Limits...
Characterizing the anti-HIV activity of papuamide A.

Andjelic CD, Planelles V, Barrows LR - Mar Drugs (2008)

Bottom Line: This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding.Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A.We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity.

View Article: PubMed Central - PubMed

Affiliation: University of Utah, Department of Pharmacology and Toxicology, 30 S. 2000 E. Rm.201, Salt Lake City, UT 84112, USA.

ABSTRACT
Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A's action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity.

Show MeSH

Related in: MedlinePlus

Inhibition of NL4-3 (X4 tropic virus) entry in the presence of papuamide A (Pap A), the known fusion inhibitor C34 and reverse transcriptase inhibitor azidothymidine (AZT) at final concentrations of 710 nM, 233 nM and 18 μM, respectively. Control represents uninfected cells and NL4-3 HIV represents untreated infected cells. Viral entry is reported as a fluorescence ratio calculated from the amount of blue versus green fluorescence, normalized to control. Results graphed as the mean ± standard error, n≥4. Fluorescence ratio was significantly different (p < 0.05) from control (*) or from untreated HIV infected cells (a).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2630844&req=5

f2b-md-06-00528: Inhibition of NL4-3 (X4 tropic virus) entry in the presence of papuamide A (Pap A), the known fusion inhibitor C34 and reverse transcriptase inhibitor azidothymidine (AZT) at final concentrations of 710 nM, 233 nM and 18 μM, respectively. Control represents uninfected cells and NL4-3 HIV represents untreated infected cells. Viral entry is reported as a fluorescence ratio calculated from the amount of blue versus green fluorescence, normalized to control. Results graphed as the mean ± standard error, n≥4. Fluorescence ratio was significantly different (p < 0.05) from control (*) or from untreated HIV infected cells (a).

Mentions: Investigation of papuamide A’s ability to inhibit HIV induced T cell death lead to the testing of papuamide A’s ability to inhibit HIV entry in the virion based fusion assay developed by Cavrois et al. [21]. This assay quantifies viral entry using the chimeric protein BlaM-vpr, β-lactamase linked to vpr, which is readily incorporated into virions produced by co-transfection with proviral DNA. Initial analysis was performed using an X4 tropic virus (virus that infects cells expressing CXCR4), produced by co-transfection with HIV-1NL4-3 proviral DNA, to infect HeLa T4+ cells expressing CXCR4. Fig. 2a shows representative microscope images of this assay for control cells (non-infected cells fluoresce green), HIV infected cells (infected cells fluoresce blue) and infected cells treated with papuamide A (showing a decrease in the presence of infected cells due to inhibition of viral entry). Fig. 2b graphs the results from this experiment and shows that papuamide A at a concentration of 710 nM inhibited approximately 80% of viral entry. Controls used included C34 (a fusion inhibitor) which effectively blocked viral entry and AZT (a reverse transcriptase inhibitor) which did not prevent viral entry as expected. To determine if this activity was X4 tropic specific, the ability of papuamide A to inhibit a R5 tropic virus (virus that infects cells expressing CCR5) was evaluated using HIV-1NL(AD8), to infect the TZM-bl cells, HeLa T4+ cell line expressing CCR5. Papuamide A effectively inhibited the entry of the R5 tropic virus in a dose dependant manner (Fig. 2c). The dose response curve provides an EC50 (50% effective concentration) of approximately 114 nM (Fig. 2c). This potent effective concentration is similar to the EC50 of 71 nM obtained for papuamide A protection against HIV induced cell death determined using the cytoprotection 3-(4,5-dimethylthiazol-2-yl)-2,5diphenltetrazolium bromide (MTT) based assay of Kiser and colleagues [22] established in our laboratory (data not shown).


Characterizing the anti-HIV activity of papuamide A.

Andjelic CD, Planelles V, Barrows LR - Mar Drugs (2008)

Inhibition of NL4-3 (X4 tropic virus) entry in the presence of papuamide A (Pap A), the known fusion inhibitor C34 and reverse transcriptase inhibitor azidothymidine (AZT) at final concentrations of 710 nM, 233 nM and 18 μM, respectively. Control represents uninfected cells and NL4-3 HIV represents untreated infected cells. Viral entry is reported as a fluorescence ratio calculated from the amount of blue versus green fluorescence, normalized to control. Results graphed as the mean ± standard error, n≥4. Fluorescence ratio was significantly different (p < 0.05) from control (*) or from untreated HIV infected cells (a).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2630844&req=5

f2b-md-06-00528: Inhibition of NL4-3 (X4 tropic virus) entry in the presence of papuamide A (Pap A), the known fusion inhibitor C34 and reverse transcriptase inhibitor azidothymidine (AZT) at final concentrations of 710 nM, 233 nM and 18 μM, respectively. Control represents uninfected cells and NL4-3 HIV represents untreated infected cells. Viral entry is reported as a fluorescence ratio calculated from the amount of blue versus green fluorescence, normalized to control. Results graphed as the mean ± standard error, n≥4. Fluorescence ratio was significantly different (p < 0.05) from control (*) or from untreated HIV infected cells (a).
Mentions: Investigation of papuamide A’s ability to inhibit HIV induced T cell death lead to the testing of papuamide A’s ability to inhibit HIV entry in the virion based fusion assay developed by Cavrois et al. [21]. This assay quantifies viral entry using the chimeric protein BlaM-vpr, β-lactamase linked to vpr, which is readily incorporated into virions produced by co-transfection with proviral DNA. Initial analysis was performed using an X4 tropic virus (virus that infects cells expressing CXCR4), produced by co-transfection with HIV-1NL4-3 proviral DNA, to infect HeLa T4+ cells expressing CXCR4. Fig. 2a shows representative microscope images of this assay for control cells (non-infected cells fluoresce green), HIV infected cells (infected cells fluoresce blue) and infected cells treated with papuamide A (showing a decrease in the presence of infected cells due to inhibition of viral entry). Fig. 2b graphs the results from this experiment and shows that papuamide A at a concentration of 710 nM inhibited approximately 80% of viral entry. Controls used included C34 (a fusion inhibitor) which effectively blocked viral entry and AZT (a reverse transcriptase inhibitor) which did not prevent viral entry as expected. To determine if this activity was X4 tropic specific, the ability of papuamide A to inhibit a R5 tropic virus (virus that infects cells expressing CCR5) was evaluated using HIV-1NL(AD8), to infect the TZM-bl cells, HeLa T4+ cell line expressing CCR5. Papuamide A effectively inhibited the entry of the R5 tropic virus in a dose dependant manner (Fig. 2c). The dose response curve provides an EC50 (50% effective concentration) of approximately 114 nM (Fig. 2c). This potent effective concentration is similar to the EC50 of 71 nM obtained for papuamide A protection against HIV induced cell death determined using the cytoprotection 3-(4,5-dimethylthiazol-2-yl)-2,5diphenltetrazolium bromide (MTT) based assay of Kiser and colleagues [22] established in our laboratory (data not shown).

Bottom Line: This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding.Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A.We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity.

View Article: PubMed Central - PubMed

Affiliation: University of Utah, Department of Pharmacology and Toxicology, 30 S. 2000 E. Rm.201, Salt Lake City, UT 84112, USA.

ABSTRACT
Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A's action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity.

Show MeSH
Related in: MedlinePlus