The ESCRT-II Subunit EAP20/VPS25 and the Bro1 Domain Proteins HD-PTP and BROX Are Individually Dispensable for Herpes Simplex Virus 1 Replication

Abstract

Capsid envelopment during assembly of the neurotropic herpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) in the infected cell cytoplasm is thought to involve the late-acting cellular ESCRT (endosomal sorting complex required for transport) components ESCRT-III and VPS4 (vacuolar protein sorting 4). However, HSV-1, unlike members of many other families of enveloped viruses, does not appear to require the ESCRT-I subunit TSG101 or the Bro1 domain-containing protein ALIX (Alg-2-interacting protein X) to recruit and activate ESCRT-III. Alternative cellular factors that are known to be capable of regulating ESCRT-III function include the ESCRT-II complex and other members of the Bro1 family. We therefore used small interfering RNA (siRNA) to knock down the essential ESCRT-II subunit EAP20/VPS25 (ELL-associated protein 20/vacuolar protein sorting 25) and the Bro1 proteins HD-PTP (His domain-containing protein tyrosine phosphatase) and BROX (Bro1 domain and CAAX motif containing). We demonstrated reductions in levels of the targeted proteins by Western blotting and used quantitative microscopic assays to confirm loss of ESCRT-II and HD-PTP function. We found that in single-step replication experiments, the final yields of HSV-1 were unchanged following loss of EAP20, HD-PTP, or BROX.IMPORTANCE HSV-1 is a pathogen of the human nervous system that uses its own virus-encoded proteins and the normal cellular ESCRT machinery to drive the construction of its envelope. How HSV-1 structural proteins interact with ESCRT components and which subsets of cellular ESCRT proteins are utilized by the virus remain largely unknown. Here, we demonstrate that an essential component of the ESCRT-II complex and two ESCRT-associated Bro1 proteins are dispensable for HSV-1 replication.

Keywords: ESCRT; envelopment; herpes simplex virus; virus assembly.

FIG 1

Alphaherpesvirus envelopment and candidate ESCRT components. ESCRT complexes are shown on the left, with ESCRT-0 (composed of subunits STAM/HRS) in brown, ESCRT-I (composed of subunits TSG101/MVB12/VPS37/VPS28) in red, and ESCRT-II (EAP30/EAP45/EAP20₂) in yellow. The ESCRT-III filament is shown as a polymer of CHMP2/3/4 subunits (green) capped by nucleating CHMP6 subunits (cyan). The ESCRT-I and ESCRT-II complex EAP20 subunits can each nucleate filament assembly through recruitment of CHMP6. Members of the Bro1 family of proteins (dark-blue cylinder) are able to directly trigger CHMP4 polymerization. On the right is the generalized structure of an alphaherpesvirus cytoplasmic envelopment intermediate, showing the presumed relationship between the ESCRT-III filament and the lipid bilayer (brown line), the viral capsid (red sphere), the inner tegument (gray layer), and the outer tegument (dark-blue ovals), some of which interact with membrane-embedded envelope glycoproteins (yellow bars). Late in envelopment, ESCRT-III constricts to draw the membrane together, sealing the envelope and pinching the enveloped virus into the organellar lumen (light-blue space). Constriction and ESCRT-III disassembly are catalyzed by the ATPase VPS4 (purple spheres). The positioning of the ESCRT-0, -I, and -II complexes and the Bro1 proteins is not meant to necessarily imply they play roles in alphaherpesvirus envelopment.

