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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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Calcium dependence of RuV-membrane interactions.(A–B) Virus-liposome binding assays. Radiolabeled RuV (A) or SFV (B) was incubated with liposomes for 1 min at 37°C at the indicated pH and buffer conditions. The percentage of total virus radioactivity associated with the liposomes was determined by sucrose gradient floatation. (C–D) Calcium requirement for virus fusion at the plasma membrane. (C) Purified RuV particles were labeled with DiD (red), prebound to Vero cells on ice, and the cells were pulsed at 37°C for 2 min in fusion medium containing 2 mM CaCl2 or 1.5 mM EDTA as indicated. Cells were then fixed and nuclei were counterstained with Hoescht (blue). Images represent a maximal projection of Z-stacks, with the scale bar indicating 25 µm. (D) Quantitation of C. DiD signal and cell numbers were quantitated and normalized to control, calcium-containing conditions. (E–F) Calcium requirement for virus fusion in endosomes. DiD-labeled virus was prebound to Vero cells on ice, and the cells with bound virus were incubated at 37°C for 20 min in binding medium containing 2 mM CaCl2 or 20 mM NH4Cl as indicated. Cells were then fixed, imaged and quantitated as in (C–D). Graphs show the mean and standard deviation of 3 (A, F) or 4 (D) independent experiments. For (B), the mean and range were calculated from 2 independent experiments.
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ppat-1004530-g006: Calcium dependence of RuV-membrane interactions.(A–B) Virus-liposome binding assays. Radiolabeled RuV (A) or SFV (B) was incubated with liposomes for 1 min at 37°C at the indicated pH and buffer conditions. The percentage of total virus radioactivity associated with the liposomes was determined by sucrose gradient floatation. (C–D) Calcium requirement for virus fusion at the plasma membrane. (C) Purified RuV particles were labeled with DiD (red), prebound to Vero cells on ice, and the cells were pulsed at 37°C for 2 min in fusion medium containing 2 mM CaCl2 or 1.5 mM EDTA as indicated. Cells were then fixed and nuclei were counterstained with Hoescht (blue). Images represent a maximal projection of Z-stacks, with the scale bar indicating 25 µm. (D) Quantitation of C. DiD signal and cell numbers were quantitated and normalized to control, calcium-containing conditions. (E–F) Calcium requirement for virus fusion in endosomes. DiD-labeled virus was prebound to Vero cells on ice, and the cells with bound virus were incubated at 37°C for 20 min in binding medium containing 2 mM CaCl2 or 20 mM NH4Cl as indicated. Cells were then fixed, imaged and quantitated as in (C–D). Graphs show the mean and standard deviation of 3 (A, F) or 4 (D) independent experiments. For (B), the mean and range were calculated from 2 independent experiments.

Mentions: An early step in viral membrane fusion pathways is the insertion of the fusion peptide or fusion loops into the target membrane. This step confers virus-target membrane association and can be monitored by testing the cofloatation of virus with target liposomes [37]. To test the role of Ca2+ in E1-membrane insertion, radiolabeled RuV was mixed with liposomes and treated at neutral or low pH in the presence of EDTA or CaCl2. Liposomes plus associated virus were separated on sucrose floatation gradients (Fig. 6A). Little virus was associated with the floated liposomes when treated at either pH in the absence of Ca2+. However, efficient virus-liposome interaction (∼74% of input virus) was observed when low pH treatment was carried out in the presence of 2 mM CaCl2. Even at pH 8.0, 2 mM CaCl2 caused a small increase (∼10%) in virus-liposome association. Once the virus was associated with liposomes, stable interaction was maintained even though the gradients did not contain Ca2+. Ca2+ did not promote non-specific electrostatic interactions of virus with membranes, since SFV-liposome floatation was strictly low pH-dependent and unaltered by the presence or absence of CaCl2 (Fig. 6B).


Rubella virus: first calcium-requiring viral fusion protein.

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

Calcium dependence of RuV-membrane interactions.(A–B) Virus-liposome binding assays. Radiolabeled RuV (A) or SFV (B) was incubated with liposomes for 1 min at 37°C at the indicated pH and buffer conditions. The percentage of total virus radioactivity associated with the liposomes was determined by sucrose gradient floatation. (C–D) Calcium requirement for virus fusion at the plasma membrane. (C) Purified RuV particles were labeled with DiD (red), prebound to Vero cells on ice, and the cells were pulsed at 37°C for 2 min in fusion medium containing 2 mM CaCl2 or 1.5 mM EDTA as indicated. Cells were then fixed and nuclei were counterstained with Hoescht (blue). Images represent a maximal projection of Z-stacks, with the scale bar indicating 25 µm. (D) Quantitation of C. DiD signal and cell numbers were quantitated and normalized to control, calcium-containing conditions. (E–F) Calcium requirement for virus fusion in endosomes. DiD-labeled virus was prebound to Vero cells on ice, and the cells with bound virus were incubated at 37°C for 20 min in binding medium containing 2 mM CaCl2 or 20 mM NH4Cl as indicated. Cells were then fixed, imaged and quantitated as in (C–D). Graphs show the mean and standard deviation of 3 (A, F) or 4 (D) independent experiments. For (B), the mean and range were calculated from 2 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004530-g006: Calcium dependence of RuV-membrane interactions.(A–B) Virus-liposome binding assays. Radiolabeled RuV (A) or SFV (B) was incubated with liposomes for 1 min at 37°C at the indicated pH and buffer conditions. The percentage of total virus radioactivity associated with the liposomes was determined by sucrose gradient floatation. (C–D) Calcium requirement for virus fusion at the plasma membrane. (C) Purified RuV particles were labeled with DiD (red), prebound to Vero cells on ice, and the cells were pulsed at 37°C for 2 min in fusion medium containing 2 mM CaCl2 or 1.5 mM EDTA as indicated. Cells were then fixed and nuclei were counterstained with Hoescht (blue). Images represent a maximal projection of Z-stacks, with the scale bar indicating 25 µm. (D) Quantitation of C. DiD signal and cell numbers were quantitated and normalized to control, calcium-containing conditions. (E–F) Calcium requirement for virus fusion in endosomes. DiD-labeled virus was prebound to Vero cells on ice, and the cells with bound virus were incubated at 37°C for 20 min in binding medium containing 2 mM CaCl2 or 20 mM NH4Cl as indicated. Cells were then fixed, imaged and quantitated as in (C–D). Graphs show the mean and standard deviation of 3 (A, F) or 4 (D) independent experiments. For (B), the mean and range were calculated from 2 independent experiments.
Mentions: An early step in viral membrane fusion pathways is the insertion of the fusion peptide or fusion loops into the target membrane. This step confers virus-target membrane association and can be monitored by testing the cofloatation of virus with target liposomes [37]. To test the role of Ca2+ in E1-membrane insertion, radiolabeled RuV was mixed with liposomes and treated at neutral or low pH in the presence of EDTA or CaCl2. Liposomes plus associated virus were separated on sucrose floatation gradients (Fig. 6A). Little virus was associated with the floated liposomes when treated at either pH in the absence of Ca2+. However, efficient virus-liposome interaction (∼74% of input virus) was observed when low pH treatment was carried out in the presence of 2 mM CaCl2. Even at pH 8.0, 2 mM CaCl2 caused a small increase (∼10%) in virus-liposome association. Once the virus was associated with liposomes, stable interaction was maintained even though the gradients did not contain Ca2+. Ca2+ did not promote non-specific electrostatic interactions of virus with membranes, since SFV-liposome floatation was strictly low pH-dependent and unaltered by the presence or absence of CaCl2 (Fig. 6B).

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