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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 the pH threshold for RuV fusion.(A) Fusion-infection assay. Virus was pre-bound to Vero cells as in Fig. 1A. Cells were washed and incubated for 3 min at 37°C in calcium-containing medium at the indicated pH, and then cultured 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were detected by immunofluorescence (green), and nuclei were counterstained with Hoescht (blue). (B) Quantitation of (A). Data were normalized to maximal fusion, which was observed at pH 6.2 in each experiment. Graph shows the mean and standard deviation of 4 independent experiments. (C) Kinetics of RuV fusion. A fusion infection assay was performed as in (A), using calcium-containing buffers at either pH 7.0 or pH 6.0 and treating for the indicated time at 37°C, followed by addition of growth medium containing 20 mM NH4Cl and culture for 48 h. Graph shows the mean and standard deviation of 3 independent experiments.
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ppat-1004530-g002: Characterization of the pH threshold for RuV fusion.(A) Fusion-infection assay. Virus was pre-bound to Vero cells as in Fig. 1A. Cells were washed and incubated for 3 min at 37°C in calcium-containing medium at the indicated pH, and then cultured 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were detected by immunofluorescence (green), and nuclei were counterstained with Hoescht (blue). (B) Quantitation of (A). Data were normalized to maximal fusion, which was observed at pH 6.2 in each experiment. Graph shows the mean and standard deviation of 4 independent experiments. (C) Kinetics of RuV fusion. A fusion infection assay was performed as in (A), using calcium-containing buffers at either pH 7.0 or pH 6.0 and treating for the indicated time at 37°C, followed by addition of growth medium containing 20 mM NH4Cl and culture for 48 h. Graph shows the mean and standard deviation of 3 independent experiments.

Mentions: Given the location of bound Ca2+ between the two E1 FLs [28], we hypothesized that it plays a role in RuV fusion with target membranes, a complex process not yet fully understood. Although RuV is known to require acidification to initiate fusion [23], [24], its pH threshold has not been determined. A fusion-infection assay previously developed for alphaviruses [34] was used to measure this precisely. RuV was prebound to Vero cells on ice, pulsed for 3 min at 37°C with medium of defined pH, and the resultant infected cells scored by immunofluorescence (Fig. 2A and B). No infection was detected upon treatment at pH 7.0, indicating that virus fusion in endosomes does not occur within this time frame. Infection was observed after treatment at pH 6.2, and gradually decreased at lower pH values, presumably reflecting virus inactivation by acidic pH. The kinetics of RuV fusion were then determined by pulsing bound RuV at pH 7.0 or 6.0 for various times (Fig. 2C). Infection increased with time of treatment at pH 6.0, reaching a maximum at 4 min. Infection from pH 7.0 incubation was first detected after 5 min and increased thereafter, reflecting the time required for virus endocytic uptake and endosomal fusion. Together our data indicate that the RuV fusion reaction is optimal at 4 min of treatment at ∼pH 6.0–6.2 at 37°C.


Rubella virus: first calcium-requiring viral fusion protein.

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

Characterization of the pH threshold for RuV fusion.(A) Fusion-infection assay. Virus was pre-bound to Vero cells as in Fig. 1A. Cells were washed and incubated for 3 min at 37°C in calcium-containing medium at the indicated pH, and then cultured 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were detected by immunofluorescence (green), and nuclei were counterstained with Hoescht (blue). (B) Quantitation of (A). Data were normalized to maximal fusion, which was observed at pH 6.2 in each experiment. Graph shows the mean and standard deviation of 4 independent experiments. (C) Kinetics of RuV fusion. A fusion infection assay was performed as in (A), using calcium-containing buffers at either pH 7.0 or pH 6.0 and treating for the indicated time at 37°C, followed by addition of growth medium containing 20 mM NH4Cl and culture for 48 h. Graph shows the mean and standard deviation of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004530-g002: Characterization of the pH threshold for RuV fusion.(A) Fusion-infection assay. Virus was pre-bound to Vero cells as in Fig. 1A. Cells were washed and incubated for 3 min at 37°C in calcium-containing medium at the indicated pH, and then cultured 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were detected by immunofluorescence (green), and nuclei were counterstained with Hoescht (blue). (B) Quantitation of (A). Data were normalized to maximal fusion, which was observed at pH 6.2 in each experiment. Graph shows the mean and standard deviation of 4 independent experiments. (C) Kinetics of RuV fusion. A fusion infection assay was performed as in (A), using calcium-containing buffers at either pH 7.0 or pH 6.0 and treating for the indicated time at 37°C, followed by addition of growth medium containing 20 mM NH4Cl and culture for 48 h. Graph shows the mean and standard deviation of 3 independent experiments.
Mentions: Given the location of bound Ca2+ between the two E1 FLs [28], we hypothesized that it plays a role in RuV fusion with target membranes, a complex process not yet fully understood. Although RuV is known to require acidification to initiate fusion [23], [24], its pH threshold has not been determined. A fusion-infection assay previously developed for alphaviruses [34] was used to measure this precisely. RuV was prebound to Vero cells on ice, pulsed for 3 min at 37°C with medium of defined pH, and the resultant infected cells scored by immunofluorescence (Fig. 2A and B). No infection was detected upon treatment at pH 7.0, indicating that virus fusion in endosomes does not occur within this time frame. Infection was observed after treatment at pH 6.2, and gradually decreased at lower pH values, presumably reflecting virus inactivation by acidic pH. The kinetics of RuV fusion were then determined by pulsing bound RuV at pH 7.0 or 6.0 for various times (Fig. 2C). Infection increased with time of treatment at pH 6.0, reaching a maximum at 4 min. Infection from pH 7.0 incubation was first detected after 5 min and increased thereafter, reflecting the time required for virus endocytic uptake and endosomal fusion. Together our data indicate that the RuV fusion reaction is optimal at 4 min of treatment at ∼pH 6.0–6.2 at 37°C.

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