FIG 2

Inhibition of EGFR degradation following knockdown of the ESCRT-II subunit EAP20. HeLa cells were transfected with control siRNA (Ctrl) or an siRNA targeting EAP20. After 24 h, the transfections were repeated, and then the cells were immediately infected with HSV-1. At 13 h postinfection, the cells were serum starved for 5 h; incubated with (+) or without (−) EGF for 2 h, as indicated; and then fixed for DAPI staining and anti-EGFR immunocytochemistry or collected for Western blotting. (A) Fields of cells immunostained for EGFR (green), exhibiting UL25-mCherry fluorescence (red), or in which EGFR and UL25 fluorescence images were merged with DAPI-stained images (Merge). (B) Western blot showing levels of EAP20 or the cell recovery/loading control GAPDH protein at the time of cell recovery. (C) Mean EGFR immunofluorescence intensities from 50 microscopic fields similar to those in panel A were quantitated using NIH ImageJ software (3 to 5 cells were scored per field). Mean EGFR fluorescence intensities and standard deviations from the mean are plotted in arbitrary units (a.u.). ns, not significant (P = 0.59); ****, P < 0.0001.

FIG 3

siRNA knockdown of EAP20 or PRPF8 and effects on HSV-1 replication. (A and B) Cells were treated with control (Ctrl) siRNA or EAP20 siRNA exactly as for Fig. 2. They were infected with HSV-1 and acid washed to inactivate unpenetrated virus, and cell extracts were prepared immediately (T₀) and after 20 h (T₂₀). (A) Cell extracts were subjected to Western blotting for EAP20 and control GAPDH. (B) Plaque assays to determine PFU yields in cell extracts. The plotted values are means and standard deviations from the mean for 6 independent replicates. (C and D) Cells were treated with control (Ctrl) siRNA or PRPF8 siRNA, and the transfections were repeated after 48 h. After a further 24 h, the cells were infected with HSV-1, and extracts were prepared exactly as for the EAP20 knockdown. (C) Cell extracts were subjected to Western blotting for PRPF8 and control GAPDH. (D) Plaque assays to determine PFU yields in cell extracts. The plotted values are means and standard deviations from the mean for four independent replicates.

FIG 4

siRNA knockdown of HD-PTP and its consequences for accumulation of SMN puncta in nuclei. HeLa cells were transfected with control siRNA (Ctrl) or a mixture of two HD-PTP siRNAs, and the transfections were repeated 24 h and 48 h later. Cells were then collected for immunostaining or Western blotting. (A) Fields of cells fixed, stained with DAPI, and immunostained for SMN. (Bottom row) SMN immunostaining alone. The white arrowhead indicates a nuclear SMN punctum. (Top row) Merge of SMN and DAPI channels. (B) Western blot showing levels of HD-PTP or control GAPDH protein in the cells at the time of their recovery. (C) SMN puncta in nuclei were counted and plotted as the mean number of puncta seen per 150 cells (left graph) and the mean number of punctum-free nuclei per 150 cells (right graph). The plotted values are means and standard deviations from the mean for 3 independent experiments, with 150 cells scored per experiment ****, P < 0.0001.

FIG 5

Titers of HSV-1 grown on HD-PTP-depleted HeLa cells. The cells were treated with control siRNA (Ctrl) or HD-PTP siRNAs exactly as for Fig. 4, infected with HSV-1, and then acid washed to inactivate unpenetrated virus. Samples were collected immediately (T₀) and after 20 h (T₂₀). (A) Cell extracts were subjected to Western blotting for HD-PTP and control GAPDH. (B) Plaque assays to determine PFU yields in cell extracts. The plotted values are means and standard deviations from the mean for 6 independent replicates.

FIG 6

Titers of HSV-1 grown on BROX-depleted HeLa cells. Cells were treated with control siRNA (Ctrl) or a mixture of two BROX siRNAs. The transfection was then repeated 48 h and 96 h after the start of the experiment. After an additional 4 h, the cells were infected with HSV-1 and then acid washed to inactivate unpenetrated virus. Samples were collected immediately (T₀) and after 20 h (T₂₀). (A) Cell extracts were subjected to Western blotting for BROX and control GAPDH. (B) Plaque assays to determine PFU yields in cell extracts. The plotted values are means and ranges for 2 independent duplicate experiments.