Sub-Inhibitory Concentrations of Human α-defensin Potentiate Neutralizing Antibodies against HIV-1 gp41 Pre-Hairpin Intermediates in the Presence of Serum
Human defensins are at the forefront of the host responses to HIV and other pathogens in mucosal tissues. However, their ability to inactivate HIV in the bloodstream has been questioned due to the antagonistic effect of serum. In this study, we have examined the effect of sub-inhibitory concentrations of human α-defensin HNP-1 on the kinetics of early steps of fusion between HIV-1 and target cells in the presence of serum. Direct measurements of HIV-cell fusion using an enzymatic assay revealed that, in spite of the modest effect on the extent of fusion, HNP-1 prolonged the exposure of functionally important transitional epitopes of HIV-1 gp41 on the cell surface. The increased lifetime of gp41 intermediates in the presence of defensin was caused by a delay in the post-coreceptor binding steps of HIV-1 entry that correlated with the marked enhancement of the virus' sensitivity to neutralizing anti-gp41 antibodies. By contrast, the activity of antibodies to gp120 was not affected. HNP-1 appeared to specifically potentiate antibodies and peptides targeting the first heptad repeat domain of gp41, while its effect on inhibitors and antibodies to other gp41 domains was less prominent. Sub-inhibitory concentrations of HNP-1 also promoted inhibition of HIV-1 entry into peripheral blood mononuclear cells by antibodies and, more importantly, by HIV-1 immune serum. Our findings demonstrate that: (i) sub-inhibitory doses of HNP-1 potently enhance the activity of a number of anti-gp41 antibodies and peptide inhibitors, apparently by prolonging the lifetime of gp41 intermediates; and (ii) the efficiency of HIV-1 fusion inhibitors and neutralizing antibodies is kinetically restricted. This study thus reveals an important role of α-defensin in enhancing adaptive immune responses to HIV-1 infection and suggests future strategies to augment these responses.
References
[1]
Wyatt R, Sodroski J (1998) The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280: 1884–1888. doi: 10.1126/science.280.5371.1884
[2]
Berger EA, Murphy PM, Farber JM (1999) Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 17: 657–700. doi: 10.1146/annurev.immunol.17.1.657
[3]
Doms RW (2000) Beyond receptor expression: the influence of receptor conformation, density, and affinity in HIV-1 infection. Virology 276: 229–237. doi: 10.1006/viro.2000.0612
[4]
Eckert DM, Kim PS (2001) Mechanisms of Viral Membrane Fusion and Its Inhibition. Annu Rev Biochem 70: 777–810.
[5]
Melikyan GB (2008) Common principles and intermediates of viral protein-mediated fusion: the HIV-1 paradigm. Retrovirology 5: 111. doi: 10.1186/1742-4690-5-111
[6]
Blumenthal R, Durell S, Viard M (2012) HIV viral entry and envelope glycoprotein mediated fusion. J Biol Chem 287: 40841–9. doi: 10.1074/jbc.r112.406272
[7]
Furuta RA, Wild CT, Weng Y, Weiss CD (1998) Capture of an early fusion-active conformation of HIV-1 gp41. Nat Struct Biol 5: 276–279. doi: 10.1038/nsb0498-276
[8]
Melikyan GB, Markosyan RM, Hemmati H, Delmedico MK, Lambert DM, 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: 413–424. doi: 10.1083/jcb.151.2.413
[9]
Kilgore NR, Salzwedel K, Reddick M, Allaway GP, Wild CT (2003) Direct evidence that C-peptide inhibitors of human immunodeficiency virus type 1 entry bind to the gp41 N-helical domain in receptor-activated viral envelope. J Virol 77: 7669–7672. doi: 10.1128/jvi.77.13.7669-7672.2003
[10]
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. doi: 10.1371/journal.ppat.1000585
[11]
Gallo SA, Reeves JD, Garg H, Foley B, Doms RW, et al. (2006) Kinetic studies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion. Retrovirology 3: 90. doi: 10.1186/1742-4690-3-90
Reeves JD, Gallo SA, Ahmad N, Miamidian JL, Harvey PE, 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. doi: 10.1073/pnas.252469399
[14]
Gustchina E, Bewley CA, Clore GM (2008) Sequestering of the prehairpin intermediate of gp41 by peptide N36Mut(e,g) potentiates the human immunodeficiency virus type 1 neutralizing activity of monoclonal antibodies directed against the N-terminal helical repeat of gp41. J Virol 82: 10032–10041. doi: 10.1128/jvi.01050-08
