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Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis.

ABSTRACT: Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48-60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs derived from DNA/RNA-binding peptides, and discovered that a peptide derived from the flock house virus (FHV) coat protein was internalized most efficiently into Chinese hamster ovary (CHO-K1), HeLa, and Jurkat cells. Comparison of the factors facilitating the internalization with those of the Tat peptide revealed that the FHV peptide induces macropinocytosis much more efficiently than the Tat peptide, which leads to its high cellular uptake efficiency. Additionally, the strong adsorption of the FHV peptide on cell membranes via glycosaminoglycans (GAGs) was shown to be a key factor for induction of macropinocytosis, and these steps were successfully monitored by live imaging of the peptide internalization into cells in relation to the actin organization. The remarkable methods of FHV peptide internalization thus highlighted the critical factors for internalizations of the arginine-rich CPPs.

SUBMITTER: Nakase I

PROVIDER: S-EPMC2835031 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis.

Nakase Ikuhiko I Hirose Hisaaki H Tanaka Gen G Tadokoro Akiko A Kobayashi Sachiko S Takeuchi Toshihide T Futaki Shiroh S

Molecular therapy : the journal of the American Society of Gene Therapy 20090825 11

Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48-60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs ...[more]

PMID: 19707187

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Project description:The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Dataset Information

Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis.

Publications

Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis.

Similar Datasets

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