Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
References
[1]
Melikyan G, Markosyan R, Hemmati H, Delmedico M, Lambert D, et al. (2000) Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J Cell Biol 151: 413–423.
[2]
Chan D, Fass D, Berger J, Kim P (1997) Core structure of gp41 from the HIV envelope glycoprotein. Cell 89: 263–273.
[3]
Gallo S, Wang W, Rawat S, Jung G, Waring A, et al. (2006) Theta-defensins prevent HIV-1 env-mediated fusion by binding gp41 and blocking 6-helix bundle formation. J Biol Chem 281: 18787–18792.
[4]
He Y, Vassell R, Zaitseva M, Nguyen N, Yang Z, et al. (2003) Peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites. J Virol 77: 1666–1671.
[5]
Wild CT, Shugars DC, Greenwell TK, McDanal CB, Matthews TJ (1994) Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci U S A 91: 9770–9774.
[6]
Poveda E, Rodes B, Labernardiere JL, Benito JM, Toro C, et al. (2004) Evolution of genotypic and phenotypic resistance to enfuvirtide in HIV-infected patients experiencing prolonged virologic failure. J Med Virol 74: 21–28.
[7]
Wei X, Decker JM, Liu H, Zhang Z, Arani RB, et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46: 1896–19050.
[8]
Fuhrman C, Warren A, Waring A, Dutz S, Sharma S, et al. (2007) Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41. FEBS J 274: 6477–6487.
[9]
Cole A, Yang O, Warren A, Waring A, Lehrer R, et al. (2006) HIV-1 adapts to a retrocyclin with cationic amino acid substitutions that reduce fusion efficiency of gp41. J Immunol 176: 6900–6905.
[10]
Owen S, Rudolph D, Wang W, Cole A, Waring A, et al. (2004) RC-101, a retrocyclin-1 analogue with enhanced activity against primary HIV type 1 isolates. AIDS Res Hum Retroviruses 20: 1157–1165.
[11]
Cole A, Herasimtschuk A, Gupta P, Waring A, Lehrer R, et al. (2007) The retrocyclin analogue RC-101 prevents human immunodeficiency virus type 1 infection of a model human cervicovaginal tissue construct. Immunology 121: 140–145.
[12]
Cole A, Patton D, Rohan L, Cole A, Cosgrove-Sweeney Y, et al. (2010) The formulated microbicide RC-101 was safe and antivirally active following intravaginal application in pigtailed macaques. PLoS ONE 5: e15111.
[13]
Cole AM, Hong T, Boo LM, Nguyen T, Zhao C, et al. (2002) Retrocyclin: A primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1. Proc Natl Acad Sci U S A 99: 1813–1818.
[14]
Eade CR, Wood MP, Cole AM (2012) Mechanisms and modifications of naturally occurring host defense peptides for anti-HIV microbicide development. Curr HIV Res 10: 61–72.
[15]
Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, et al. (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59: 284–291.
[16]
Lamers RP, Eade CR, Waring AJ, Cole AL, Cole AM (2011) Characterization of the retrocyclin analogue RC-101 as a preventative of staphylococcus aureus nasal colonization. Antimicrob Agents Chemother 55: 5338–5346.
[17]
Fields GB, Noble RL (1990) Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 35: 161–214.
[18]
Micewicz E, Cole A, Jung C, Luong H, Phillips M, et al. (2010) Grifonin-1: A small HIV-1 entry inhibitor derived from the algal lectin, griffithsin. PLoS ONE 5: e14360.
[19]
Kimpton J, Emerman M (1992) Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol 66: 2232–2239.
[20]
Reed LJ, Muench H (1938) A simple method for estimating fifty percent endpoints. Am J Hygiene 27: 493–497.
[21]
Miyauchi K, Kozlov MM, Melikyan GB (2009) Early steps of HIV-1 fusion define the sensitivity to inhibitory peptides that block 6-helix bundle formation. PLoS Pathog 5: e1000585.
[22]
Barrio AM, Lagercrantz E, Sperber GO, Blomberg J, Bongcam-Rudloff E (2009) Annotation and visualization of endogenous retroviral sequences using the distributed annotation system (DAS) and eBioX. BMC Bioinformatics 10 Suppl 6S18.
[23]
Baldwin C, Berkhout B (2008) Mechanistic studies of a T20-dependent human immunodeficiency virus type 1 variant. J Virol 82: 7735–7740.
[24]
Baldwin C, Sanders R, Deng Y, Jurriaans S, Lange J, et al. (2004) Emergence of a drug-dependent human immunodeficiency virus type 1 variant during therapy with the T20 fusion inhibitor. J Virol 78: 12428–12437.
[25]
Caffrey M (2001) Model for the structure of the HIV gp41 ectodomain: Insight into the intermolecular interactions of the gp41 loop. Biochim Biophys Acta 1536: 116–122.
[26]
Chang T, Vargas J, DelPortillo A, Klotman M (2005) Dual role of alpha-defensin-1 in anti-HIV-1 innate immunity. J Clin Invest 115: 765–773.
[27]
Furci L, Sironi F, Tolazzi M, Vassena L, Lusso P (2007) Alpha-defensins block the early steps of HIV-1 infection: Interference with the binding of gp120 to CD4. Blood 109: 2928–2935.
[28]
Trivedi VD, Cheng SF, Wu CW, Karthikeyan R, Chen CJ, et al. (2003) The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide, T20. Protein Eng 16: 311–317.
[29]
Eggink D, Baldwin CE, Deng Y, Langedijk JP, Lu M, et al. (2008) Selection of T1249-resistant human immunodeficiency virus type 1 variants. J Virol 82: 6678–6688.
[30]
Liu Z, Shan M, Li L, Lu L, Meng S, et al. (2011) In vitro selection and characterization of HIV-1 variants with increased resistance to sifuvirtide, a novel HIV-1 fusion inhibitor. J.Biol.Chem. 286: 3277–3287.
[31]
Ray N, Blackburn L, Doms R (2009) HR-2 mutations in human immunodeficiency virus type 1 gp41 restore fusion kinetics delayed by HR-1 mutations that cause clinical resistance to enfuvirtide. J Virol 83: 2989–2995.
[32]
Reeves J, Gallo S, Ahmad N, Miamidian J, Harvey P, et al. (2002) Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc Natl Acad Sci U S A 99: 16249–16254.
