. The x axes indicate fractions.

FIG. 3

Characterization of HSV-1 capsids isolated from virions in vitro. (A) Coomassie blue staining of an SDS–10% PAGE analysis of the capsids. The capsids were isolated as a light-scattering zone in the sucrose gradient (left lane). Capsids were treated with 10 μg of trypsin per ml at 37°C for 5 min (right lane). The HSV-1 proteins identified by molecular weight are indicated on the left. (B) Western blot of the in vitro-isolated capsids (left lane) and capsids treated with 10 μg of trypsin per ml (right lane). The proteins identified with specific antibodies are indicated on the right.

FIG. 4

HSV-1 capsids bind to rat liver nuclei in vitro. (A) Confocal immunofluorescence microscopy of isolated HSV-1 capsids bound to purified rat liver nuclei in the presence of rat liver cytosol (control panel). The capsids were detected with RomV antibodies, which were generated against capsids isolated from virions. The background panel indicates a sample where capsids were omitted. Binding to nuclei pretreated with MAb 414, WGA, anti-importin β (anti-p97), or an ATP-depletion system (no ATP) is indicated in the other panels. (B) Conventional immunofluorescence microscopy of capsids binding to rat liver nuclei in the absence of cytosol (No cyt.), in the presence of 0.1 μM importin β (Imp β), or with 0.25 μM importin β and 10 μM RanQ69L (Imp β + RanQ69L). The capsids were detected as described for panel A. (C) Inhibition of capsid binding to nuclei pretreated with the indicated inhibitors in the presence of cytosol was quantitated as detailed in Materials and Methods. Inhibition of binding is shown relative to that of the control sample (without inhibitors) which represents the zero level in the graph. The mean values of at least triplicate samples with standard deviations are shown. (D) Stimulation of capsid binding to rat liver nuclei in vitro. Capsid binding to nuclei in the absence of cytosol (normalized to zero) or after addition of the components indicated on the right was quantitated as detailed in Materials and Methods. The mean values of at least triplicate samples with standard deviations are shown.

FIG. 5

Preferred binding of HSV-1 capsids to NPCs. An electron micrograph of capsids (arrowheads) binding to rat liver nuclei in vitro is shown. Small arrows indicate the cytoplasmic fibrils emanating from the NPC, and large arrows show the additional electron-dense material at the capsid vertices.

FIG. 6

Trypsin-treated capsids show reduced binding to nuclei in vitro. The figure shows confocal images of isolated, intact HSV-1 capsids (control) and trypsin-treated capsids (trypsin) bound to purified rat liver nuclei in the presence of rat liver cytosol.

FIG. 7

Uncoating of HSV-1 capsids in vitro. [³H]thymidine-labeled HSV-1 capsids were mixed either with BSA (column 1), BSA and rat liver nuclei (column 2), or BSA and rabbit reticulocyte lysate (column 3) or with BSA, reticulocyte lysate, and rat liver nuclei (column 4) in the presence of an ATP-regenerating system. The release of DNA was measured by assaying for DNase sensitivity using TCA precipitation. In untreated capsids (column 1), the DNA was completely protected, and in SDS-disrupted capsids (column 5), it was fully digested. The mean values of triplicate samples with standard deviations of at least three independent experiments are shown.

FIG. 8

Inhibition of in vitro uncoating of HSV-1 capsids. [³H]thymidine-labeled HSV-1 capsids were mixed with BSA, reticulocyte lysate, and rat liver nuclei in the presence of an ATP-regenerating system (column 1). The release of DNA was measured by assaying for DNase sensitivity using TCA precipitation. The inhibitors were tested by pretreating the nuclei with MAb 414 (column 2), anti-importin β (column 3), WGA (100 μg/ml) (column 4), hexokinase and glucose for ATP depletion (column 5), and GTPγS (1 mM) (column 6). See Materials and Methods for the details. The mean values of triplicate samples with standard deviations of at least three independent experiments are shown.