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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 diminishes the infection enhancing property of semen.(A) SEVI fibrils were formed in an artificial semen simulant (AS) (Owen and Katz, 2005). The resulting fibrils were then diluted to 20 µM in AS and treated with 200 µM CLR01 or CLR03, or with buffer for 2 hr. Fibril integrity was assessed using ThT fluorescence. Values represent means ±SEM (n = 4). 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 (ns denotes not significant; ** denotes p < 0.01). (B) CLR01 abrogates semen-mediated enhancement of HIV infection. Seminal plasma (10%) or cell culture medium containing CLR01 or CLR03 was mixed with CCR5-tropic HIV-1 or transmitter/founder HIV-1 CH058. After 10 min, TZM-bl cells were infected and infectivity was measured 3 day post infection. Shown are the n-fold increased infection rates obtained for semen-treated virus relative to those of medium-treated virus. Values represent means ±SD (n = 4). Unpaired t-tests were used to compare the buffer control (0 µM compound) to the CLR03 or CLR01 condition at each concentration (ns denotes not significant; * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.05397.028
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fig9: CLR01 diminishes the infection enhancing property of semen.(A) SEVI fibrils were formed in an artificial semen simulant (AS) (Owen and Katz, 2005). The resulting fibrils were then diluted to 20 µM in AS and treated with 200 µM CLR01 or CLR03, or with buffer for 2 hr. Fibril integrity was assessed using ThT fluorescence. Values represent means ±SEM (n = 4). 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 (ns denotes not significant; ** denotes p < 0.01). (B) CLR01 abrogates semen-mediated enhancement of HIV infection. Seminal plasma (10%) or cell culture medium containing CLR01 or CLR03 was mixed with CCR5-tropic HIV-1 or transmitter/founder HIV-1 CH058. After 10 min, TZM-bl cells were infected and infectivity was measured 3 day post infection. Shown are the n-fold increased infection rates obtained for semen-treated virus relative to those of medium-treated virus. Values represent means ±SD (n = 4). Unpaired t-tests were used to compare the buffer control (0 µM compound) to the CLR03 or CLR01 condition at each concentration (ns denotes not significant; * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.05397.028

Mentions: CLR01 binds exposed lysine and arginine residues in amyloidogenic peptides (Sinha et al., 2011; Attar et al., 2012; Prabhudesai et al., 2012; Sinha et al., 2012; Acharya et al., 2014; Ferreira et al., 2014; Zheng et al., 2015). In vivo, the amyloids we examined are present in the complex environment of human semen. We wondered whether the interactions between CLR01 and seminal peptides might be hindered in conditions resembling those in seminal fluid. To test whether this is the case, lyophilized PAP248-286 was dissolved in an artificial semen simulant (AS) (Owen and Katz, 2005; Olsen et al., 2012) containing 50 mg/ml BSA and agitated until fibril formation was complete. These PAP248-286(AS) fibrils were then diluted in AS and incubated with CLR01. A reduction in ThT fluorescence intensity to 43% of the initial value was detected (Figure 9A). This finding confirms that CLR01 maintains its amyloid-remodeling activity in a complex solution resembling seminal fluid.10.7554/eLife.05397.028Figure 9.CLR01 diminishes the infection enhancing property of semen.


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 diminishes the infection enhancing property of semen.(A) SEVI fibrils were formed in an artificial semen simulant (AS) (Owen and Katz, 2005). The resulting fibrils were then diluted to 20 µM in AS and treated with 200 µM CLR01 or CLR03, or with buffer for 2 hr. Fibril integrity was assessed using ThT fluorescence. Values represent means ±SEM (n = 4). 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 (ns denotes not significant; ** denotes p < 0.01). (B) CLR01 abrogates semen-mediated enhancement of HIV infection. Seminal plasma (10%) or cell culture medium containing CLR01 or CLR03 was mixed with CCR5-tropic HIV-1 or transmitter/founder HIV-1 CH058. After 10 min, TZM-bl cells were infected and infectivity was measured 3 day post infection. Shown are the n-fold increased infection rates obtained for semen-treated virus relative to those of medium-treated virus. Values represent means ±SD (n = 4). Unpaired t-tests were used to compare the buffer control (0 µM compound) to the CLR03 or CLR01 condition at each concentration (ns denotes not significant; * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.05397.028
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Related In: Results  -  Collection

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fig9: CLR01 diminishes the infection enhancing property of semen.(A) SEVI fibrils were formed in an artificial semen simulant (AS) (Owen and Katz, 2005). The resulting fibrils were then diluted to 20 µM in AS and treated with 200 µM CLR01 or CLR03, or with buffer for 2 hr. Fibril integrity was assessed using ThT fluorescence. Values represent means ±SEM (n = 4). 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 (ns denotes not significant; ** denotes p < 0.01). (B) CLR01 abrogates semen-mediated enhancement of HIV infection. Seminal plasma (10%) or cell culture medium containing CLR01 or CLR03 was mixed with CCR5-tropic HIV-1 or transmitter/founder HIV-1 CH058. After 10 min, TZM-bl cells were infected and infectivity was measured 3 day post infection. Shown are the n-fold increased infection rates obtained for semen-treated virus relative to those of medium-treated virus. Values represent means ±SD (n = 4). Unpaired t-tests were used to compare the buffer control (0 µM compound) to the CLR03 or CLR01 condition at each concentration (ns denotes not significant; * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.05397.028
Mentions: CLR01 binds exposed lysine and arginine residues in amyloidogenic peptides (Sinha et al., 2011; Attar et al., 2012; Prabhudesai et al., 2012; Sinha et al., 2012; Acharya et al., 2014; Ferreira et al., 2014; Zheng et al., 2015). In vivo, the amyloids we examined are present in the complex environment of human semen. We wondered whether the interactions between CLR01 and seminal peptides might be hindered in conditions resembling those in seminal fluid. To test whether this is the case, lyophilized PAP248-286 was dissolved in an artificial semen simulant (AS) (Owen and Katz, 2005; Olsen et al., 2012) containing 50 mg/ml BSA and agitated until fibril formation was complete. These PAP248-286(AS) fibrils were then diluted in AS and incubated with CLR01. A reduction in ThT fluorescence intensity to 43% of the initial value was detected (Figure 9A). This finding confirms that CLR01 maintains its amyloid-remodeling activity in a complex solution resembling seminal fluid.10.7554/eLife.05397.028Figure 9.CLR01 diminishes the infection enhancing property of semen.

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