An engineered HIV-1 gp41 trimeric coiled coil with increased stability and anti-HIV-1 activity: implication for developing anti-HIV microbicides.

Abstract

OBJECTIVES:

We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy.

METHODS:

Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay.

RESULTS:

At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity.

CONCLUSIONS:

The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.

• Authors: Pei Tong, Zhifeng Lu, Xi Chen, Qian Wang, Fei Yu, Peng Zou, Xiaoxia Yu, Yu Li, Lu Lu, Ying-Hua Chen, Shibo Jiang
• Journal: The Journal of antimicrobial chemotherapy (J Antimicrob Chemother) Vol. 68 Issue 11 Pg. 2533-44 (Nov 2013) ISSN: 1460-2091 [Electronic] England
• PMID: 23794600 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Anti-Infective Agents
• Biological Products
• HIV Envelope Protein gp41
• HIV Fusion Inhibitors
• Recombinant Proteins
• gp41 protein, Human immunodeficiency virus 1

Topics

• Anti-Infective Agents (isolation & purification, pharmacology)
• Biological Products (isolation & purification, pharmacology)
• Circular Dichroism
• HIV Envelope Protein gp41 (chemistry, genetics, pharmacology)
• HIV Fusion Inhibitors (isolation & purification, pharmacology)
• HIV-1
• Humans
• Microbial Sensitivity Tests
• Protein Engineering
• Protein Stability
• Protein Structure, Secondary
• Recombinant Proteins (chemistry, genetics, pharmacology)
• Ultracentrifugation