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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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Characterization of RuV E1 N88A,D136A virus.(A) Cell binding. Radiolabeled WT RuV or N88A,D136A mutant was bound to Vero cells on ice. Binding efficiency was calculated relative to WT RuV. (B) Stability of the RuV E2-E1 heterodimer. Radiolabeled WT RuV or N88A,D136A mutant viruses were lysed in detergent, E1 was immunoprecipitated, and co-retrieval of E2 was determined by SDS-PAGE and autoradiography. (C) E1 trypsin resistance. Radiolabeled WT RuV or N88A,D136A mutant was treated for 1 min at 37°C and the indicated pH, and E1 trypsin-resistance quantitated as in Fig. 5C. (D) Virus-liposome association. Liposome association of radiolabeled WT RuV or N88A,D136A mutant was determined as in Fig. 6A in presence of 2 mM CaCl2. Graphs show the mean and standard deviation of 5 (A) or 3 (C and D) independent experiments.
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ppat-1004530-g008: Characterization of RuV E1 N88A,D136A virus.(A) Cell binding. Radiolabeled WT RuV or N88A,D136A mutant was bound to Vero cells on ice. Binding efficiency was calculated relative to WT RuV. (B) Stability of the RuV E2-E1 heterodimer. Radiolabeled WT RuV or N88A,D136A mutant viruses were lysed in detergent, E1 was immunoprecipitated, and co-retrieval of E2 was determined by SDS-PAGE and autoradiography. (C) E1 trypsin resistance. Radiolabeled WT RuV or N88A,D136A mutant was treated for 1 min at 37°C and the indicated pH, and E1 trypsin-resistance quantitated as in Fig. 5C. (D) Virus-liposome association. Liposome association of radiolabeled WT RuV or N88A,D136A mutant was determined as in Fig. 6A in presence of 2 mM CaCl2. Graphs show the mean and standard deviation of 5 (A) or 3 (C and D) independent experiments.

Mentions: Fusion-infection assays of the mutants revealed that their infectivity was not rescued even by low pH-treatment in the presence of 20 mM CaCl2 (Fig. S6). We therefore tested specific steps of entry and fusion for the N88A,D136A double mutant. Binding studies showed that the efficiency of radiolabeled WT and mutant virus binding to Vero cells on ice was comparable (Fig. 8A). Co-immunoprecipitation analysis of solubilised virus showed that both the WT and mutant E1 proteins were efficiently recognized by a commercial mAb to RuV E1 and that similar levels of E2 were retrieved, indicating comparable stability of the E2-E1 dimer interaction (Fig. 8B). Together with the efficient assembly of the mutant (Fig. 7D), these results thus suggest that the folding of N88A,D136A E1 is not aberrant.


Rubella virus: first calcium-requiring viral fusion protein.

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

Characterization of RuV E1 N88A,D136A virus.(A) Cell binding. Radiolabeled WT RuV or N88A,D136A mutant was bound to Vero cells on ice. Binding efficiency was calculated relative to WT RuV. (B) Stability of the RuV E2-E1 heterodimer. Radiolabeled WT RuV or N88A,D136A mutant viruses were lysed in detergent, E1 was immunoprecipitated, and co-retrieval of E2 was determined by SDS-PAGE and autoradiography. (C) E1 trypsin resistance. Radiolabeled WT RuV or N88A,D136A mutant was treated for 1 min at 37°C and the indicated pH, and E1 trypsin-resistance quantitated as in Fig. 5C. (D) Virus-liposome association. Liposome association of radiolabeled WT RuV or N88A,D136A mutant was determined as in Fig. 6A in presence of 2 mM CaCl2. Graphs show the mean and standard deviation of 5 (A) or 3 (C and D) independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004530-g008: Characterization of RuV E1 N88A,D136A virus.(A) Cell binding. Radiolabeled WT RuV or N88A,D136A mutant was bound to Vero cells on ice. Binding efficiency was calculated relative to WT RuV. (B) Stability of the RuV E2-E1 heterodimer. Radiolabeled WT RuV or N88A,D136A mutant viruses were lysed in detergent, E1 was immunoprecipitated, and co-retrieval of E2 was determined by SDS-PAGE and autoradiography. (C) E1 trypsin resistance. Radiolabeled WT RuV or N88A,D136A mutant was treated for 1 min at 37°C and the indicated pH, and E1 trypsin-resistance quantitated as in Fig. 5C. (D) Virus-liposome association. Liposome association of radiolabeled WT RuV or N88A,D136A mutant was determined as in Fig. 6A in presence of 2 mM CaCl2. Graphs show the mean and standard deviation of 5 (A) or 3 (C and D) independent experiments.
Mentions: Fusion-infection assays of the mutants revealed that their infectivity was not rescued even by low pH-treatment in the presence of 20 mM CaCl2 (Fig. S6). We therefore tested specific steps of entry and fusion for the N88A,D136A double mutant. Binding studies showed that the efficiency of radiolabeled WT and mutant virus binding to Vero cells on ice was comparable (Fig. 8A). Co-immunoprecipitation analysis of solubilised virus showed that both the WT and mutant E1 proteins were efficiently recognized by a commercial mAb to RuV E1 and that similar levels of E2 were retrieved, indicating comparable stability of the E2-E1 dimer interaction (Fig. 8B). Together with the efficient assembly of the mutant (Fig. 7D), these results thus suggest that the folding of N88A,D136A E1 is not aberrant.

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