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A molecular tweezer antagonizes seminal amyloids and HIV infection.

Lump E, Castellano LM, Meier C, Seeliger J, Erwin N, Sperlich B, Stürzel CM, Usmani S, Hammond RM, von Einem J, Gerold G, Kreppel F, Bravo-Rodriguez K, Pietschmann T, Holmes VM, Palesch D, Zirafi O, Weissman D, Sowislok A, Wettig B, Heid C, Kirchhoff F, Weil T, Klärner FG, Schrader T, Bitan G, Sanchez-Garcia E, Winter R, Shorter J, Münch J - Elife (2015)

Bottom Line: In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils.We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism.CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

ABSTRACT
Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

No MeSH data available.


Related in: MedlinePlus

CLR01 destroys retroviral particles and selectively disrupts raft-rich membranes.(A) CLR01 releases p24 capsid antigen from HIV particles. HIV-1 was incubated with PBS, 100 µM CLR03, or 100 µM CLR01 and centrifuged at 20,000×g and 4°C for 1 hr. The p24 content of the supernatant was determined via p24 ELISA. Values represent means ±SD. Unpaired t-tests were used to compare the buffer control to the CLR03 or CLR01 condition (ns denotes not significant; ** denotes p < 0.01). (B) HIV-1 was incubated at 37°C with 10 µM CLR01 or buffer control. Aliquots were taken after different time points and analyzed regarding their infectivity using TZM-bl reporter cells. Values represent normalized mean infection rates derived from triplicate measurements ±SD compared to the buffer control (100%). Unpaired t-tests were used to compare the buffer control to the CLR01 condition at each time point (*** denotes p < 0.001). (C) CLR01 destroys retroviral particles. Images obtained by atomic force microscopy (AFM) show single MLV and glycoprotein-deficient HIV particles before and after treatment with 100 µM CLR01. Scale bar: 100 nm. (D) CLR01 destroys MLV particles. Height distribution of MLV particles after treatment with buffer (left panel) or 100 µM CLR01 (right panel). Values were derived from AFM images shown in the insets. Scale bar: 2 µm. (E) CLR01 selectively destroys membranes with high lipid raft content. Giant unilamellar vesicles (GUVs) consisting of pure DOPC were labeled with N-Rh-DHPE (red). GUVs containing a mixture of DOPC, SM and Chol (45/25/30 mol%) were labeled with N-Rh-DHPE (red) and Bodipy-Chol (green). Both types of GUVs were filled with buffer containing the fluorophore ATTO 647 (blue) and treated with 150 µM CLR01 for the indicated times before images were taken by confocal microscopy. Note that ATTO 647 remains inside the DOPC GUVs treated with CLR01, but escapes the DOPC/SM/Chol GUVs treated with CLR01. Scale bar: 10 μm. (F) Upper panel: AFM images (10 µm scans) of a pure DOPC lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm. The thickness of the hydrated membrane is 3.7 nm. Lower panel: AFM image (10 µm scan) of a DOPC/SM/Chol (45/25/30 mol%) lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm and indicating a homogeneous lipid bilayer with coexisting domains in lo (liquid-ordered) and ld (liquid-disordered domain) phase. The height difference between domains is 1 nm; the ld phase has a thickness of 4.0 nm.DOI:http://dx.doi.org/10.7554/eLife.05397.022
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fig6: CLR01 destroys retroviral particles and selectively disrupts raft-rich membranes.(A) CLR01 releases p24 capsid antigen from HIV particles. HIV-1 was incubated with PBS, 100 µM CLR03, or 100 µM CLR01 and centrifuged at 20,000×g and 4°C for 1 hr. The p24 content of the supernatant was determined via p24 ELISA. Values represent means ±SD. Unpaired t-tests were used to compare the buffer control to the CLR03 or CLR01 condition (ns denotes not significant; ** denotes p < 0.01). (B) HIV-1 was incubated at 37°C with 10 µM CLR01 or buffer control. Aliquots were taken after different time points and analyzed regarding their infectivity using TZM-bl reporter cells. Values represent normalized mean infection rates derived from triplicate measurements ±SD compared to the buffer control (100%). Unpaired t-tests were used to compare the buffer control to the CLR01 condition at each time point (*** denotes p < 0.001). (C) CLR01 destroys retroviral particles. Images obtained by atomic force microscopy (AFM) show single MLV and glycoprotein-deficient HIV particles before and after treatment with 100 µM CLR01. Scale bar: 100 nm. (D) CLR01 destroys MLV particles. Height distribution of MLV particles after treatment with buffer (left panel) or 100 µM CLR01 (right panel). Values were derived from AFM images shown in the insets. Scale bar: 2 µm. (E) CLR01 selectively destroys membranes with high lipid raft content. Giant unilamellar vesicles (GUVs) consisting of pure DOPC were labeled with N-Rh-DHPE (red). GUVs containing a mixture of DOPC, SM and Chol (45/25/30 mol%) were labeled with N-Rh-DHPE (red) and Bodipy-Chol (green). Both types of GUVs were filled with buffer containing the fluorophore ATTO 647 (blue) and treated with 150 µM CLR01 for the indicated times before images were taken by confocal microscopy. Note that ATTO 647 remains inside the DOPC GUVs treated with CLR01, but escapes the DOPC/SM/Chol GUVs treated with CLR01. Scale bar: 10 μm. (F) Upper panel: AFM images (10 µm scans) of a pure DOPC lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm. The thickness of the hydrated membrane is 3.7 nm. Lower panel: AFM image (10 µm scan) of a DOPC/SM/Chol (45/25/30 mol%) lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm and indicating a homogeneous lipid bilayer with coexisting domains in lo (liquid-ordered) and ld (liquid-disordered domain) phase. The height difference between domains is 1 nm; the ld phase has a thickness of 4.0 nm.DOI:http://dx.doi.org/10.7554/eLife.05397.022

