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Inner tegument protein pUL37 of herpes simplex virus type 1 is involved in directing capsids to the trans-Golgi network for envelopment.

Pasdeloup D, Beilstein F, Roberts AP, McElwee M, McNab D, Rixon FJ - J. Gen. Virol. (2010)

Bottom Line: Although wild-type capsids accumulated at the TGN, capsids of the pUL37(-) mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles.This was in contrast to capsids from a gD(-)gE(-) mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment.We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, University of Glasgow, Glasgow G11 5JR, UK. pasdeloup@vms.cnrs-gif.fr

ABSTRACT
Secondary envelopment of herpes simplex virus type 1 has been demonstrated as taking place at the trans-Golgi network (TGN). The inner tegument proteins pUL36 and pUL37 and the envelope glycoproteins gD and gE are known to be important for secondary envelopment. We compared the cellular localizations of capsids from a virus mutant lacking the UL37 gene with those of a virus mutant lacking the genes encoding gD and gE. Although wild-type capsids accumulated at the TGN, capsids of the pUL37(-) mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles. This was in contrast to capsids from a gD(-)gE(-) mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment. We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

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Characterization of the vFRΔ37-VP26GFP and vgD-gE-VP26RFP viruses. (a) RS cells were mock-infected (lane 1) or infected with 5 p.f.u. of vFRΔ37-VP26GFP (lane 2), vVP26GFP (lane 3) or WT HSV-1 (lane 4) per cell and harvested after 24 h. Proteins were analysed by Western blotting using the pUL37 antibody, together with either the GFP-specific antibody (left panel) or the VP26-specific antibody (right panel). Note that a viral protein migrating at approximately 75 kDa is recognized non-specifically by the pUL37 antibody. Molecular mass markers (in kDa) are indicated to the left of the figure. (b) Vero cells were infected with 5 p.f.u. of WT HSV-1 (lane 1), vRR1097-gEβ (lane 2), vUL35RFP1D1 (lane 3), vgD-VP26RFP (lane 4) or vgD-gE-VP26RFP (lane 5) per cell or were mock-infected (lane 6), and harvested after 24 h. Proteins were analysed by Western blotting using a gD antibody, a gE antibody and a VP26-specific antibody. Molecular mass markers (in kDa) are indicated to the right of the figure. (c) Growth of viruses on complementing (filled bars) and non-complementing (empty bars) cell lines. Concentrated stocks of virus were titrated on RS cells (vVP26GFP), Vero cells (vUL35RFP1D1, vgD-VP26RFP, vgD-gE-VP26RFP), 80C02 cells [a clone of RS cells expressing UL37 (Roberts et al., 2009)] (vFRΔ37-VP26GFP) or VD60 cells [a clone of Vero cells expressing gD (Ligas & Johnson, 1988)] (vgD-VP26RFP, vgD-gE-VP26RFP). *vVP26GFP and vUL35RFP1D1 were not tested on complementing cells. **The titres of vFRΔ37-VP26GFP and vgD-gE-VP26RFP on non-complementing cells were assigned as <103 because the input virus caused severe cytopathic effects at lower dilutions.
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f1: Characterization of the vFRΔ37-VP26GFP and vgD-gE-VP26RFP viruses. (a) RS cells were mock-infected (lane 1) or infected with 5 p.f.u. of vFRΔ37-VP26GFP (lane 2), vVP26GFP (lane 3) or WT HSV-1 (lane 4) per cell and harvested after 24 h. Proteins were analysed by Western blotting using the pUL37 antibody, together with either the GFP-specific antibody (left panel) or the VP26-specific antibody (right panel). Note that a viral protein migrating at approximately 75 kDa is recognized non-specifically by the pUL37 antibody. Molecular mass markers (in kDa) are indicated to the left of the figure. (b) Vero cells were infected with 5 p.f.u. of WT HSV-1 (lane 1), vRR1097-gEβ (lane 2), vUL35RFP1D1 (lane 3), vgD-VP26RFP (lane 4) or vgD-gE-VP26RFP (lane 5) per cell or were mock-infected (lane 6), and harvested after 24 h. Proteins were analysed by Western blotting using a gD antibody, a gE antibody and a VP26-specific antibody. Molecular mass markers (in kDa) are indicated to the right of the figure. (c) Growth of viruses on complementing (filled bars) and non-complementing (empty bars) cell lines. Concentrated stocks of virus were titrated on RS cells (vVP26GFP), Vero cells (vUL35RFP1D1, vgD-VP26RFP, vgD-gE-VP26RFP), 80C02 cells [a clone of RS cells expressing UL37 (Roberts et al., 2009)] (vFRΔ37-VP26GFP) or VD60 cells [a clone of Vero cells expressing gD (Ligas & Johnson, 1988)] (vgD-VP26RFP, vgD-gE-VP26RFP). *vVP26GFP and vUL35RFP1D1 were not tested on complementing cells. **The titres of vFRΔ37-VP26GFP and vgD-gE-VP26RFP on non-complementing cells were assigned as <103 because the input virus caused severe cytopathic effects at lower dilutions.

