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Rubella virus: first calcium-requiring viral fusion protein.

Dubé M, Rey FA, Kielian M - PLoS Pathog. (2014)

Bottom Line: Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world.Other tested cations did not substitute.Alanine substitution of N88 or D136 was lethal.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

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Effect of Ca2+ on RuV fusion.(A and B) Fusion-infection assays were performed with (A) RuV or (B) SFV as in Fig. 2A, but treating at the indicated pH for 4 min at 37°C in calcium-free fusion medium supplemented where indicated with 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.2-treated samples in medium plus CaCl2, and are the mean and standard deviation of 3 independent experiments. (C) Modified fusion-infection assay in which RuV was pre-bound to Vero cells in binding buffer containing 1.5 mM EDTA or 2 mM CaCl2. Cells were then washed to remove unbound virus and treated for 4 min at 37°C with calcium-free fusion medium at the indicated pH, supplemented with either 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.0 samples in which both the binding and fusion media contained calcium, and are the mean and range of 2 independent experiments.
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ppat-1004530-g003: Effect of Ca2+ on RuV fusion.(A and B) Fusion-infection assays were performed with (A) RuV or (B) SFV as in Fig. 2A, but treating at the indicated pH for 4 min at 37°C in calcium-free fusion medium supplemented where indicated with 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.2-treated samples in medium plus CaCl2, and are the mean and standard deviation of 3 independent experiments. (C) Modified fusion-infection assay in which RuV was pre-bound to Vero cells in binding buffer containing 1.5 mM EDTA or 2 mM CaCl2. Cells were then washed to remove unbound virus and treated for 4 min at 37°C with calcium-free fusion medium at the indicated pH, supplemented with either 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.0 samples in which both the binding and fusion media contained calcium, and are the mean and range of 2 independent experiments.

Mentions: We then used this assay to determine the role of Ca2+ in RuV fusion (Fig. 3A). Treatment for 4 min at neutral pH did not allow RuV fusion whether Ca2+ was present or not. In contrast, fusion at pH 6.0 or 5.8 was strongly dependent on Ca2+, with increases of up to 46-fold with 2 mM CaCl2 compared to calcium-free treatment. Addition of the calcium chelator EDTA completely abrogated fusion at both pH 5.8 and 6.0. Treatment at very low pH (5.0) did not rescue fusion in the absence of Ca2+ (Fig. S1), indicating that the fusion defect is not due to an acid-shift in the RuV pH threshold. A similar calcium dependence for RuV fusion was observed using HeLa cells or primary human umbilical vein endothelial cells as targets (Fig. S2). As predicted [34], [35], SFV fusion was strongly pH-dependent but calcium-independent (Fig. 3B).


Rubella virus: first calcium-requiring viral fusion protein.

Dubé M, Rey FA, Kielian M - PLoS Pathog. (2014)

Effect of Ca2+ on RuV fusion.(A and B) Fusion-infection assays were performed with (A) RuV or (B) SFV as in Fig. 2A, but treating at the indicated pH for 4 min at 37°C in calcium-free fusion medium supplemented where indicated with 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.2-treated samples in medium plus CaCl2, and are the mean and standard deviation of 3 independent experiments. (C) Modified fusion-infection assay in which RuV was pre-bound to Vero cells in binding buffer containing 1.5 mM EDTA or 2 mM CaCl2. Cells were then washed to remove unbound virus and treated for 4 min at 37°C with calcium-free fusion medium at the indicated pH, supplemented with either 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.0 samples in which both the binding and fusion media contained calcium, and are the mean and range of 2 independent experiments.
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ppat-1004530-g003: Effect of Ca2+ on RuV fusion.(A and B) Fusion-infection assays were performed with (A) RuV or (B) SFV as in Fig. 2A, but treating at the indicated pH for 4 min at 37°C in calcium-free fusion medium supplemented where indicated with 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.2-treated samples in medium plus CaCl2, and are the mean and standard deviation of 3 independent experiments. (C) Modified fusion-infection assay in which RuV was pre-bound to Vero cells in binding buffer containing 1.5 mM EDTA or 2 mM CaCl2. Cells were then washed to remove unbound virus and treated for 4 min at 37°C with calcium-free fusion medium at the indicated pH, supplemented with either 1.5 mM EDTA or 2 mM CaCl2. Data were normalized to the pH 6.0 samples in which both the binding and fusion media contained calcium, and are the mean and range of 2 independent experiments.
Mentions: We then used this assay to determine the role of Ca2+ in RuV fusion (Fig. 3A). Treatment for 4 min at neutral pH did not allow RuV fusion whether Ca2+ was present or not. In contrast, fusion at pH 6.0 or 5.8 was strongly dependent on Ca2+, with increases of up to 46-fold with 2 mM CaCl2 compared to calcium-free treatment. Addition of the calcium chelator EDTA completely abrogated fusion at both pH 5.8 and 6.0. Treatment at very low pH (5.0) did not rescue fusion in the absence of Ca2+ (Fig. S1), indicating that the fusion defect is not due to an acid-shift in the RuV pH threshold. A similar calcium dependence for RuV fusion was observed using HeLa cells or primary human umbilical vein endothelial cells as targets (Fig. S2). As predicted [34], [35], SFV fusion was strongly pH-dependent but calcium-independent (Fig. 3B).

Bottom Line: Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world.Other tested cations did not substitute.Alanine substitution of N88 or D136 was lethal.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

Show MeSH
Related in: MedlinePlus