Volume 113, Issue 7, Pages 1425-1439 (October 2017) A Degraded Fragment of HIV-1 Gp120 in Rat Hepatocytes Forms Fibrils and Enhances HIV-1 Infection  Jinquan Chen, Ruxia Ren, Fei Yu, Chunyan Wang, Xuanxuan Zhang, Wenjuan Li, Suiyi Tan, Shibo Jiang, Shuwen Liu, Lin Li  Biophysical Journal  Volume 113, Issue 7, Pages 1425-1439 (October 2017) DOI: 10.1016/j.bpj.2017.08.005 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 DPF1 forms fibrils by self-assembly. The spontaneous formation of DPF1 fibrils at different concentrations was assessed by ThT fluorescence (A) and Congo Red staining assays (B). Here, the samples of DPF1 and DPF1scr (8, 4, 2, 1, 0.5, 0.25, and 0 mg/mL) were collected immediately after being diluted into deionized water. (C) DPF1 (2 mg/mL) rapidly formed fibrils at the indicated time points, as shown by ThT assay. (D) The spectra of DPF1 (250 μg/mL) were observed by CD. EP2 (250 μg/mL) and DPF1scr (250 μg/mL) were also observed by CD as controls. (E) DPF1 fibrils were visualized by negative-stain TEM. The scale bar represents 1 μm (magnification: 20,000×). EP2 and DPF1scr were used as positive and negative controls. (F) A low concentration of DPF1 fibrils (10 μg/mL) was imaged by AFM. The scale bar represents 1 μm. DPF1scr was used as a negative control. The hydrodynamic size (G) and the ζ-potential (H) of DPF1 fibrils (1 mg/mL) were determined by a Zetasizer Nano ZS instrument. Readings for the blank control were subtracted from all samples. To see this figure in color, go online. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 DPF1 fibrils enhance HIV-1 infection in vitro. DPF1 promoted the infectivity of the R5-tropic (A), X4-tropic (B), and dual-tropic (C) strains of HIV-1. TZM-bl cells were infected with different HIV-1 strains in the presence of a range of concentrations of DPF1. After 48 h of infection, the infectivity was determined by measuring luciferase activity. (D) Shown here is the viability of HIV-1 target cells exposed to DPF1 fibrils. Exponentially growing cells were treated with serially diluted DPF1 fibrils for 48 h. Cell viability was determined by an MTT assay. Average values (±SD) were calculated from triplicate measurements, and the data represent one representative trial of three independent experiments. Compared to mock-treated cells, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by one-way ANOVA. RLU/s, relative light units/s. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Infection of CEMx174 5.25M7 cells with HIV-1SF-162 in the presence of DPF1. (A) Given here are fluorescence microscopy and (B) flow cytometric analyses. The scale bar in (A) is 100 μm. The numbers in (B) indicate the percentages of HIV-1-infected (GFP+) cells. Values for the mean fluorescence intensity of GFP+ CEMx174 5.25M7 cells are indicated. To see this figure in color, go online. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 DPF1 promotes the binding of HIV-1 virions to the target cells. Proteostat-stained DPF1 (100 μg/mL, red) was mixed with eGFP-labeled HIV-1 virions (green, R5 strains) for 3 h. Images were acquired via fluorescence confocal microscopy. The scale bar represents 5 μm. To see this figure in color, go online. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 DPF1 promotes PAP248–286 and SEM186–107 amyloid fibril formation, as shown by ThT and Congo Red assays. DPF1 (200 μg/mL) reduced the lag time of PAP248-286 amyloid fibril formation, as monitored by both ThT fluorescence (A) and Congo Red (B) assays. DPF1 (200 μg/mL) reduced the lag time of SEM186–107 amyloid fibril formation, as monitored by both ThT fluorescence (C) and Congo Red (D) assays. Readings for the blank control were subtracted from all samples. DPF1 promoted amyloid fibril formation by PAP248–286 (E) or SEM186–107 (F) in a concentration-dependent manner, as detected by ThT assay. DPF1 (200 μg/mL) accelerated SEVI (G) and SEM1 (H) amyloid fibril formation in SF, as shown by ThT assays. To see this figure in color, go online. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 DPF1 accelerates SEVI and SEM1 amyloid fibril formation, as shown by TEM. Negative-stain TEM was used to visualize 300 μg/mL SEVI amyloid fibrils (A) or SEM1 (B) collected at different time points after agitation with or without DPF1 (20 μg/mL). Images are shown relative to scale bars (= 1 μm; magnification = 20,000×). Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 DPF1 accelerates β-sheet formation of PAP 248–286 and SEM186–107 as shown by CD spectra. PAP248–286 or SEM186–107 (3 mg/mL) was agitated at 37°C and 1400 rpm in the presence or absence of DPF1 (200 μg/mL). The spectra of PAP248–286 (200 μg/mL) in the presence or absence of DPF1 (16.67 μg/mL) were observed at different time points after agitation: (A) 0 h, (B) 4 h, and (C) 48 h. Similarly, the spectra of SEM186–107 (200 μg/mL) in the presence or absence of DPF1 (16.67 μg/mL) were observed at different time points after agitation: (D) 0 h, (E) 1 h, and (F) 48 h. Readings for the blank control were subtracted from all samples. To see this figure in color, go online. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 8 DPF1-enhanced SEVI and SEM1 amyloid fibrils retain the ability to enhance HIV infection. Fibril samples (SEVI at 30 μg/mL and DPF1 at 2 μg/mL) promoted the infectivity of (A) R5-tropic, (B) X4-tropic, and (C) dual-tropic HIV-1 strains. Similarly, fibril samples (SEM1 amyloid fibrils at 30 μg/mL and DPF1 at 2 μg/mL) promoted the infectivity of (D) R5-tropic, (E) X4-tropic, and (F) dual-tropic HIV-1 strains. The means of triplicate samples (±SDs) are shown. One-way ANOVA and Dunnett’s post hoc multiple comparisons test were used to statistically analyze differences between the group of viruses treated with PAP248–286 amyloid fibrils and the group of viruses treated with amyloid fibrils plus DPF1 (∗p < 0.05; ∗∗p < 0.01). RLU/s, relative light units/s. To see this figure in color, go online. Biophysical Journal 2017 113, 1425-1439DOI: (10.1016/j.bpj.2017.08.005) Copyright © 2017 Biophysical Society Terms and Conditions