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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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Effect of Ca2+ on RuV infection.(A) RuV or SFV was pre-bound to Vero cells on ice for 90 min in binding medium. Cells were shifted to 37°C for 20 min in medium containing the indicated concentrations of CaCl2, and then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were scored by immunofluorescence. Infectivity was normalized to that observed at 2 mM CaCl2, which was ∼15% infected cells. (B) RuV was prebound to Vero cells as in panel A and incubated at 37°C for 20 min in medium with or without 2 mM CaCl2 (Uptake). The cells were then incubated for 1 h at 37°C in medium with or without 2 mM CaCl2, to test if infection could be rescued by the addition of Ca2+ (Rescue). The cells were then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl, scored by immunofluorescence, and infectivity normalized to that observed when 2 mM CaCl2 was present throughout the experiment. Data in A are the mean and range of 2 independent experiments. Data in B are the mean and standard deviation from 3 independent experiments. Statistical analysis was performed in (B) using a paired Student's t test.
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ppat-1004530-g001: Effect of Ca2+ on RuV infection.(A) RuV or SFV was pre-bound to Vero cells on ice for 90 min in binding medium. Cells were shifted to 37°C for 20 min in medium containing the indicated concentrations of CaCl2, and then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were scored by immunofluorescence. Infectivity was normalized to that observed at 2 mM CaCl2, which was ∼15% infected cells. (B) RuV was prebound to Vero cells as in panel A and incubated at 37°C for 20 min in medium with or without 2 mM CaCl2 (Uptake). The cells were then incubated for 1 h at 37°C in medium with or without 2 mM CaCl2, to test if infection could be rescued by the addition of Ca2+ (Rescue). The cells were then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl, scored by immunofluorescence, and infectivity normalized to that observed when 2 mM CaCl2 was present throughout the experiment. Data in A are the mean and range of 2 independent experiments. Data in B are the mean and standard deviation from 3 independent experiments. Statistical analysis was performed in (B) using a paired Student's t test.

Mentions: The importance of calcium during RuV entry was tested by pre-binding virus to target Vero cells on ice and then incubating at 37°C in medium containing various concentrations of CaCl2. To avoid possible effects of calcium deprivation on endosomal acidification [33], the internalization period was limited to 20 min. Cells were then incubated in growth medium containing NH4Cl to neutralize endosomal pH, and infected cells scored at 48 h post-infection by immunofluorescence (Fig. 1A). RuV infection was strongly dependent on the concentration of Ca2+, with infection efficiency decreased by more than 90% in the presence of 30 µM CaCl2 vs. 2 mM CaCl2. Infection by the low pH-dependent alphavirus Semliki Forest Virus (SFV) was independent of Ca2+ under these conditions, excluding aberrant endosomal acidification. Addition of CaCl2 after the 20 min internalization period but prior to endosomal neutralization by NH4Cl did not cause significant rescue of RuV infection (p>0.05) (Fig. 1B). This result suggests that the internalized RuV particles were inactivated by endosomal acidity during the 20 min uptake period. While the lack of rescue could be due to several factors such as rapid acid-inactivation and/or an inability of added calcium to access the virus-containing endosomes, it demonstrates that Ca2+ deprivation did not simply delay RuV endocytosis. Together our data support a critical and specific role of Ca2+ during productive RuV entry.


Rubella virus: first calcium-requiring viral fusion protein.

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

Effect of Ca2+ on RuV infection.(A) RuV or SFV was pre-bound to Vero cells on ice for 90 min in binding medium. Cells were shifted to 37°C for 20 min in medium containing the indicated concentrations of CaCl2, and then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were scored by immunofluorescence. Infectivity was normalized to that observed at 2 mM CaCl2, which was ∼15% infected cells. (B) RuV was prebound to Vero cells as in panel A and incubated at 37°C for 20 min in medium with or without 2 mM CaCl2 (Uptake). The cells were then incubated for 1 h at 37°C in medium with or without 2 mM CaCl2, to test if infection could be rescued by the addition of Ca2+ (Rescue). The cells were then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl, scored by immunofluorescence, and infectivity normalized to that observed when 2 mM CaCl2 was present throughout the experiment. Data in A are the mean and range of 2 independent experiments. Data in B are the mean and standard deviation from 3 independent experiments. Statistical analysis was performed in (B) using a paired Student's t test.
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Related In: Results  -  Collection

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ppat-1004530-g001: Effect of Ca2+ on RuV infection.(A) RuV or SFV was pre-bound to Vero cells on ice for 90 min in binding medium. Cells were shifted to 37°C for 20 min in medium containing the indicated concentrations of CaCl2, and then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl to prevent secondary infection. Infected cells were scored by immunofluorescence. Infectivity was normalized to that observed at 2 mM CaCl2, which was ∼15% infected cells. (B) RuV was prebound to Vero cells as in panel A and incubated at 37°C for 20 min in medium with or without 2 mM CaCl2 (Uptake). The cells were then incubated for 1 h at 37°C in medium with or without 2 mM CaCl2, to test if infection could be rescued by the addition of Ca2+ (Rescue). The cells were then cultured for 48 h at 37°C in growth medium plus 20 mM NH4Cl, scored by immunofluorescence, and infectivity normalized to that observed when 2 mM CaCl2 was present throughout the experiment. Data in A are the mean and range of 2 independent experiments. Data in B are the mean and standard deviation from 3 independent experiments. Statistical analysis was performed in (B) using a paired Student's t test.
Mentions: The importance of calcium during RuV entry was tested by pre-binding virus to target Vero cells on ice and then incubating at 37°C in medium containing various concentrations of CaCl2. To avoid possible effects of calcium deprivation on endosomal acidification [33], the internalization period was limited to 20 min. Cells were then incubated in growth medium containing NH4Cl to neutralize endosomal pH, and infected cells scored at 48 h post-infection by immunofluorescence (Fig. 1A). RuV infection was strongly dependent on the concentration of Ca2+, with infection efficiency decreased by more than 90% in the presence of 30 µM CaCl2 vs. 2 mM CaCl2. Infection by the low pH-dependent alphavirus Semliki Forest Virus (SFV) was independent of Ca2+ under these conditions, excluding aberrant endosomal acidification. Addition of CaCl2 after the 20 min internalization period but prior to endosomal neutralization by NH4Cl did not cause significant rescue of RuV infection (p>0.05) (Fig. 1B). This result suggests that the internalized RuV particles were inactivated by endosomal acidity during the 20 min uptake period. While the lack of rescue could be due to several factors such as rapid acid-inactivation and/or an inability of added calcium to access the virus-containing endosomes, it demonstrates that Ca2+ deprivation did not simply delay RuV endocytosis. Together our data support a critical and specific role of Ca2+ during productive RuV entry.

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