Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

<div><p>Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.</p> </div

Generation of filamentous M13 phages displaying cell-penetrating 3D8 VL transbody (3D8 VL-M13) and TAT peptide (TAT-M13).

<p>As a control, anti-DR4 hAY4 scFv without cell-penetrating ability was also employed; the phage particles are designated as hAY4 scFv-M13. (<b>A</b>) Phage titers obtained from 100-ml culture supernatants of recombinant phagemid-transformed bacteria by VCSM13 helper phage superinfection under optimal culture conditions as described in the text. Phage titers were determined by CFU assay. Data represent mean ± S.E. (<i>error bars</i>) of 5 independent experiments. (<b>B</b>) Western blot analysis of display efficiency of fusion proteins (insert-pIII) (<i>filled arrow</i>) <i>versus</i> full-length pIII (<i>open arrow</i>) from VCSM13 helper phage on the recombinant M13 filamentous phage particles. Equal titers (10⁹ or 10¹⁰ CFU) of phage particles prepared as described in (A) were western blotted with anti-myc antibody to detect only pIII-fusion proteins (3D8 VL-pIII, and TAT-pIII, and hAY4 scFv-pIII) from the phagemid vectors or anti-M13 pIII antibody to detect both pIII-fusion proteins from the phagemid vectors and full-length pIII from the helper phage. The positions of molecular size marker are indicated. (<b>C</b>) Phage ELISA on DR4 or DNA to examine antigen-binding specificity of the recombinant phages. Various titers (10⁷∼10¹¹ CFU) of recombinant phages or VCSM13 helper phage were applied to each well, precoated with the indicated antigen. Bound phages were detected with HRP-conjugated anti-M13 antibody. Data represent mean ± S.E. (<i>error bars</i>) of three independent experiments carried out in triplicate.</p

A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

<div><p>Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an <i>in vivo</i> mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses <i>in vitro</i> in HeLa cells and in an <i>in vivo</i> mouse system.</p></div

3D8 VL-M13 is internalized by caveolae-mediated endocytosis, whereas TAT-M13 by clathrin- and caveolae-mediated endocytosis.

<p>Unless otherwise specified, HeLa cells (1×10⁶ cells) in serum-free medium were treated at 37°C with 10¹² CFU of 3D8 VL-M13 for 6 h or 10¹³ CFU of TAT-M13 for 2 h. (<b>A</b>) Effect of pre-treatment of specific endocytosis inhibitors on the internalization of 3D8 VL-M13 or TAT-M13. HeLa cells were pre-treated with CPZ (1 µg/ml), MβCD (5 mM), or Cyt-D (1 µg/ml) for 30 min and then incubated with 3D8 VL-M13 or TAT-M13. Internalized phages were visualized by confocal immunofluorescence microscopy using primary anti-pVIII antibody and secondary TRITC-anti-mouse antibody or quantified by the CFU assay and represented as mean ± S.E. (<i>error bars</i>) of 3 independent experiments. Images show the merging of phages (<i>red</i>) and DAPI-stained nuclei (<i>blue</i>) at the centered single confocal section. (<b>B</b>) Co-localization of internalized 3D8 VL-M13 or TAT-M13 with intracellular endocytosis markers. Cells were co-treated for 2 h with the recombinant phages and endocytosis markers, 10 µg/ml Alexa 488-transferrin (TF, <i>green</i>), Alexa 488-choleratoxin-B (Ctx-B, green), or FITC-dextran (Dextran, green), and then analyzed by confocal microscopy after staining for internalized phages (TRITC, <i>red</i>) as described in (A). The lower panels show enlarged images of the boxed region in the upper panels. (<b>C</b>) Knockdown of clathrin, caveolin-1, or dynamin by specific siRNA, monitored by Western blotting (<i>left panels</i>), and the effects on internalization of 3D8 VL-M13 and TAT-M13 (<i>right panels</i>). HeLa cells were transfected with the indicated siRNA for 48 h and then incubated with 3D8 VL-M13 and TAT-M13 prior to phage visualization by confocal immunofluorescence microscopy as described in (A). ‘Control siRNA’ means a scrambled siRNA used as a control. In (A–C), image magnification, ×400 and scale bar, 5 µm.</p

3D8 VL-M13 and TAT-M13 mainly interact with HSPG and CSPG, respectively, as a cell surface receptor for the cellular internalization.

