ms2 coat protein sequence  (GenScript corporation)

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: Bioconjugation of the B19 viral protein 1 unique region (VP1u) with the bacteriophage MS2 capsid, and internalization of the bioconjugate on erythroleukemic UT7/Epo cells: ( A ) Schematic depiction of the bioconjugation and labeling strategy of the bacteriophage MS2 capsid (PDB ID: 1MST ). The recombinant ∆C126 VP1u protein with a C-terminal metal affinity tag (MAT), FLAG tag, and a unique cysteine was coupled to the MS2 capsid using the heterobifunctional maleimide-PEG 24 -N-hydroxysuccinimide ester (NHS-PEG 24 -maleimide) crosslinker. The NHS-ester was conjugated to the surface lysines of the MS2 capsid and the maleimide group reacted with the unique cysteine in the VP1u. In addition, the capsid was labeled with the NHS-Atto 488 (green) and NHS-Atto 633 (blue) dyes; ( B ) Migration of the wild-type (WT) and bioconjugated MS2 capsids by agarose gel electrophoresis and detection of encapsidated RNA with GelRed. Capsid proteins are labeled with NHS-Atto dyes (green-blue); ( C ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the MS2 capsids, recombinant VP1u proteins, and the bioconjugates. Recombinant VP1u and MS2-VP1u conjugates were detected with an anti-FLAG antibody. In the Western blot, the transfer of low molecular weights proteins was less efficient; therefore, the detected signal did not quantitatively reflect the original and relative input of the proteins; ( D ) Uptake of the fluorescent MS2-∆C126 VP1u into UT7/Epo cells and detection of the internalized capsids in living cells by fluorescence microscopy and fluorescence-activated cell sorting (FACS); ( E ) Fluorescence microscopy images of the MS2-∆C126 VP1u and the Atto-labeled but internalization-deficient controls MS2-∆N30/∆C126 VP1u and MS2 (63× magnification). Scale bar: 15 µm.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Labeling, Recombinant, FLAG-tag, Migration, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Fluorescence, Microscopy, FACS

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: Competition of native B19V internalization with the MS2-VP1u bioconjugate. ( A ) Schematic comparison of the native B19V (three VP1u) with the MS2-∆C126 VP1u bioconjugate (20–30 PEG 24 -linked ∆C126 VP1u per capsid). Nucleic acids are encapsidated in the icosahedral assembled coat proteins (blue). The VP1u contains a phospholipase A 2 (PLA 2 ) motif (green) and a receptor-binding domain (red); ( B ) Internalization of B19V into UT7/Epo cells was measured in presence of increasing concentrations of MS2-VP1u as competitor. After 30 min incubation at 37 °C, cells were washed, trypsinized and internalized B19V DNA was quantified by quantitative polymersaase chain reaction (qPCR; ( C ) Competition of B19V uptake in presence of equimolar concentrations of MS2-∆C126 or internalization-deficient MS2-∆N30/∆C126 ( n = 3). Samples kept at 4 °C instead of incubation at 37 °C show the background signal without internalization.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Comparison, Binding Assay, Incubation

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: Native B19V and MS2-VP1u specifically target erythroid cell lines. ( A ) MS2-∆C126 VP1u and native B19V were incubated with the same mixed cell culture of non-adherent UT7/Epo cells, and adherent HeLa and MRC-5 cells. Cells were fixed and immunostained with an anti-B19V capsid antibody (860-55D ). Cell-specific internalization of MS2-∆C126 VP1u (green) and native B19V (magenta) was visualized by fluorescence microscopy. Scale bar: 100 µm (20× magnification), 15 µm (63× magnification); ( B ) Cellular uptake of native B19V into the different cell lines measured by qPCR ( n = 3); ( C ) Internalization of MS2-VP1u on normal and phorbol myristate acetate (PMA)-differentiated erythroleukemic K562 cells, visualized by fluorescence microscopy. Scale bar: 30 µm (63× magnification).

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Incubation, Cell Culture, Fluorescence, Microscopy

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: VP1u receptor expression correlates with B19V internalization and infection along the erythroid differentiation: ( A ) cluster of differentiation (CD) 34+ hematopoietic stem cells were expanded ex vivo to differentiate into the erythroid lineage and tested for MS2-∆C126 VP1u uptake (63× magnification). MS2-∆C126 VP1u internalization was further tested on peripheral blood cells, containing mature erythrocytes and ~1% reticulocytes, representing the final erythroid differentiation stages; ( B ) Immunostaining of glycophorin A (63× magnification) and May–Grünwald–Giemsa staining (100× magnification) indicates the successful transition to the terminal erythroid differentiation; B19V internalization ( C ) and infection ( D ) along the erythroid differentiation was quantified by qPCR ( n = 3); ( E ) Internalization of B19V into erythroid progenitor/precursor cells (day 8) was competed with ∆C126 VP1u or dysfunctional ∆N30/∆C126 VP1u; ( F ) After VP1u-competed uptake of B19V, cells (day 8) were incubated for further 24 h to measure the effect of the competition on the viral infection ( n = 3). Notably, B19V internalization was performed with more cells (2×) and higher multiplicity of infection (MOI) (7.5×) compared with infectivity assays, explaining the relatively high detection signal of uptake versus infection. Scale bar: 15 µm.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Expressing, Infection, Ex Vivo, Immunostaining, Staining, Incubation

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: The VP1u receptor is expressed on erythropoietin (EPO)-dependent erythroid differentiation stages: CD34+ hematopoietic stem cells were expanded in StemCell medium and then stimulated with 3 U/mL EPO. VP1u receptor expression, B19V uptake and infection were tested at different days after EPO-stimulation. ( A ) Fluorescence microscopy images and ( B ) flow cytometry analysis of MS2-∆C126 VP1u uptake into differentiated cells; ( C ) Histograms of the FACS data, showing the mean fluorescence of MS2-VP1u internalization per cell and ( D ) number of VP1u receptor-positive cells; ( E ) Quantification of B19V uptake and ( F ) infection before (day 0) or after stimulation with EPO ( n = 3). Scale bar: 15 µm.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Expressing, Infection, Fluorescence, Microscopy, Flow Cytometry