Mentions: To define the underlying mechanism of this antiviral activity, we tested whether CLR01 disrupts the integrity of the viral membrane, leading to the release of the inner viral p24 capsid protein. HIV virions were exposed to buffer, CLR01 or CLR03 and then separated by centrifugation into a soluble fraction (containing free p24) and a sedimentable fraction (containing intact viral particles). ELISA measurements demonstrated that the amount of p24 was increased in the soluble fraction of CLR01-treated samples as compared to samples treated with CLR03 or buffer (Figure 6A). Time course experiments revealed that a 5-min incubation of virus with 10 µM CLR01 resulted in a 62% decrease in HIV infectivity, and a 10-min incubation achieved almost a 100% reduction (Figure 6B). Atomic force microscopy (AFM) of mouse leukemia virus particles (MLV) confirmed that treatment with CLR01 destroyed virion architecture (Figure 6C,D). This effect was independent of the presence of viral glycoproteins, since CLR01 also destroyed HIV-1 particles lacking gp120/41 (∆env) (Figure 6C), suggesting that CLR01 disrupts the integrity of the viral membrane.10.7554/eLife.05397.022Figure 6.CLR01 destroys retroviral particles and selectively disrupts raft-rich membranes.


A molecular tweezer antagonizes seminal amyloids and HIV infection.

Lump E, Castellano LM, Meier C, Seeliger J, Erwin N, Sperlich B, Stürzel CM, Usmani S, Hammond RM, von Einem J, Gerold G, Kreppel F, Bravo-Rodriguez K, Pietschmann T, Holmes VM, Palesch D, Zirafi O, Weissman D, Sowislok A, Wettig B, Heid C, Kirchhoff F, Weil T, Klärner FG, Schrader T, Bitan G, Sanchez-Garcia E, Winter R, Shorter J, Münch J - Elife (2015)

