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Ivanovic, Tijana

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Ivanovic

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Tijana

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Ivanovic, Tijana
Author's Publications: search.filters.isAuthorOfPublication.01452f64-e795-437f-8fdb-e2b34af47066

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    Kinetics of Proton Transport into Influenza Virions by the Viral M2 Channel
    (Public Library of Science, 2012) Ivanovic, Tijana; Rozendaal, Rutger; Floyd, Daniel Lee; Popovic, Milos; van Oijen, Antoine M.; Harrison, Stephen
    M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a \(pK_m\) of ~4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.
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    Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates
    (eLife Sciences Publications, Ltd, 2013) Ivanovic, Tijana; Choi, Jason L; Whelan, Sean; van Oijen, Antoine M; Harrison, Stephen
    Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism.