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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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Concentration-dependence and specificity of RuV Ca2+ requirement.(A) RuV fusion infection assay was performed as in Fig. 3A, in the presence of CaCl2, MgCl2 or Ca(C2H3O2)2 buffered with 1 mM EGTA to produce the indicated concentrations of free Ca2+ or Mg2+. Data were normalized to the pH 6.0, 2 mM CaCl2 sample, and are the mean and standard deviation of 3 independent experiments. (B) RuV and SFV fusion infection assays were performed as in Fig. 3A, with the fusion media supplemented with 0.5 mM CaCl2 and 0.2–20 mM MgCl2. Data were normalized to pH 6.0 with 0.5 mM CaCl2 and no added MgCl2, and represent the mean and range of 2 independent experiments.
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ppat-1004530-g004: Concentration-dependence and specificity of RuV Ca2+ requirement.(A) RuV fusion infection assay was performed as in Fig. 3A, in the presence of CaCl2, MgCl2 or Ca(C2H3O2)2 buffered with 1 mM EGTA to produce the indicated concentrations of free Ca2+ or Mg2+. Data were normalized to the pH 6.0, 2 mM CaCl2 sample, and are the mean and standard deviation of 3 independent experiments. (B) RuV and SFV fusion infection assays were performed as in Fig. 3A, with the fusion media supplemented with 0.5 mM CaCl2 and 0.2–20 mM MgCl2. Data were normalized to pH 6.0 with 0.5 mM CaCl2 and no added MgCl2, and represent the mean and range of 2 independent experiments.

Mentions: To more rigorously determine the Ca2+ concentration requirement for RuV fusion, we performed fusion-infection assays in which Ca2+ was buffered with EGTA, a more selective Ca2+ chelator. RuV fusion in this assay was maximal at 2 mM CaCl2 and showed a gradual reduction at decreasing Ca2+ concentrations (Fig. 4A). Mg2+, Mn2+ and Zn2+ did not substitute for Ca2+ even at concentrations of 2 mM (Fig. 4A, Fig. S3). When fusion was triggered using a sub-optimal concentration of Ca2+ (0.5 mM), the addition of Mg2+ at concentrations up to 20 mM neither substituted nor competed with Ca2+ (Fig. 4B). The structure of the RuV E1 homotrimer shows that the bound Ca2+ is coordinated in part by an acetate ion [28]. However, when tested in the fusion-infection assay CaCl2 and Ca(C2H3O2)2 promoted fusion with a similar concentration dependence (Fig. 4A), implying that acetate is not critical for the role of Ca2+ during fusion.


Rubella virus: first calcium-requiring viral fusion protein.

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

Concentration-dependence and specificity of RuV Ca2+ requirement.(A) RuV fusion infection assay was performed as in Fig. 3A, in the presence of CaCl2, MgCl2 or Ca(C2H3O2)2 buffered with 1 mM EGTA to produce the indicated concentrations of free Ca2+ or Mg2+. Data were normalized to the pH 6.0, 2 mM CaCl2 sample, and are the mean and standard deviation of 3 independent experiments. (B) RuV and SFV fusion infection assays were performed as in Fig. 3A, with the fusion media supplemented with 0.5 mM CaCl2 and 0.2–20 mM MgCl2. Data were normalized to pH 6.0 with 0.5 mM CaCl2 and no added MgCl2, and represent the mean and range of 2 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256232&req=5

ppat-1004530-g004: Concentration-dependence and specificity of RuV Ca2+ requirement.(A) RuV fusion infection assay was performed as in Fig. 3A, in the presence of CaCl2, MgCl2 or Ca(C2H3O2)2 buffered with 1 mM EGTA to produce the indicated concentrations of free Ca2+ or Mg2+. Data were normalized to the pH 6.0, 2 mM CaCl2 sample, and are the mean and standard deviation of 3 independent experiments. (B) RuV and SFV fusion infection assays were performed as in Fig. 3A, with the fusion media supplemented with 0.5 mM CaCl2 and 0.2–20 mM MgCl2. Data were normalized to pH 6.0 with 0.5 mM CaCl2 and no added MgCl2, and represent the mean and range of 2 independent experiments.
Mentions: To more rigorously determine the Ca2+ concentration requirement for RuV fusion, we performed fusion-infection assays in which Ca2+ was buffered with EGTA, a more selective Ca2+ chelator. RuV fusion in this assay was maximal at 2 mM CaCl2 and showed a gradual reduction at decreasing Ca2+ concentrations (Fig. 4A). Mg2+, Mn2+ and Zn2+ did not substitute for Ca2+ even at concentrations of 2 mM (Fig. 4A, Fig. S3). When fusion was triggered using a sub-optimal concentration of Ca2+ (0.5 mM), the addition of Mg2+ at concentrations up to 20 mM neither substituted nor competed with Ca2+ (Fig. 4B). The structure of the RuV E1 homotrimer shows that the bound Ca2+ is coordinated in part by an acetate ion [28]. However, when tested in the fusion-infection assay CaCl2 and Ca(C2H3O2)2 promoted fusion with a similar concentration dependence (Fig. 4A), implying that acetate is not critical for the role of Ca2+ during fusion.

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