CLR01 destroys retroviral particles and selectively disrupts raft-rich membranes.(A) CLR01 releases p24 capsid antigen from HIV particles. HIV-1 was incubated with PBS, 100 µM CLR03, or 100 µM CLR01 and centrifuged at 20,000×g and 4°C for 1 hr. The p24 content of the supernatant was determined via p24 ELISA. Values represent means ±SD. Unpaired t-tests were used to compare the buffer control to the CLR03 or CLR01 condition (ns denotes not significant; ** denotes p < 0.01). (B) HIV-1 was incubated at 37°C with 10 µM CLR01 or buffer control. Aliquots were taken after different time points and analyzed regarding their infectivity using TZM-bl reporter cells. Values represent normalized mean infection rates derived from triplicate measurements ±SD compared to the buffer control (100%). Unpaired t-tests were used to compare the buffer control to the CLR01 condition at each time point (*** denotes p < 0.001). (C) CLR01 destroys retroviral particles. Images obtained by atomic force microscopy (AFM) show single MLV and glycoprotein-deficient HIV particles before and after treatment with 100 µM CLR01. Scale bar: 100 nm. (D) CLR01 destroys MLV particles. Height distribution of MLV particles after treatment with buffer (left panel) or 100 µM CLR01 (right panel). Values were derived from AFM images shown in the insets. Scale bar: 2 µm. (E) CLR01 selectively destroys membranes with high lipid raft content. Giant unilamellar vesicles (GUVs) consisting of pure DOPC were labeled with N-Rh-DHPE (red). GUVs containing a mixture of DOPC, SM and Chol (45/25/30 mol%) were labeled with N-Rh-DHPE (red) and Bodipy-Chol (green). Both types of GUVs were filled with buffer containing the fluorophore ATTO 647 (blue) and treated with 150 µM CLR01 for the indicated times before images were taken by confocal microscopy. Note that ATTO 647 remains inside the DOPC GUVs treated with CLR01, but escapes the DOPC/SM/Chol GUVs treated with CLR01. Scale bar: 10 μm. (F) Upper panel: AFM images (10 µm scans) of a pure DOPC lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm. The thickness of the hydrated membrane is 3.7 nm. Lower panel: AFM image (10 µm scan) of a DOPC/SM/Chol (45/25/30 mol%) lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm and indicating a homogeneous lipid bilayer with coexisting domains in lo (liquid-ordered) and ld (liquid-disordered domain) phase. The height difference between domains is 1 nm; the ld phase has a thickness of 4.0 nm.DOI:http://dx.doi.org/10.7554/eLife.05397.022
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fig6: CLR01 destroys retroviral particles and selectively disrupts raft-rich membranes.(A) CLR01 releases p24 capsid antigen from HIV particles. HIV-1 was incubated with PBS, 100 µM CLR03, or 100 µM CLR01 and centrifuged at 20,000×g and 4°C for 1 hr. The p24 content of the supernatant was determined via p24 ELISA. Values represent means ±SD. Unpaired t-tests were used to compare the buffer control to the CLR03 or CLR01 condition (ns denotes not significant; ** denotes p < 0.01). (B) HIV-1 was incubated at 37°C with 10 µM CLR01 or buffer control. Aliquots were taken after different time points and analyzed regarding their infectivity using TZM-bl reporter cells. Values represent normalized mean infection rates derived from triplicate measurements ±SD compared to the buffer control (100%). Unpaired t-tests were used to compare the buffer control to the CLR01 condition at each time point (*** denotes p < 0.001). (C) CLR01 destroys retroviral particles. Images obtained by atomic force microscopy (AFM) show single MLV and glycoprotein-deficient HIV particles before and after treatment with 100 µM CLR01. Scale bar: 100 nm. (D) CLR01 destroys MLV particles. Height distribution of MLV particles after treatment with buffer (left panel) or 100 µM CLR01 (right panel). Values were derived from AFM images shown in the insets. Scale bar: 2 µm. (E) CLR01 selectively destroys membranes with high lipid raft content. Giant unilamellar vesicles (GUVs) consisting of pure DOPC were labeled with N-Rh-DHPE (red). GUVs containing a mixture of DOPC, SM and Chol (45/25/30 mol%) were labeled with N-Rh-DHPE (red) and Bodipy-Chol (green). Both types of GUVs were filled with buffer containing the fluorophore ATTO 647 (blue) and treated with 150 µM CLR01 for the indicated times before images were taken by confocal microscopy. Note that ATTO 647 remains inside the DOPC GUVs treated with CLR01, but escapes the DOPC/SM/Chol GUVs treated with CLR01. Scale bar: 10 μm. (F) Upper panel: AFM images (10 µm scans) of a pure DOPC lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm. The thickness of the hydrated membrane is 3.7 nm. Lower panel: AFM image (10 µm scan) of a DOPC/SM/Chol (45/25/30 mol%) lipid membrane on mica before injection (0 min) and 1 min and 60 min after injection of 800 µl of 150 µM CLR01 in 10 mM NaH2PO4, pH 7.6 into the AFM fluid cell. The whole scan area is shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm and indicating a homogeneous lipid bilayer with coexisting domains in lo (liquid-ordered) and ld (liquid-disordered domain) phase. The height difference between domains is 1 nm; the ld phase has a thickness of 4.0 nm.DOI:http://dx.doi.org/10.7554/eLife.05397.022
Mentions: To define the underlying mechanism of this antiviral activity, we tested whether CLR01 disrupts the integrity of the viral membrane, leading to the release of the inner viral p24 capsid protein. HIV virions were exposed to buffer, CLR01 or CLR03 and then separated by centrifugation into a soluble fraction (containing free p24) and a sedimentable fraction (containing intact viral particles). ELISA measurements demonstrated that the amount of p24 was increased in the soluble fraction of CLR01-treated samples as compared to samples treated with CLR03 or buffer (Figure 6A). Time course experiments revealed that a 5-min incubation of virus with 10 µM CLR01 resulted in a 62% decrease in HIV infectivity, and a 10-min incubation achieved almost a 100% reduction (Figure 6B). Atomic force microscopy (AFM) of mouse leukemia virus particles (MLV) confirmed that treatment with CLR01 destroyed virion architecture (Figure 6C,D). This effect was independent of the presence of viral glycoproteins, since CLR01 also destroyed HIV-1 particles lacking gp120/41 (∆env) (Figure 6C), suggesting that CLR01 disrupts the integrity of the viral membrane.10.7554/eLife.05397.022Figure 6.CLR01 destroys retroviral particles and selectively disrupts raft-rich membranes.

Bottom Line: In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils.We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism.CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

ABSTRACT
Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

No MeSH data available.


Related in: MedlinePlus