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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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Association of capsids with the TGN. (a) HeLa cells were infected with 5 p.f.u. of vVP26GFP or vFRΔ37-VP26GFP per cell. At 15 h post-infection, the cells were fixed and stained with anti-TGN46 antibody and a GAR568 antibody (red). Capsids were visualized through direct GFP fluorescence (green). Bar, 10 μm. (b) HeLa cells were infected with 5 p.f.u. of vUL35RFP1D1 or vgD-gE-VP26RFP per cell. At 15 h post-infection, cells were fixed and labelled with anti-TGN46 antibody and a GARCy5 antibody (pseudo-coloured in green). Capsids were visualized through direct RFP fluorescence (red). In all cases, nuclei were stained with DAPI (blue). The boxed regions in the Merge+DAPI images are shown enlarged in the final panel and the positions of some TGN-derived vesicles are indicated by arrowheads. (c) Quantification of the amount of TGN signal that colocalizes with capsid signal for the four different viruses in HeLa cells (1, vVP26GFP; 2, vFRΔ37-VP26GFP; 3, vUL35RFP1D1; 4, vgD-gE-VP26RFP). The numbers of fields of view analysed are indicated above each bar. (d) HFFF2 cells were infected with 5 p.f.u. of vFRΔ37-VP26GFP per cell and labelled as above. A capsid aggregate is indicated by an arrow and a TGN vesicle by an arrowhead. Bars, 5 μm.
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f2: Association of capsids with the TGN. (a) HeLa cells were infected with 5 p.f.u. of vVP26GFP or vFRΔ37-VP26GFP per cell. At 15 h post-infection, the cells were fixed and stained with anti-TGN46 antibody and a GAR568 antibody (red). Capsids were visualized through direct GFP fluorescence (green). Bar, 10 μm. (b) HeLa cells were infected with 5 p.f.u. of vUL35RFP1D1 or vgD-gE-VP26RFP per cell. At 15 h post-infection, cells were fixed and labelled with anti-TGN46 antibody and a GARCy5 antibody (pseudo-coloured in green). Capsids were visualized through direct RFP fluorescence (red). In all cases, nuclei were stained with DAPI (blue). The boxed regions in the Merge+DAPI images are shown enlarged in the final panel and the positions of some TGN-derived vesicles are indicated by arrowheads. (c) Quantification of the amount of TGN signal that colocalizes with capsid signal for the four different viruses in HeLa cells (1, vVP26GFP; 2, vFRΔ37-VP26GFP; 3, vUL35RFP1D1; 4, vgD-gE-VP26RFP). The numbers of fields of view analysed are indicated above each bar. (d) HFFF2 cells were infected with 5 p.f.u. of vFRΔ37-VP26GFP per cell and labelled as above. A capsid aggregate is indicated by an arrow and a TGN vesicle by an arrowhead. Bars, 5 μm.

Mentions: To examine the effect of the UL37 and gD–gE mutations on capsid association with the TGN, HFFF2 (human fetal foreskin fibroblasts; European Collection of Cell Cultures) and HeLa cells were infected for 15, 18 or 24 h with 5 p.f.u. of vVP26GFP, vFRΔ37-VP26GFP, vUL35RFP1D1 or vgD-gE-VP26RFP per cell, and stained for the TGN using the anti-TGN46 antibody (Fig. 2) or for the Golgi with the anti-giantin antibody (see Supplementary Fig. S2, available in JGV Online). Similar results were observed at all times post-infection and only the 15 h images are shown. The TGN is disrupted into small vesicles upon infection by HSV-1, as described previously (Campadelli et al., 1993). In all cases, the patterns of association between capsids and TGN vesicles were the same in HFFF2 and HeLa cells. However, while cytoplasmic capsids formed aggregates in vFRΔ37-VP26GFP-infected HFFF2 cells, as described previously for other UL37− mutants (Desai et al., 2001; Klupp et al., 2001; Roberts et al., 2009), aggregates were not seen in HeLa cells, where their absence made it easier to observe the behaviour of individual capsids. Many of the capsids in the control vVP26GFP- and vUL35RFP1D1-infected cells colocalized with TGN vesicles, although they were also present in other regions of the cytoplasm (Fig. 2a, b). In the case of vgD-gE-VP26RFP, capsids accumulated in clusters adjacent to, but separated from, TGN vesicles (Fig. 2b). The tendency of capsids to aggregate in the absence of gD and gE was described previously for the parental mutant, vRR1097-gEβ (Farnsworth et al., 2003). In contrast to the juxtaposition of capsids and TGN seen with vgD-gE-VP26RFP, vFRΔ37-VP26GFP capsids accumulated throughout the cytoplasm of infected cells, without exhibiting any association with TGN46- or giantin-positive vesicles (Fig. 2a; Supplementary Fig. S2). The differing behaviours of vFRΔ37-VP26GFP and vgD-gE-VP26RFP were not due to capsid aggregation, as vFRΔ37-VP26GFP capsids also failed to associate with TGN in HFFF2 cells, where large aggregates formed readily (Fig. 2d). Quantification of the fluorescence signals confirmed that there was a significant decrease (approx. 90 %) in the level of colocalization of vFRΔ37-VP26GFP and vgD-gE-VP26RFP capsids with TGN, compared with their corresponding controls (Fig. 2c). To confirm that the fluorescent protein tags on VP26 were not influencing the behaviour of capsids, the experiments were repeated using the original untagged versions of the mutants, vFRΔUL37 and vRR1097-gEβ (see Supplementary Fig. S3, available in JGV Online). In agreement with previous results, WT capsids were largely colocalized with TGN vesicles and vRR1097-gEβ capsids were aggregated and juxtaposed to TGN vesicles, whereas vFRΔUL37 capsids were widely dispersed and showed no association with the TGN.


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)

Association of capsids with the TGN. (a) HeLa cells were infected with 5 p.f.u. of vVP26GFP or vFRΔ37-VP26GFP per cell. At 15 h post-infection, the cells were fixed and stained with anti-TGN46 antibody and a GAR568 antibody (red). Capsids were visualized through direct GFP fluorescence (green). Bar, 10 μm. (b) HeLa cells were infected with 5 p.f.u. of vUL35RFP1D1 or vgD-gE-VP26RFP per cell. At 15 h post-infection, cells were fixed and labelled with anti-TGN46 antibody and a GARCy5 antibody (pseudo-coloured in green). Capsids were visualized through direct RFP fluorescence (red). In all cases, nuclei were stained with DAPI (blue). The boxed regions in the Merge+DAPI images are shown enlarged in the final panel and the positions of some TGN-derived vesicles are indicated by arrowheads. (c) Quantification of the amount of TGN signal that colocalizes with capsid signal for the four different viruses in HeLa cells (1, vVP26GFP; 2, vFRΔ37-VP26GFP; 3, vUL35RFP1D1; 4, vgD-gE-VP26RFP). The numbers of fields of view analysed are indicated above each bar. (d) HFFF2 cells were infected with 5 p.f.u. of vFRΔ37-VP26GFP per cell and labelled as above. A capsid aggregate is indicated by an arrow and a TGN vesicle by an arrowhead. Bars, 5 μm.
