Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of mutation of the RuV E1 Ca2+-coordinating residues on virus replication.(A) Close-up view of the tip of RuV E1 domain II [28] (PDB 4B3V). The two fusion loops are shown in cyan, the bound Ca2+ cation as an orange sphere, and the Ca2+-coordinating residues D136 and N88 in red. (B) Growth kinetics. BHK-21 cells were electroporated with the indicated viral RNAs, the growth medium collected at the indicated time points, and the progeny virus quantitated by infectious center (IC) assays. Data shown are the mean and range of 2 independent experiments. (C–D) Assembly assays. BHK-21 cells were electroporated with the indicated viral RNAs, and the growth medium and cell lysates harvested at 48 h. Virus particles in the medium and viral proteins in the lysates were visualized by SDS-PAGE and Western blot. (D) Quantitation of assembly assays performed as in (C). Graphs show the mean and standard deviation of 3 independent experiments, with release normalized to that of WT RuV.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256232&req=5

ppat-1004530-g007: Effect of mutation of the RuV E1 Ca2+-coordinating residues on virus replication.(A) Close-up view of the tip of RuV E1 domain II [28] (PDB 4B3V). The two fusion loops are shown in cyan, the bound Ca2+ cation as an orange sphere, and the Ca2+-coordinating residues D136 and N88 in red. (B) Growth kinetics. BHK-21 cells were electroporated with the indicated viral RNAs, the growth medium collected at the indicated time points, and the progeny virus quantitated by infectious center (IC) assays. Data shown are the mean and range of 2 independent experiments. (C–D) Assembly assays. BHK-21 cells were electroporated with the indicated viral RNAs, and the growth medium and cell lysates harvested at 48 h. Virus particles in the medium and viral proteins in the lysates were visualized by SDS-PAGE and Western blot. (D) Quantitation of assembly assays performed as in (C). Graphs show the mean and standard deviation of 3 independent experiments, with release normalized to that of WT RuV.

Mentions: The RuV E1 trimer structure shows that Ca2+ is coordinated by residues N88 and D136 in FL 1 and 2, respectively [28] (Fig. 7A). Such polar/charged residues are commonly involved in coordination of Ca2+, but are usually absent from fusion peptides [38], having the potential to hinder their insertion into the target membrane. We tested whether we could overcome the RuV calcium requirement by substituting these residues with alanine, an uncharged residue that is common in fusion loops [38]. Mutant viral RNAs were transcribed from the RuV infectious clone and electroporated into BHK-21 cells. Cells electroporated with WT RuV RNA produced infectious particles as soon as 24 h, and reached a maximum titer of 1×107 IC/ml at 72 h post-electroporation (Fig. 7B). In contrast, neither the single N88A or D136A mutants nor the double mutant N88A,D136A produced detectable infectious virus under the same conditions. Equivalent expression of E1, E2 and capsid was detected in WT vs. mutant-infected cells, ruling out a problem in mutant viral protein expression (Fig. 7C). The amount of virus particles pelleted from the medium at 48 h post-electroporation was comparable between WT and mutants (Fig. 7C). The efficiency of particle release (normalized to expression levels in the cell lysates) was equivalent between WT and mutants (Fig. 7D). Thus, mutation of the Ca2+-coordinating residues in RuV E1 does not overcome the requirement for calcium. Instead, these mutations are lethal to RuV infection without affecting virus synthesis and assembly.


Rubella virus: first calcium-requiring viral fusion protein.

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

Effect of mutation of the RuV E1 Ca2+-coordinating residues on virus replication.(A) Close-up view of the tip of RuV E1 domain II [28] (PDB 4B3V). The two fusion loops are shown in cyan, the bound Ca2+ cation as an orange sphere, and the Ca2+-coordinating residues D136 and N88 in red. (B) Growth kinetics. BHK-21 cells were electroporated with the indicated viral RNAs, the growth medium collected at the indicated time points, and the progeny virus quantitated by infectious center (IC) assays. Data shown are the mean and range of 2 independent experiments. (C–D) Assembly assays. BHK-21 cells were electroporated with the indicated viral RNAs, and the growth medium and cell lysates harvested at 48 h. Virus particles in the medium and viral proteins in the lysates were visualized by SDS-PAGE and Western blot. (D) Quantitation of assembly assays performed as in (C). Graphs show the mean and standard deviation of 3 independent experiments, with release normalized to that of WT RuV.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004530-g007: Effect of mutation of the RuV E1 Ca2+-coordinating residues on virus replication.(A) Close-up view of the tip of RuV E1 domain II [28] (PDB 4B3V). The two fusion loops are shown in cyan, the bound Ca2+ cation as an orange sphere, and the Ca2+-coordinating residues D136 and N88 in red. (B) Growth kinetics. BHK-21 cells were electroporated with the indicated viral RNAs, the growth medium collected at the indicated time points, and the progeny virus quantitated by infectious center (IC) assays. Data shown are the mean and range of 2 independent experiments. (C–D) Assembly assays. BHK-21 cells were electroporated with the indicated viral RNAs, and the growth medium and cell lysates harvested at 48 h. Virus particles in the medium and viral proteins in the lysates were visualized by SDS-PAGE and Western blot. (D) Quantitation of assembly assays performed as in (C). Graphs show the mean and standard deviation of 3 independent experiments, with release normalized to that of WT RuV.
Mentions: The RuV E1 trimer structure shows that Ca2+ is coordinated by residues N88 and D136 in FL 1 and 2, respectively [28] (Fig. 7A). Such polar/charged residues are commonly involved in coordination of Ca2+, but are usually absent from fusion peptides [38], having the potential to hinder their insertion into the target membrane. We tested whether we could overcome the RuV calcium requirement by substituting these residues with alanine, an uncharged residue that is common in fusion loops [38]. Mutant viral RNAs were transcribed from the RuV infectious clone and electroporated into BHK-21 cells. Cells electroporated with WT RuV RNA produced infectious particles as soon as 24 h, and reached a maximum titer of 1×107 IC/ml at 72 h post-electroporation (Fig. 7B). In contrast, neither the single N88A or D136A mutants nor the double mutant N88A,D136A produced detectable infectious virus under the same conditions. Equivalent expression of E1, E2 and capsid was detected in WT vs. mutant-infected cells, ruling out a problem in mutant viral protein expression (Fig. 7C). The amount of virus particles pelleted from the medium at 48 h post-electroporation was comparable between WT and mutants (Fig. 7C). The efficiency of particle release (normalized to expression levels in the cell lysates) was equivalent between WT and mutants (Fig. 7D). Thus, mutation of the Ca2+-coordinating residues in RuV E1 does not overcome the requirement for calcium. Instead, these mutations are lethal to RuV infection without affecting virus synthesis and assembly.

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