Mentions: To screen vFRΔ37-VP26GFP for fusion of GFP to VP26 and for the absence of pUL37, RS cells were infected for 24 h with WT HSV-1, vVP26GFP or vFRΔ37-VP26GFP. The cells were then harvested and analysed by Western blot analysis using GFP-, VP26- and UL37-specific antibodies [see Supplementary Methods (available in JGV Online) for antibody details]. Fig. 1(a) shows that pUL37 is present in WT HSV-1-infected and vVP26GFP-infected cells (lanes 4 and 3, respectively), but is missing from vFRΔ37-VP26GFP-infected cells (lane 2). The VP26-specific antibody recognizes a band of 14 kDa in WT HSV-1-infected cells (lane 4, right), but this band is missing in vFRΔ37-VP26GFP-infected and vVP26GFP-infected cells (lanes 2 and 3, right), where a band of approximately 40–45 kDa is recognized by both VP26- and GFP-specific antibodies. This band is of the approximate size expected for the GFP–VP26 fusion protein (39 kDa).


Inner tegument protein pUL37 of herpes simplex virus type 1 is involved in directing capsids to the trans-Golgi network for envelopment.

Pasdeloup D, Beilstein F, Roberts AP, McElwee M, McNab D, Rixon FJ - J. Gen. Virol. (2010)

