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Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase.

Paskaleva EE, Lin X, Duus K, McSharry JJ, Veille JC, Thornber C, Liu Y, Lee DY, Canki M - Virol. J. (2008)

Bottom Line: That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract.We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT.We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA. paskale@mail.amc.edu

ABSTRACT
Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 mug/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

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Inhibition of HIV-1 binding and replication. GHOST cells were plate at 1 × 105/well in 12-well plates and incubated at 37°C in CO2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.
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Figure 4: Inhibition of HIV-1 binding and replication. GHOST cells were plate at 1 × 105/well in 12-well plates and incubated at 37°C in CO2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.

Mentions: In addition to demonstrating inhibition of HIV-1 fusion by SP4-2-CD4 interaction, we were interested to define mechanism of this inhibition by investigating whether treatment with S. fusiforme prevents virus binding to the cell surface receptors in culture (Fig. 4). Cells that are infected at 4°C allow only HIV-1 binding to the cell surface receptor but not fusion or entry. Except for 2 h SP4-2 pretreatment of cells that was done at 37°C to allow for SP4-2-CD4 interaction, we performed all the subsequent steps, including HIV-1 infection at 4°C. GHOST X4/R5 expressing cells were treated with increasing concentrations of SP4-2 (0–20 μg), and then washed three times with warm media to remove any unbound SP4-2. Next, cells were cooled and infected at 4°C with NL4-3 for 2 h, washed three times to remove any unbound virus, and bound HIV-1 was quantified from replicates (n = 6) by HIV-1 core antigen p24 ELISA (Fig. 4A). Treatment with 0, 12, 16, and 20 μg/ml SP4-2, resulted in a dose dependent decrease of HIV-1 bound to cells, which measured 860, 805, 435, and 331 pg/ml p24, respectively. The percent decrease in bound virus was calculated comparative to 100% bound virus (860 pg/ml p24), which was 6.3, 49.4, and 61.5%, respectively. Treatment with both 16 and 20 μg SP4-2 led to statistically significant decrease (p ≤ 0.0001) compared to no treatment (0 μg). To test whether HIV-1 bound at 4°C was capable of membrane fusion and replication, in a parallel experiment performed under same conditions, we returned the infected and washed cell cultures to 37°C for 48 h, and quantified virus replication by monitoring HIV-1 p24 production (Fig. 4B). Cell cultures pretreated with 0, 4, 8, 12, and 24 μg/ml SP4-2, replicated HIV-1 in a dose dependent manner that produced 1061, 807, 544, 352, and 148 p24 pg/ml, respectively. The HIV-1 inhibition was calculated to be 23.9, 48.7, 66.8, and 86%.


Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase.

Paskaleva EE, Lin X, Duus K, McSharry JJ, Veille JC, Thornber C, Liu Y, Lee DY, Canki M - Virol. J. (2008)

Inhibition of HIV-1 binding and replication. GHOST cells were plate at 1 × 105/well in 12-well plates and incubated at 37°C in CO2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Inhibition of HIV-1 binding and replication. GHOST cells were plate at 1 × 105/well in 12-well plates and incubated at 37°C in CO2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.
Mentions: In addition to demonstrating inhibition of HIV-1 fusion by SP4-2-CD4 interaction, we were interested to define mechanism of this inhibition by investigating whether treatment with S. fusiforme prevents virus binding to the cell surface receptors in culture (Fig. 4). Cells that are infected at 4°C allow only HIV-1 binding to the cell surface receptor but not fusion or entry. Except for 2 h SP4-2 pretreatment of cells that was done at 37°C to allow for SP4-2-CD4 interaction, we performed all the subsequent steps, including HIV-1 infection at 4°C. GHOST X4/R5 expressing cells were treated with increasing concentrations of SP4-2 (0–20 μg), and then washed three times with warm media to remove any unbound SP4-2. Next, cells were cooled and infected at 4°C with NL4-3 for 2 h, washed three times to remove any unbound virus, and bound HIV-1 was quantified from replicates (n = 6) by HIV-1 core antigen p24 ELISA (Fig. 4A). Treatment with 0, 12, 16, and 20 μg/ml SP4-2, resulted in a dose dependent decrease of HIV-1 bound to cells, which measured 860, 805, 435, and 331 pg/ml p24, respectively. The percent decrease in bound virus was calculated comparative to 100% bound virus (860 pg/ml p24), which was 6.3, 49.4, and 61.5%, respectively. Treatment with both 16 and 20 μg SP4-2 led to statistically significant decrease (p ≤ 0.0001) compared to no treatment (0 μg). To test whether HIV-1 bound at 4°C was capable of membrane fusion and replication, in a parallel experiment performed under same conditions, we returned the infected and washed cell cultures to 37°C for 48 h, and quantified virus replication by monitoring HIV-1 p24 production (Fig. 4B). Cell cultures pretreated with 0, 4, 8, 12, and 24 μg/ml SP4-2, replicated HIV-1 in a dose dependent manner that produced 1061, 807, 544, 352, and 148 p24 pg/ml, respectively. The HIV-1 inhibition was calculated to be 23.9, 48.7, 66.8, and 86%.

Bottom Line: That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract.We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT.We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA. paskale@mail.amc.edu

ABSTRACT
Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 mug/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

Show MeSH
Related in: MedlinePlus