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F-actin modulates measles virus cell-cell fusion and assembly by altering the interaction between the matrix protein and the cytoplasmic tail of hemagglutinin.

ABSTRACT: Actin filament (F-actin) is believed to be involved in measles virus (MV) assembly as a cellular factor, but the precise roles remain unknown. Here we show that Phe at position 50 of the MV matrix (M) protein is important for its association with F-actin, through which the function of the M protein is regulated. In plasmid-expressed or MV-infected cells, a coimmunoprecipitation study revealed that the wild-type M (M-WT) protein associated strongly with F-actin but only weakly with the cytoplasmic tail of the hemagglutinin (H) protein. Since the F50P mutation allowed the M protein the enhanced interaction with the H protein in return for the sharply declined association with F-actin, the mutant M (M-F50P) protein strongly inhibited MV cell-cell fusion and promoted the uptake of the H protein into virus particles. The abundantly incorporated H protein resulted in the increase in infectivity of the F50P virus, although the virus contained a level of genome RNA equal to that of the WT virus. When the structure of F-actin was disrupted with cytochalasin D, the M-WT protein liberated from F-actin interacted with the H protein as tightly as the M-F50P protein, suppressing cell-cell fusion and promoting virus assembly comparably efficiently as the M-F50P protein. The cell-cell fusion activity of the WT virus appeared to be upheld by F-actin, which prevents the M protein interaction with the H protein. Our results indicate that F-actin in association with the M protein alters the interaction between the M and H proteins, thereby modulating MV cell-cell fusion and assembly.

SUBMITTER: Wakimoto H

PROVIDER: S-EPMC3571460 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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F-actin modulates measles virus cell-cell fusion and assembly by altering the interaction between the matrix protein and the cytoplasmic tail of hemagglutinin.

Wakimoto Hiroshi H Shimodo Masakatsu M Satoh Yuto Y Kitagawa Yoshinori Y Takeuchi Kaoru K Gotoh Bin B Itoh Masae M

Journal of virology 20121205 4

Actin filament (F-actin) is believed to be involved in measles virus (MV) assembly as a cellular factor, but the precise roles remain unknown. Here we show that Phe at position 50 of the MV matrix (M) protein is important for its association with F-actin, through which the function of the M protein is regulated. In plasmid-expressed or MV-infected cells, a coimmunoprecipitation study revealed that the wild-type M (M-WT) protein associated strongly with F-actin but only weakly with the cytoplasmi ...[more]

PMID: 23221571

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Project description:Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.

Project description:The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incorporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult. In direct binding assays, we show here that MA binds to Env CTs. Using MA mutants, matrix-capsid (MACA) proteins, and MA proteins incubated in the presence of inositol polyphosphate, we show a correlation between MA trimerization and CT binding. RNA ligands with high affinities for MA reduced MA-CT binding levels, suggesting that MA-RNA binding interferes with trimerization and/or directly or indirectly blocks MA-CT binding. Rough-mapping studies indicate that C-terminal CT helices are involved in MA binding and are in agreement with cell culture studies with replication-competent viruses. Our results support a model in which full-length HIV-1 Env trimers are captured in assembling PrGag lattices by virtue of their binding to MA trimers.IMPORTANCE The mechanism by which HIV-1 envelope (Env) protein trimers assemble into virus particles is poorly understood but involves an interaction between Env cytoplasmic tails (CTs) and the matrix (MA) domain of the structural precursor Gag (PrGag) proteins. We show here that direct binding of MA to Env CTs correlates with MA trimerization, suggesting models where MA lattices regulate CT interactions and/or MA-CT trimer-trimer associations increase the avidity of MA-CT binding. We also show that MA binding to RNA ligands impairs MA-CT binding, potentially by interfering with MA trimerization and/or directly or allosterically blocking MA-CT binding sites. Rough mapping implicated CT C-terminal helices in MA binding, in agreement with cell culture studies on MA-CT interactions. Our results indicate that targeting HIV-1 MA-CT interactions may be a promising avenue for antiviral therapy.

Project description:Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

Dataset Information

F-actin modulates measles virus cell-cell fusion and assembly by altering the interaction between the matrix protein and the cytoplasmic tail of hemagglutinin.

Publications

F-actin modulates measles virus cell-cell fusion and assembly by altering the interaction between the matrix protein and the cytoplasmic tail of hemagglutinin.

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