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The soluble amino-terminal region of HVEM mediates efficient herpes simplex virus type 1 infection of gD receptor-negative cells.

Baek H, Kim JH, Noh YT, Kwon H - Virol. J. (2012)

Bottom Line: Previous studies from our own and other labs reported the surprising finding that the soluble V domain of the herpes simplex virus type 1 (HSV-1) entry receptor nectin-1 can both block HSV infection of receptor-bearing cells and mediate infection of receptor-deficient cells.Here we show that this property is not unique to nectin-1.We found that sHveA102-mediated infection involves pH-independent endocytosis whereas HSV infection of HVEM-expressing CHO-K1 cells is known to be pH-dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, 215-4, Gongneung-Dong, Nowon-Ku, Seoul 139-706, South Korea.

ABSTRACT

Background: Previous studies from our own and other labs reported the surprising finding that the soluble V domain of the herpes simplex virus type 1 (HSV-1) entry receptor nectin-1 can both block HSV infection of receptor-bearing cells and mediate infection of receptor-deficient cells. Here we show that this property is not unique to nectin-1. We generated a pair of truncated, soluble forms of the other major HSV-1 entry receptor, herpes virus entry mediator (HVEM or HveA), and examined its effects on HSV-1 infection of receptor-deficient cells.

Results: In cultures of CHO-K1 cells, sHveA102 comprising the two amino-terminal cysteine-rich pseudorepeats (CRPs) of HVEM enabled infection of greater than 80% of the cells at an MOI of 3, while sHveA162 comprising the complete ectodomain failed to mediate infection. Both sHveA102 and sHveA162 blocked infection of CHO-K1 cells stably expressing HVEM in a dose-dependent manner, indicating that both were capable of binding to viral gD. We found that sHveA102-mediated infection involves pH-independent endocytosis whereas HSV infection of HVEM-expressing CHO-K1 cells is known to be pH-dependent.

Conclusions: Our results suggest that the C-terminal portion of the soluble HVEM ectodomain inhibits gD activation and that this effect is neutralized in the full-length form of HVEM in normal infection.

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Structure and expression of soluble HVEM proteins. (A) Schematic representations of full-length HVEM (HveA) and the truncated HVEM proteins produced for this study. Lollipops indicate glycosylation sites and numbers indicate amino acid positions relative to position 1 of mature HVEM. CRP, cysteine-rich pseudorepeat; H6, six-histidine tag. (B) Silver-stained SDS-PAGE gels of soluble protein samples. (C) Western blot detection of purified recombinant soluble proteins using anti-His tag antibody. Lane 1, sHA102; lane 2, sHA162; lane 3, sNec1123 used in Figure 2D; lane 4, sgD287 used in Figure 3.
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Figure 1: Structure and expression of soluble HVEM proteins. (A) Schematic representations of full-length HVEM (HveA) and the truncated HVEM proteins produced for this study. Lollipops indicate glycosylation sites and numbers indicate amino acid positions relative to position 1 of mature HVEM. CRP, cysteine-rich pseudorepeat; H6, six-histidine tag. (B) Silver-stained SDS-PAGE gels of soluble protein samples. (C) Western blot detection of purified recombinant soluble proteins using anti-His tag antibody. Lane 1, sHA102; lane 2, sHA162; lane 3, sNec1123 used in Figure 2D; lane 4, sgD287 used in Figure 3.

Mentions: We constructed expression plasmids for two His6-tagged soluble HVEM proteins, sHA102 containing the gD-binding first two CRPs (102 aa), and sHA162 comprising the full ectodomain (162 aa) (Figure 1A). Soluble proteins were produced by transfection of 293 T cells and purified from the culture supernatant, as previously described [26]. The molecular size sHA102 was approximately 15 kDa (Figure 1B, C), somewhat larger than the predicted size of 11 kDa most likely due to N-linked glycosylation in CRP2 [28]. sHA162 migrated predominantly as a 38 kDa species, significantly larger than the predicted 18 kDa for the monomeric form, suggesting incomplete denaturation of the dimeric form that predominates in solution [28].


The soluble amino-terminal region of HVEM mediates efficient herpes simplex virus type 1 infection of gD receptor-negative cells.

Baek H, Kim JH, Noh YT, Kwon H - Virol. J. (2012)

Structure and expression of soluble HVEM proteins. (A) Schematic representations of full-length HVEM (HveA) and the truncated HVEM proteins produced for this study. Lollipops indicate glycosylation sites and numbers indicate amino acid positions relative to position 1 of mature HVEM. CRP, cysteine-rich pseudorepeat; H6, six-histidine tag. (B) Silver-stained SDS-PAGE gels of soluble protein samples. (C) Western blot detection of purified recombinant soluble proteins using anti-His tag antibody. Lane 1, sHA102; lane 2, sHA162; lane 3, sNec1123 used in Figure 2D; lane 4, sgD287 used in Figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285515&req=5

Figure 1: Structure and expression of soluble HVEM proteins. (A) Schematic representations of full-length HVEM (HveA) and the truncated HVEM proteins produced for this study. Lollipops indicate glycosylation sites and numbers indicate amino acid positions relative to position 1 of mature HVEM. CRP, cysteine-rich pseudorepeat; H6, six-histidine tag. (B) Silver-stained SDS-PAGE gels of soluble protein samples. (C) Western blot detection of purified recombinant soluble proteins using anti-His tag antibody. Lane 1, sHA102; lane 2, sHA162; lane 3, sNec1123 used in Figure 2D; lane 4, sgD287 used in Figure 3.
Mentions: We constructed expression plasmids for two His6-tagged soluble HVEM proteins, sHA102 containing the gD-binding first two CRPs (102 aa), and sHA162 comprising the full ectodomain (162 aa) (Figure 1A). Soluble proteins were produced by transfection of 293 T cells and purified from the culture supernatant, as previously described [26]. The molecular size sHA102 was approximately 15 kDa (Figure 1B, C), somewhat larger than the predicted size of 11 kDa most likely due to N-linked glycosylation in CRP2 [28]. sHA162 migrated predominantly as a 38 kDa species, significantly larger than the predicted 18 kDa for the monomeric form, suggesting incomplete denaturation of the dimeric form that predominates in solution [28].

Bottom Line: Previous studies from our own and other labs reported the surprising finding that the soluble V domain of the herpes simplex virus type 1 (HSV-1) entry receptor nectin-1 can both block HSV infection of receptor-bearing cells and mediate infection of receptor-deficient cells.Here we show that this property is not unique to nectin-1.We found that sHveA102-mediated infection involves pH-independent endocytosis whereas HSV infection of HVEM-expressing CHO-K1 cells is known to be pH-dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, 215-4, Gongneung-Dong, Nowon-Ku, Seoul 139-706, South Korea.

ABSTRACT

Background: Previous studies from our own and other labs reported the surprising finding that the soluble V domain of the herpes simplex virus type 1 (HSV-1) entry receptor nectin-1 can both block HSV infection of receptor-bearing cells and mediate infection of receptor-deficient cells. Here we show that this property is not unique to nectin-1. We generated a pair of truncated, soluble forms of the other major HSV-1 entry receptor, herpes virus entry mediator (HVEM or HveA), and examined its effects on HSV-1 infection of receptor-deficient cells.

Results: In cultures of CHO-K1 cells, sHveA102 comprising the two amino-terminal cysteine-rich pseudorepeats (CRPs) of HVEM enabled infection of greater than 80% of the cells at an MOI of 3, while sHveA162 comprising the complete ectodomain failed to mediate infection. Both sHveA102 and sHveA162 blocked infection of CHO-K1 cells stably expressing HVEM in a dose-dependent manner, indicating that both were capable of binding to viral gD. We found that sHveA102-mediated infection involves pH-independent endocytosis whereas HSV infection of HVEM-expressing CHO-K1 cells is known to be pH-dependent.

Conclusions: Our results suggest that the C-terminal portion of the soluble HVEM ectodomain inhibits gD activation and that this effect is neutralized in the full-length form of HVEM in normal infection.

Show MeSH
Related in: MedlinePlus