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f2: Association of capsids with the TGN. (a) HeLa cells were infected with 5 p.f.u. of vVP26GFP or vFRΔ37-VP26GFP per cell. At 15 h post-infection, the cells were fixed and stained with anti-TGN46 antibody and a GAR568 antibody (red). Capsids were visualized through direct GFP fluorescence (green). Bar, 10 μm. (b) HeLa cells were infected with 5 p.f.u. of vUL35RFP1D1 or vgD-gE-VP26RFP per cell. At 15 h post-infection, cells were fixed and labelled with anti-TGN46 antibody and a GARCy5 antibody (pseudo-coloured in green). Capsids were visualized through direct RFP fluorescence (red). In all cases, nuclei were stained with DAPI (blue). The boxed regions in the Merge+DAPI images are shown enlarged in the final panel and the positions of some TGN-derived vesicles are indicated by arrowheads. (c) Quantification of the amount of TGN signal that colocalizes with capsid signal for the four different viruses in HeLa cells (1, vVP26GFP; 2, vFRΔ37-VP26GFP; 3, vUL35RFP1D1; 4, vgD-gE-VP26RFP). The numbers of fields of view analysed are indicated above each bar. (d) HFFF2 cells were infected with 5 p.f.u. of vFRΔ37-VP26GFP per cell and labelled as above. A capsid aggregate is indicated by an arrow and a TGN vesicle by an arrowhead. Bars, 5 μm.
Mentions: To examine the effect of the UL37 and gD–gE mutations on capsid association with the TGN, HFFF2 (human fetal foreskin fibroblasts; European Collection of Cell Cultures) and HeLa cells were infected for 15, 18 or 24 h with 5 p.f.u. of vVP26GFP, vFRΔ37-VP26GFP, vUL35RFP1D1 or vgD-gE-VP26RFP per cell, and stained for the TGN using the anti-TGN46 antibody (Fig. 2) or for the Golgi with the anti-giantin antibody (see Supplementary Fig. S2, available in JGV Online). Similar results were observed at all times post-infection and only the 15 h images are shown. The TGN is disrupted into small vesicles upon infection by HSV-1, as described previously (Campadelli et al., 1993). In all cases, the patterns of association between capsids and TGN vesicles were the same in HFFF2 and HeLa cells. However, while cytoplasmic capsids formed aggregates in vFRΔ37-VP26GFP-infected HFFF2 cells, as described previously for other UL37− mutants (Desai et al., 2001; Klupp et al., 2001; Roberts et al., 2009), aggregates were not seen in HeLa cells, where their absence made it easier to observe the behaviour of individual capsids. Many of the capsids in the control vVP26GFP- and vUL35RFP1D1-infected cells colocalized with TGN vesicles, although they were also present in other regions of the cytoplasm (Fig. 2a, b). In the case of vgD-gE-VP26RFP, capsids accumulated in clusters adjacent to, but separated from, TGN vesicles (Fig. 2b). The tendency of capsids to aggregate in the absence of gD and gE was described previously for the parental mutant, vRR1097-gEβ (Farnsworth et al., 2003). In contrast to the juxtaposition of capsids and TGN seen with vgD-gE-VP26RFP, vFRΔ37-VP26GFP capsids accumulated throughout the cytoplasm of infected cells, without exhibiting any association with TGN46- or giantin-positive vesicles (Fig. 2a; Supplementary Fig. S2). The differing behaviours of vFRΔ37-VP26GFP and vgD-gE-VP26RFP were not due to capsid aggregation, as vFRΔ37-VP26GFP capsids also failed to associate with TGN in HFFF2 cells, where large aggregates formed readily (Fig. 2d). Quantification of the fluorescence signals confirmed that there was a significant decrease (approx. 90 %) in the level of colocalization of vFRΔ37-VP26GFP and vgD-gE-VP26RFP capsids with TGN, compared with their corresponding controls (Fig. 2c). To confirm that the fluorescent protein tags on VP26 were not influencing the behaviour of capsids, the experiments were repeated using the original untagged versions of the mutants, vFRΔUL37 and vRR1097-gEβ (see Supplementary Fig. S3, available in JGV Online). In agreement with previous results, WT capsids were largely colocalized with TGN vesicles and vRR1097-gEβ capsids were aggregated and juxtaposed to TGN vesicles, whereas vFRΔUL37 capsids were widely dispersed and showed no association with the TGN.

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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Related in: MedlinePlus