<p>In all experiments, wild-type CHO-K1 and the mutant cells (1×10⁶ cells) in serum-free medium were treated at 37°C with 10¹² CFU of 3D8 VL-M13 for 6 h or 10¹³ CFU of TAT-M13 for 2 h. (<b>A</b>) Effects of the presence of soluble GAGs on internalization of 3D8 VL-M13 and TAT-M13. CHO-K1 cells were pre-treated with 100 IU/ml of heparin, or 50 µg/ml of CS-A, CS-B, and CS-C for 30 min and then incubated with 3D8 VL-M13 or TAT-M13. (<b>B</b>) Effects of treatment of cells with GAG lyases on the internalization of 3D8 VL-M13 and TAT-M13. CHO-K1 cells were pre-treated with 5 mIU/ml of heparinase III (Hep III) or 20 mIU/ml of chondroitinase ABC (Chon ABC) for 2 h at 37°C and then incubated with 3D8 VL-M13 or TAT-M13. (<b>C</b>) Internalization of 3D8 VL-M13 and TAT-M13 into CHO-K1 mutant cells genetically defective in GAG biosynthesis. Wild-type CHO-K1, HS-deficient pgsD-677 (no HS, 3 times more CS), or HS/CS-deficient pgsA-745 (no proteoglycans) cells were incubated with 3D8 VL-M13 or TAT-M13. In (A–C), internalized phages were visualized by confocal immunofluorescence microscopy using primary anti-pVIII antibody and secondary TRITC-anti-mouse antibody (A and B, <i>red</i>) or FITC-anti-mouse antibody (C, <i>green</i>) or quantified by the CFU assay and represented as mean ± S.E. (<i>error bars</i>) of three independent experiments. Image magnification, ×400; scale bar, 5 µm.</p

3D8 VL-M13 and TAT-M13 phages penetrate living cells in an energy-dependent manner and in the presence of serum proteins, and can be rescued in their infective form in proportion to the input titer.

<p>Unless otherwise specified, HeLa cells (1×10⁶ cells) in serum-free medium were treated at 37°C with 10¹² CFU of VCSM13, 3D8 VL-M13, or hAY4 scFv-M13 for 6 h or 10¹³ CFU of TAT-M13 for 2 h. (<b>A</b>) Internalization and subcellular localization of phage particles in HeLa cells, untreated (‘control’) or treated with VCSM13 helper phage or the indicated recombinant phages and analyzed by confocal immunofluorescence microscopy. Internalized phages were detected by primary anti-pVIII antibody and secondary FITC-anti-mouse antibody. Images show the merging of phages (<i>green</i>) and DAPI-stained nuclei (<i>blue</i>) at the centered single confocal section. (<b>B</b>) Effect of input phage titer on the recovery of internalized recombinant phages. HeLa cells were incubated with 10¹⁰, 10¹¹, or 10¹² CFU of phages (input phage titer), thoroughly washed with low pH glycine buffer to remove surface bound phages, and lysed. Bacteria were infected with the cell lysates to determine output phage titer by CFU assay. Data represent mean ± S.E. of 3 independent experiments. (<b>C</b>) Plasmid DNA analysis from the recovered phage particles. Cell lysates prepared as described in (B) were transformed into bacteria and then plasmid DNA was extracted from randomly chosen two colonies (<i>#1</i>, <i>#2</i>). The recovered plasmid DNA was digested with <i>Sfi</i>I to excise the DNA insert [3D8 VL (366 bp) or TAT (62 bp)] prior to agarose gel electrophoresis and visualization by ethidium bromide staining. ‘C’ indicates the original phagemid vector carrying the 3D8 VL or TAT gene. ‘M’, DNA size marker. (<b>D</b>) Effects of incubation time, temperature, and presence of serum proteins on internalization of 3D8 VL-M13 and TAT-M13 phages. HeLa cells were incubated with 3D8 VL-M13 or TAT-M13 for the indicated periods (<i>left panels</i>), at 4°C or 37°C (<i>middle panels</i>), or at 37°C in the presence or absence of 10% fetal bovine serum (<i>right panels</i>). Internalized phages were quantified by the CFU assay and represented as mean ± S.E. of 2 independent experiments as described in (B) and visualized by confocal immunofluorescence microscopy as described in (A). In (A) and (D), image magnification, ×400; scale bar, 5 µm.</p

STG90 exhibits antiviral effects against PRV.

