Negatively Charged Peptide Nanofibrils from Immunoglobulin Light Chain Sequester Viral Particles but Lack Cell-Binding and Viral Transduction-Enhancing Properties

Abstract

Positively charged naturally occurring or engineered peptide nanofibrils (PNF) are effective enhancers of lentiviral and retroviral transduction, an often rate-limiting step in gene transfer and gene therapy approaches. These polycationic PNF are thought to bridge the electrostatic repulsions between negatively charged membranes of virions and cells, thereby enhancing virion attachment to and infection of target cells. Here, we analyzed PNF, which are formed by the peptide AL1, that represents a fragment of an immunoglobulin light chain that causes systemic AL amyloidosis. We found that negatively charged AL1 PNF interact with viral particles to a comparable extent as positively charged PNF. However, AL1 PNF lacked cell-binding activity, and consequently, did not enhance retroviral infection. These findings show that virion capture and cell binding of PNF are mediated by different mechanisms, offering avenues for the design of advanced PNF with selective functions.

Figure 1

AL1 peptide forms negatively charged fibrils that do not enhance infection. (A) Transmission electron microscopy image of AL1 PNF; scale bar: 250 nm. (B) ThT emission spectrum of indicated concentrations of preformed AL1 PNF. (C) ζ-Potential of freshly dissolved and fibrillar PAP248-286 and AL1 peptides, and fibrillar EF-C PNF. (D) Metabolic activity of TZM-bl cells incubated for 3 days with indicated concentrations of AL1 PNF or buffer only. Values represent mean values ± standard deviation (SD) relative to the untreated control. (E) AL1 PNF lack HIV-1-enhancing activity. SEVI, EF-C, and AL1 PNF were incubated with HIV-1, and mixtures were added to TZM-bl reporter cells. After 3 days, infection rates were determined. Values represent ß-galactosidase activities obtained from triplicate infections ±SD (RLU/s: relative light units per second).

Figure 2

AL1 PNF bind retroviral particles. (A): Scanning electron microscopy and (B): Cryo-TEM of AL1 PNF (white arrows) incubated with retroviral MLV-Gag particles (blue; not all particles are colored to see detailed structures). Scale bars: 500 nm (A) and 100 nm (B). (C): confocal microscopy images of AL1 PNF stained with Proteostat dye (red) and incubated for 5 min with retroviral MLV-Gag-YFP particles (green); scale bar: 5 μm. (D, E) Representative fluorescence-activated cell sorting (FACS) histograms (D) and mean fluorescence intensities (MFI) (E) of fibrils incubated with serial MLV-Gag-YFP dilutions. The fibrils (each 100 μg/mL) were incubated with either phosphate-buffered saline (PBS) (no virus) or serial 10-fold dilutions of fluorescent MLV-Gag-YFP particles for 10 min and then analyzed by flow cytometry (see also Figure S1).

Figure 3

AL1 PNF lack cell-binding activity. (A) Representative confocal microscopy images of TZM-bl HeLa cells incubated with Proteostat-labeled (red) EF-C, SEVI, and AL1 PNF (each 5 μg/mL). After 1 h, the cells were imaged, washed three times in PBS, and imaged again. Nuclei were stained with Hoechst (blue). (B) Quantitative evaluation of Proteostat-labeled fibril binding using ImageJ from two individual experiments with three to four images each using ImageJ. Fluorescence intensity was normalized to intensities obtained before washing. (C) Flow cytometry evaluation of Proteostat-labeled fibril binding to TZM-bl HeLa cells. TZM-bl HeLa cells were incubated with the Proteostat-labeled fibrils, washed, trypsinized, and analyzed by flow cytometry. Shown are mean (±SD) of two independent experiments. Asterisks indicate background signal only. (D) TZM-bl HeLa cells were inoculated with HIV-1 that was exposed to indicated concentration of the fibrils. After 3 h, the cells were washed three times, lysed, and analyzed for viral capsid antigen by p24 enzyme-linked immunosorbent (ELISA). Shown are mean (±SD) of two independent experiments.