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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

Lysine or poly-L-lysine, but not BSA or preclearing CLR01 solutions, antagonize the ability of CLR01 to inhibit seeded assembly of SEVI fibrils.PAP248-286 (1 mM) was incubated with agitation at 37°C for 24 hr with preformed SEVI fibrils (2% wt/wt) plus CLR01 (100 µM) or buffer in the presence or absence of lysine (20 mM), poly-L-lysine (1 mM), or BSA (10 mg/ml). In some reactions, CLR01 was first cleared by centrifugation at 16,100×g for 20 min at 25°C (precleared). Fibrillization was assessed via ThT fluorescence. Values represent means ±SEM (n = 3). A one-way ANOVA with the post hoc Dunnett's multiple comparisons test was used to compare the buffer alone control to the other conditions (*** denotes p < 0.0001).DOI:http://dx.doi.org/10.7554/eLife.05397.009
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fig2s5: Lysine or poly-L-lysine, but not BSA or preclearing CLR01 solutions, antagonize the ability of CLR01 to inhibit seeded assembly of SEVI fibrils.PAP248-286 (1 mM) was incubated with agitation at 37°C for 24 hr with preformed SEVI fibrils (2% wt/wt) plus CLR01 (100 µM) or buffer in the presence or absence of lysine (20 mM), poly-L-lysine (1 mM), or BSA (10 mg/ml). In some reactions, CLR01 was first cleared by centrifugation at 16,100×g for 20 min at 25°C (precleared). Fibrillization was assessed via ThT fluorescence. Values represent means ±SEM (n = 3). A one-way ANOVA with the post hoc Dunnett's multiple comparisons test was used to compare the buffer alone control to the other conditions (*** denotes p < 0.0001).DOI:http://dx.doi.org/10.7554/eLife.05397.009

Mentions: The addition of a small amount of preformed SEVI fibrils to soluble PAP248-286 seeds polymerization and eliminates the lag phase for assembly (Ye et al., 2009) (compare Figure 2A with Figure 2J). Remarkably, in addition to obstructing unseeded PAP248-286 assembly (Figure 2A), CLR01 also completely inhibited seeded fibrillization (Figure 2J,K). Dose–response analysis established an IC50 of ∼15.3 µM for CLR01 inhibition of seeded PAP248-286 assembly (Figure 2L) compared to ∼1.19 µM for spontaneous PAP248-286 assembly (Figure 2D). Thus, higher concentrations of CLR01 are required to inhibit PAP248-286 assembly once SEVI fibrils have formed. Interestingly, the Hill slope for inhibition of seeded PAP248-286 assembly was ∼3.1 indicating positive co-operativity and was steeper than the Hill slope of ∼0.6 for inhibition of spontaneous PAP248-286 fibrillization (Figure 2D,L). This dissimilarity might indicate a mechanistic difference in how CLR01 inhibits seeded and spontaneous PAP248-286 fibrillization. However, this difference is not likely to be due colloidal CLR01 aggregates, as inhibition of seeded assembly by CLR01 was unaffected by BSA or by preclearing CLR01 solutions via centrifugation (Figure 2—figure supplement 5). Rather, we suggest that the CLR01-mediated inhibition of fibril elongation in seeded SEVI assembly has requirements distinct from the CLR01-mediated inhibition of initial fibril nucleation in spontaneous, unseeded SEVI assembly.


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)

Lysine or poly-L-lysine, but not BSA or preclearing CLR01 solutions, antagonize the ability of CLR01 to inhibit seeded assembly of SEVI fibrils.PAP248-286 (1 mM) was incubated with agitation at 37°C for 24 hr with preformed SEVI fibrils (2% wt/wt) plus CLR01 (100 µM) or buffer in the presence or absence of lysine (20 mM), poly-L-lysine (1 mM), or BSA (10 mg/ml). In some reactions, CLR01 was first cleared by centrifugation at 16,100×g for 20 min at 25°C (precleared). Fibrillization was assessed via ThT fluorescence. Values represent means ±SEM (n = 3). A one-way ANOVA with the post hoc Dunnett's multiple comparisons test was used to compare the buffer alone control to the other conditions (*** denotes p < 0.0001).DOI:http://dx.doi.org/10.7554/eLife.05397.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536748&req=5

fig2s5: Lysine or poly-L-lysine, but not BSA or preclearing CLR01 solutions, antagonize the ability of CLR01 to inhibit seeded assembly of SEVI fibrils.PAP248-286 (1 mM) was incubated with agitation at 37°C for 24 hr with preformed SEVI fibrils (2% wt/wt) plus CLR01 (100 µM) or buffer in the presence or absence of lysine (20 mM), poly-L-lysine (1 mM), or BSA (10 mg/ml). In some reactions, CLR01 was first cleared by centrifugation at 16,100×g for 20 min at 25°C (precleared). Fibrillization was assessed via ThT fluorescence. Values represent means ±SEM (n = 3). A one-way ANOVA with the post hoc Dunnett's multiple comparisons test was used to compare the buffer alone control to the other conditions (*** denotes p < 0.0001).DOI:http://dx.doi.org/10.7554/eLife.05397.009
Mentions: The addition of a small amount of preformed SEVI fibrils to soluble PAP248-286 seeds polymerization and eliminates the lag phase for assembly (Ye et al., 2009) (compare Figure 2A with Figure 2J). Remarkably, in addition to obstructing unseeded PAP248-286 assembly (Figure 2A), CLR01 also completely inhibited seeded fibrillization (Figure 2J,K). Dose–response analysis established an IC50 of ∼15.3 µM for CLR01 inhibition of seeded PAP248-286 assembly (Figure 2L) compared to ∼1.19 µM for spontaneous PAP248-286 assembly (Figure 2D). Thus, higher concentrations of CLR01 are required to inhibit PAP248-286 assembly once SEVI fibrils have formed. Interestingly, the Hill slope for inhibition of seeded PAP248-286 assembly was ∼3.1 indicating positive co-operativity and was steeper than the Hill slope of ∼0.6 for inhibition of spontaneous PAP248-286 fibrillization (Figure 2D,L). This dissimilarity might indicate a mechanistic difference in how CLR01 inhibits seeded and spontaneous PAP248-286 fibrillization. However, this difference is not likely to be due colloidal CLR01 aggregates, as inhibition of seeded assembly by CLR01 was unaffected by BSA or by preclearing CLR01 solutions via centrifugation (Figure 2—figure supplement 5). Rather, we suggest that the CLR01-mediated inhibition of fibril elongation in seeded SEVI assembly has requirements distinct from the CLR01-mediated inhibition of initial fibril nucleation in spontaneous, unseeded SEVI assembly.

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