<p><b>A</b>. The expression levels of PRV gpD RNA in the muscle and brain of WT-Mock, WT-PRV, STG69-PRV, STG90-PRV, and STG135-PRV mice were investigated. Only live STG90-PRV mice did not show PRV gpD expression. <b>B</b>. Immunohistochemistry was performed to detect PRV in PRV target organs; brain (upper panel: ×100) and femoral muscle (bottom panel: ×400). The PRV gpD protein was stained with a monoclonal anti-PRV antibody and visualized with DAB. The Purkinje layer cells in the WT-PRV group stained brown for the gpD protein, whereas no staining was observed in the other groups.</p

New antiviral mechanism by 3D8 scFv protein.

<p><b>A</b>. Model of the HSV-I replication cycle. Virus infection begins with binding of the virus to the cell surface. The viral envelope fuses with the cell membrane and delivers the viral capsid into the cytoplasm. Viral DNA synthesis begins shortly after the appearance of the beta proteins and the temporal program of viral gene expression ends with the appearance of the gamma or late proteins, which constitute the structural proteins of the virus. Finally, the virus undergoes a lytic cycle. <b>B</b>. Expression model of 3D8 scFv proteins. 3D8 scFv proteins were localized in cytosol using a vector system and targeted in the nucleus by nuclear localization signal. Therefore, 3D8 scFv proteins in SCH07072 were present in both the cytosol and nucleus. <b>C</b>. 3D8 scFv has a unique dual and stereoscopic protection mechanism that includes DNase activity in the nucleus and RNase activity in the cytoplasm. 3D8 scFv acts by inhibiting (1) viral DNA replication and RNA transcription in the nucleus via viral DNA degradation and (2) translation in the cytoplasm via viral RNA degradation. In other words, 3D8 scFv targets the viral DNA genome itself or its RNA transcripts spatially in two different subcellular spaces (nucleus and cytoplasm) and at two different times.</p

Internalized 3D8 VL-M13 phage routes to the cytosol and remains stable without further trafficking to other subcellular compartments, whereas TAT-M13 phage is routed to other subcellular compartments before rapid degradation in the lysosome.

<p>In the following pulse-chase experiments, HeLa cells (1×10⁶ cells) in serum-free medium were treated at 37°C with 10¹² CFU of 3D8 VL-M13 for 2 h or 10¹³ CFU of TAT-M13 for 30 min. Then surface bound phages were removed by multiple washes with low pH glycine buffer and then internalized phages were chased at 0, 2, 6, and 18 h. (<b>A</b>) Time-course intracellular localization of internalized phages was visualized by confocal immunofluorescence microscopy or time-course output phages were quantified by the CFU assay and represented as mean ± S.E. (<i>error bars</i>) of three independent experiments. (<b>B</b>) Time-course intracellular trafficking of internalized phages monitored by co-localization with transferrin (TF, <i>a</i>), caveolin-1 (<i>b</i>), early endosome marker EEA-1 (<i>c</i>), late endosome/lysosome tracker LysoTracker (<i>d</i>), ER marker calnexin (<i>e</i>), or Golgi marker 58K Golgi protein (<i>f</i>), as visualized by confocal immunofluorescence microscopy. In (A) and (B), internalized phages were visualized by confocal immunofluorescence microscopy with primary anti-pVIII antibody and secondary FITC-anti-mouse antibody (A and B, <i>a</i>, <i>green</i>) or TRITC-anti-mouse antibody (B, <i>b-f</i>, <i>green</i>). In (A) and (B), magnification, ×400; scale bar, 5 µm.</p

3D8 scFv inhibits HSV-1 encoded gene expression by DNase and RNase activity.

<p><b>A</b>. Schematic diagram for identification of viral gene expression pattern by HSV-1 challenging. <b>B</b>. Wild-type HeLa, SCH07072, and muSCH cells were infected with HSV::GFV at an MOI of 0.5 and then incubated for 2, 6.5, and 25 hr in the presence or absence of PAA 1 hr after virus challenge. ICP4 was used for the immediate early stage, UL9 for the early stage, and UL19 for the late stage of viral infection. Data bars show mean ± standard error. ** and *** indicate significant differences from HSV-1 viral DNA at <i>p<0.01</i> and <i>p<0.001</i>, respectively (one-way analysis of variance and Tukey's post hoc <i>t</i>-test). <b>C</b>. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression of immediate early genes (ICP0 and ICP4), early genes (UL9 and UL29), and late genes (UL19 and UL38). The relative concentrations of HSV mRNAs were calculated after normalization to the GAPDH gene using the delta delta C_T method. Data bars represent mean ± standard error. *, **, *** indicate significant differences from HeLa cells at <i>p<0.05, p<0.01</i>, and <i>p<0.001</i>, respectively (one-way analysis of variance and Tukey's post hoc <i>t</i>-test).</p