Site-specific proteolytic cleavage of the amino terminus of herpes simplex virus glycoprotein K on virion particles inhibits virus entry

Abstract

Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310-6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.

Fig. 1.

Schematic representation of mutant viral genomes. (A) HSV-1(F)-YE102-VC1 recombinant virus generated from pYEbac102, carrying an in-frame insertion of the V5 epitope and enterokinase recognition site inserted immediately after amino acid 68 of gK, as well as an in-frame insertion of the 3×FLAG epitope at the amino terminus of UL20. (B) Schematic representation of recombinant viruses constructed for TEV-specific proteolytic activity. The V5 epitope tag and the TEV protease site (ENLYFQG) were inserted immediately after amino acid 68 of gK to generate mutant virus gK-V5-TEV.

Fig. 2.

Replication kinetics and plaque morphology of the YE102-VC1 virus in comparison to those of HSV-1(F)-YE102. (A) Representative viral plaques of HSV-1(F)-YE102 and HSV-1(F)-YE102-VC1 on Vero cells at 48 hpi. Viral plaques were visualized by phase-contrast microscopy after immunostaining with anti-HSV-1 polyclonal antibody. (B) One-step replication kinetics of HSV-1(F)-YE102 and HSV-1(F)-YE102-VC1 at an MOI of 0.2 on Vero cells. The x axis shows hours postinfection, and the y axis shows PFU/ml. All experiments were performed in triplicate. Error bars represent standard errors of the means.

Fig. 3.

Detection of gK and UL20 on gradient-purified virions. Western immunoblot analysis of double-gradient-purified virions was performed using anti-V5 (gK) and anti-FLAG (UL20) antibodies. Individual lanes are labeled as follows: C, positive control for GAPDH; W, lysates from purified wild-type virus [HSV-1(F)-YE102]; M, lysates from purified mutant virus YE102-VC1; L, lysates from infected Vero cells; V, samples derived from purified virions. Individual blots were probed with anti-GAPDH, anti-ICP8, anti-gB, anti-gD, anti-gC, anti-FLAG (UL20), or anti-V5 (gK) antibody as indicated. The panel labeled at the top as IP:V5Ab shows immunoblots of immunoprecipitations with anti-V5 (gK) antibody probed with either anti-FLAG (UL20) or anti-V5 (gK) antibodies. In this panel, lanes are immunoprecipitates from mock-infected Vero cells (U), HSV-1(F)-YE102 purified virions (W), or YE102-VC1 purified virions (M). Molecular mass standards are shown with dots on each panel (250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa; Precision Plus protein standards; Bio-Rad). HRP-conjugated goat anti-mouse (HRP-GAb; IgG) was used for all data, except data shown on the panel labeled IP:V5Ab, wherein the F(ab)₂- and Fc-purified portions of the HRP-GAb IgG were used for the α-V5 and α-FLAG panels, respectively.

Fig. 4.

Transmission immunogold electron microscopy to detect gK and gB on virion particles. (A) HSV-1(F)-YE102 virions stained with anti-gK (anti-V5) antibody; (B) YE102-VC1 stained with anti-gK (anti-V5) antibody; (C) gK-V5-TEV stained with anti-gK (anti-V5) antibody.

Fig. 5.

Assessment of the efficiencies of gK-null and gKΔ31–68 virus entry into Vero cells. (A) The relative efficiency of entry is shown as the percentage of cells expressing ICP4 at 12 hpi. Mean values and standard deviations for three independent experiments were calculated as shown. (B) Representative kinetics of virus HSV-1(F), gK-null, and gKΔ31-68 entry into Vero cells. Vero cell monolayers were infected with an MOI of 1. At different times postentry at 34°C (10, 20, 30, and 60 min), viruses that had not entered yet were inactivated with low pH, and the kinetics of virus entry were determined by monitoring ICP4 expression at 12 hpi using flow cytometry. For each sample, the percent ICP4-positive cells in a population of 5,000 Vero cells was measured.

Fig. 6.

Construction and characterization of recombinant viruses containing TEV modifications. (A) Representative plaque morphology of the gDΔTEV and gK-V5-TEV viruses in comparison to those of their parental HSV-1(F)-YK608 virus at 48 hpi. Viral plaques were visualized after immunohistochemical staining using phase-contrast microscopy and by fluorescence microscopy. (B) Replication kinetics of HSV-1(F) YK608, gDΔTEV, and gK-V5-TEV viruses on Vero cells infected with an MOI of 0.2. The x axis shows hours postinfection, and the y axis shows PFU/ml. All experiments were performed in triplicate. Error bars represent standard errors of the means.

Fig. 7.

Treatment of infected cell extracts. (A) Cell extracts from gK-V5-TEV-infected cells were immunoprecipitated with anti-V5 antibody and mock or TEV treated for 1 h at 30°C. Immunoprecipitates were tested for the presence of gK using anti-V5 antibody. Lane 1 (PBS), cell extracts mock digested in PBS buffer; lane 2 (PBS + E), cell extracts digested with AcTEV in PBS; lane 3 [PBS + INACT.E (CP)], PBS plus inactivated enzyme passed through a desalting column; lane 4 [PBS + E (CP)], PBS plus enzyme passed through a desalting column. (B) Densitometric analysis of immunoblots shown in panel A.

Fig. 8.

The effect of TEV protease treatment on virion infectivity. gDΔTEV (A) or gK-V5-TEV (B) virus obtained from supernatants of infected cells was treated with PBS, AcTEV protease (PBS + E), or SDS-inactivated AcTEV protease [PBS + INACT.E (CP)], and the numbers of resultant PFU were determined. Similarly, the relative kinetics of viral entry were determined for gDΔTEV (C) or gK-V5-TEV (D) in the presence (PBS + E) or absence (PBS) of the AcTEV protease. These experiments were done in triplicate, and error bars represent standard errors of the means. Asterisks in panel B designate statistically significant differences as calculated by one-way analysis of variance (ANOVA) (P < 0.05).