Characterization of the vFRΔ37-VP26GFP and vgD-gE-VP26RFP viruses. (a) RS cells were mock-infected (lane 1) or infected with 5 p.f.u. of vFRΔ37-VP26GFP (lane 2), vVP26GFP (lane 3) or WT HSV-1 (lane 4) per cell and harvested after 24 h. Proteins were analysed by Western blotting using the pUL37 antibody, together with either the GFP-specific antibody (left panel) or the VP26-specific antibody (right panel). Note that a viral protein migrating at approximately 75 kDa is recognized non-specifically by the pUL37 antibody. Molecular mass markers (in kDa) are indicated to the left of the figure. (b) Vero cells were infected with 5 p.f.u. of WT HSV-1 (lane 1), vRR1097-gEβ (lane 2), vUL35RFP1D1 (lane 3), vgD-VP26RFP (lane 4) or vgD-gE-VP26RFP (lane 5) per cell or were mock-infected (lane 6), and harvested after 24 h. Proteins were analysed by Western blotting using a gD antibody, a gE antibody and a VP26-specific antibody. Molecular mass markers (in kDa) are indicated to the right of the figure. (c) Growth of viruses on complementing (filled bars) and non-complementing (empty bars) cell lines. Concentrated stocks of virus were titrated on RS cells (vVP26GFP), Vero cells (vUL35RFP1D1, vgD-VP26RFP, vgD-gE-VP26RFP), 80C02 cells [a clone of RS cells expressing UL37 (Roberts et al., 2009)] (vFRΔ37-VP26GFP) or VD60 cells [a clone of Vero cells expressing gD (Ligas & Johnson, 1988)] (vgD-VP26RFP, vgD-gE-VP26RFP). *vVP26GFP and vUL35RFP1D1 were not tested on complementing cells. **The titres of vFRΔ37-VP26GFP and vgD-gE-VP26RFP on non-complementing cells were assigned as <103 because the input virus caused severe cytopathic effects at lower dilutions.
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f1: Characterization of the vFRΔ37-VP26GFP and vgD-gE-VP26RFP viruses. (a) RS cells were mock-infected (lane 1) or infected with 5 p.f.u. of vFRΔ37-VP26GFP (lane 2), vVP26GFP (lane 3) or WT HSV-1 (lane 4) per cell and harvested after 24 h. Proteins were analysed by Western blotting using the pUL37 antibody, together with either the GFP-specific antibody (left panel) or the VP26-specific antibody (right panel). Note that a viral protein migrating at approximately 75 kDa is recognized non-specifically by the pUL37 antibody. Molecular mass markers (in kDa) are indicated to the left of the figure. (b) Vero cells were infected with 5 p.f.u. of WT HSV-1 (lane 1), vRR1097-gEβ (lane 2), vUL35RFP1D1 (lane 3), vgD-VP26RFP (lane 4) or vgD-gE-VP26RFP (lane 5) per cell or were mock-infected (lane 6), and harvested after 24 h. Proteins were analysed by Western blotting using a gD antibody, a gE antibody and a VP26-specific antibody. Molecular mass markers (in kDa) are indicated to the right of the figure. (c) Growth of viruses on complementing (filled bars) and non-complementing (empty bars) cell lines. Concentrated stocks of virus were titrated on RS cells (vVP26GFP), Vero cells (vUL35RFP1D1, vgD-VP26RFP, vgD-gE-VP26RFP), 80C02 cells [a clone of RS cells expressing UL37 (Roberts et al., 2009)] (vFRΔ37-VP26GFP) or VD60 cells [a clone of Vero cells expressing gD (Ligas & Johnson, 1988)] (vgD-VP26RFP, vgD-gE-VP26RFP). *vVP26GFP and vUL35RFP1D1 were not tested on complementing cells. **The titres of vFRΔ37-VP26GFP and vgD-gE-VP26RFP on non-complementing cells were assigned as <103 because the input virus caused severe cytopathic effects at lower dilutions.
Mentions: To screen vFRΔ37-VP26GFP for fusion of GFP to VP26 and for the absence of pUL37, RS cells were infected for 24 h with WT HSV-1, vVP26GFP or vFRΔ37-VP26GFP. The cells were then harvested and analysed by Western blot analysis using GFP-, VP26- and UL37-specific antibodies [see Supplementary Methods (available in JGV Online) for antibody details]. Fig. 1(a) shows that pUL37 is present in WT HSV-1-infected and vVP26GFP-infected cells (lanes 4 and 3, respectively), but is missing from vFRΔ37-VP26GFP-infected cells (lane 2). The VP26-specific antibody recognizes a band of 14 kDa in WT HSV-1-infected cells (lane 4, right), but this band is missing in vFRΔ37-VP26GFP-infected and vVP26GFP-infected cells (lanes 2 and 3, right), where a band of approximately 40–45 kDa is recognized by both VP26- and GFP-specific antibodies. This band is of the approximate size expected for the GFP–VP26 fusion protein (39 kDa).

Bottom Line: Although wild-type capsids accumulated at the TGN, capsids of the pUL37(-) mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles.This was in contrast to capsids from a gD(-)gE(-) mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment.We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, University of Glasgow, Glasgow G11 5JR, UK. pasdeloup@vms.cnrs-gif.fr

ABSTRACT
Secondary envelopment of herpes simplex virus type 1 has been demonstrated as taking place at the trans-Golgi network (TGN). The inner tegument proteins pUL36 and pUL37 and the envelope glycoproteins gD and gE are known to be important for secondary envelopment. We compared the cellular localizations of capsids from a virus mutant lacking the UL37 gene with those of a virus mutant lacking the genes encoding gD and gE. Although wild-type capsids accumulated at the TGN, capsids of the pUL37(-) mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles. This was in contrast to capsids from a gD(-)gE(-) mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment. We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

Show MeSH
Related in: MedlinePlus