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Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion.

Liao M, Kielian M - J. Cell Biol. (2005)

Bottom Line: During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides.Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion.These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Alphaviruses and flaviviruses infect cells through low pH-dependent membrane fusion reactions mediated by their structurally similar viral fusion proteins. During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides. We demonstrate that exogenous domain III can function as a dominant-negative inhibitor of alphavirus and flavivirus membrane fusion and infection. Domain III binds stably to the fusion protein, thus preventing the foldback reaction and blocking the lipid mixing step of fusion. Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion. These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

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Related in: MedlinePlus

SFV E1 domain III proteins inhibit alphavirus infection in the endocytic pathway. SFV, SIN, VSV, and DV2 were diluted in medium of pH 7.2, containing the indicated concentrations of SFV domain III proteins and incubated with BHK cells for 1 h at 20°C to allow endocytic uptake. Infection was blocked by addition of medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Data are shown as a percentage of control infection in the absence of domain III proteins. Error bars are the mean ± SD. n = 3.
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fig4: SFV E1 domain III proteins inhibit alphavirus infection in the endocytic pathway. SFV, SIN, VSV, and DV2 were diluted in medium of pH 7.2, containing the indicated concentrations of SFV domain III proteins and incubated with BHK cells for 1 h at 20°C to allow endocytic uptake. Infection was blocked by addition of medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Data are shown as a percentage of control infection in the absence of domain III proteins. Error bars are the mean ± SD. n = 3.

Mentions: Because alphavirus receptor binding is not mediated by the E1 protein, we used this system to test the ability of domain III proteins to inhibit virus fusion from within the endosome, which is the physiological route of virus infection. We infected BHK cells with either SFV, SIN, vesicular stomatitis virus (VSV), or DV2, in the presence or absence of 20 μM His-DIIIS or 40 μM DIIIS. VSV, an unrelated rhabdovirus, and DV2 are important controls because these viruses also infect cells by endocytosis and low pH-triggered fusion (Matlin et al., 1982; Heinz and Allison, 2001). After a 1-h endocytic uptake period, NH4Cl was added to prevent further infection, and the primary infected cells were quantitated by immunofluorescence. Infection by both alphaviruses was significantly inhibited by the inclusion of either His-DIIIS or DIIIS (Fig. 4). In contrast, VSV and DV2 infection was not inhibited. Compared with the FIA, inhibition of alphavirus endocytic infection required a higher concentration of His-DIIIS and also showed lower efficacy versus untagged DIIIS. This could reflect relatively inefficient endocytic uptake of His-DIIIS by the cells or differential routing of virus and domain III within the endocytic pathway. Although its targeting to the endosomal site of virus fusion is probably not optimized, it is already clear that domain III protein can block fusion and infection under physiological virus entry conditions.


Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion.

Liao M, Kielian M - J. Cell Biol. (2005)

SFV E1 domain III proteins inhibit alphavirus infection in the endocytic pathway. SFV, SIN, VSV, and DV2 were diluted in medium of pH 7.2, containing the indicated concentrations of SFV domain III proteins and incubated with BHK cells for 1 h at 20°C to allow endocytic uptake. Infection was blocked by addition of medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Data are shown as a percentage of control infection in the absence of domain III proteins. Error bars are the mean ± SD. n = 3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171229&req=5

fig4: SFV E1 domain III proteins inhibit alphavirus infection in the endocytic pathway. SFV, SIN, VSV, and DV2 were diluted in medium of pH 7.2, containing the indicated concentrations of SFV domain III proteins and incubated with BHK cells for 1 h at 20°C to allow endocytic uptake. Infection was blocked by addition of medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Data are shown as a percentage of control infection in the absence of domain III proteins. Error bars are the mean ± SD. n = 3.
Mentions: Because alphavirus receptor binding is not mediated by the E1 protein, we used this system to test the ability of domain III proteins to inhibit virus fusion from within the endosome, which is the physiological route of virus infection. We infected BHK cells with either SFV, SIN, vesicular stomatitis virus (VSV), or DV2, in the presence or absence of 20 μM His-DIIIS or 40 μM DIIIS. VSV, an unrelated rhabdovirus, and DV2 are important controls because these viruses also infect cells by endocytosis and low pH-triggered fusion (Matlin et al., 1982; Heinz and Allison, 2001). After a 1-h endocytic uptake period, NH4Cl was added to prevent further infection, and the primary infected cells were quantitated by immunofluorescence. Infection by both alphaviruses was significantly inhibited by the inclusion of either His-DIIIS or DIIIS (Fig. 4). In contrast, VSV and DV2 infection was not inhibited. Compared with the FIA, inhibition of alphavirus endocytic infection required a higher concentration of His-DIIIS and also showed lower efficacy versus untagged DIIIS. This could reflect relatively inefficient endocytic uptake of His-DIIIS by the cells or differential routing of virus and domain III within the endocytic pathway. Although its targeting to the endosomal site of virus fusion is probably not optimized, it is already clear that domain III protein can block fusion and infection under physiological virus entry conditions.

Bottom Line: During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides.Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion.These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Alphaviruses and flaviviruses infect cells through low pH-dependent membrane fusion reactions mediated by their structurally similar viral fusion proteins. During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides. We demonstrate that exogenous domain III can function as a dominant-negative inhibitor of alphavirus and flavivirus membrane fusion and infection. Domain III binds stably to the fusion protein, thus preventing the foldback reaction and blocking the lipid mixing step of fusion. Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion. These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

Show MeSH
Related in: MedlinePlus