[15]
Miyauchi K, Kim Y, Latinovic O, Morozov V, Melikyan GB (2009) HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes. Cell 137: 433–444. doi: 10.1016/j.cell.2009.02.046
[16]
Markosyan RM, Cohen FS, Melikyan GB (2003) HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation. Mol Biol Cell 14: 926–938. doi: 10.1091/mbc.e02-09-0573
[17]
de la Vega M, Marin M, Kondo N, Miyauchi K, Kim Y, et al. (2011) Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion. Retrovirology 8: 99. doi: 10.1186/1742-4690-8-99
[18]
Labrijn AF, Poignard P, Raja A, Zwick MB, Delgado K, et al. (2003) Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77: 10557–10565. doi: 10.1128/jvi.77.19.10557-10565.2003
[19]
Hamburger AE, Kim S, Welch BD, Kay MS (2005) Steric accessibility of the HIV-1 gp41 N-trimer region. J Biol Chem 280: 12567–12572. doi: 10.1074/jbc.m412770200
[20]
Eckert DM, Shi Y, Kim S, Welch BD, Kang E, et al. (2008) Characterization of the steric defense of the HIV-1 gp41 N-trimer region. Protein Sci 17: 2091–2100. doi: 10.1110/ps.038273.108
[21]
Chen W, Zhu Z, Feng Y, Dimitrov DS (2008) Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers. Proc Natl Acad Sci U S A 105: 17121–17126. doi: 10.1073/pnas.0805297105
[22]
Frey G, Peng H, Rits-Volloch S, Morelli M, Cheng Y, et al. (2008) A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies. Proc Natl Acad Sci U S A 105: 3739–3744. doi: 10.1073/pnas.0800255105
[23]
Peachman KK, Wieczorek L, Polonis VR, Alving CR, Rao M (2010) The effect of sCD4 on the binding and accessibility of HIV-1 gp41 MPER epitopes to human monoclonal antibodies. Virology 408: 213–223. doi: 10.1016/j.virol.2010.09.029
[24]
Luftig MA, Mattu M, Di Giovine P, Geleziunas R, Hrin R, et al. (2006) Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody. Nat Struct Mol Biol 13: 740–747. doi: 10.1038/nsmb1127
[25]
Steckbeck JD, Orlov I, Chow A, Grieser H, Miller K, et al. (2005) Kinetic rates of antibody binding correlate with neutralization sensitivity of variant simian immunodeficiency virus strains. J Virol 79: 12311–12320. doi: 10.1128/jvi.79.19.12311-12320.2005
[26]
Haim H, Steiner I, Panet A (2007) Time frames for neutralization during the human immunodeficiency virus type 1 entry phase, as monitored in synchronously infected cell cultures. J Virol 81: 3525–3534. doi: 10.1128/jvi.02293-06
[27]
Ketas TJ, Kuhmann SE, Palmer A, Zurita J, He W, et al. (2007) Cell surface expression of CCR5 and other host factors influence the inhibition of HIV-1 infection of human lymphocytes by CCR5 ligands. Virology 364: 281–290. doi: 10.1016/j.virol.2007.02.022
[28]
Choudhry V, Zhang MY, Harris I, Sidorov IA, Vu B, et al. (2006) Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration. Biochem Biophys Res Commun 348: 1107–1115. doi: 10.1016/j.bbrc.2006.07.163
[29]
Demirkhanyan LH, Marin M, Padilla-Parra S, Zhan C, Miyauchi K, et al. (2012) Multifaceted mechanisms of HIV-1 entry inhibition by human alpha-defensin. J Biol Chem 287: 28821–38. doi: 10.1074/jbc.m112.375949
[30]
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. doi: 10.1182/blood-2006-05-024489
[31]
Chang TL, Vargas J Jr, DelPortillo A, Klotman ME (2005) Dual role of alpha-defensin-1 in anti-HIV-1 innate immunity. J Clin Invest 115: 765–773. doi: 10.1172/jci21948
[32]
Leikina E, Delanoe-Ayari H, Melikov K, Cho MS, Chen A, et al. (2005) Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins. Nat Immunol 6: 995–1001. doi: 10.1038/ni1248
[33]
Gallo SA, Puri A, Blumenthal R (2001) HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process. Biochemistry 40: 12231–12236. doi: 10.1021/bi0155596
[34]
Lin PF, Blair W, Wang T, Spicer T, Guo Q, et al. (2003) A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding. Proc Natl Acad Sci U S A 100: 11013–11018. doi: 10.1073/pnas.1832214100
[35]
Si Z, Madani N, Cox JM, Chruma JJ, Klein JC, et al. (2004) Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins. Proc Natl Acad Sci U S A 101: 5036–5041. doi: 10.1073/pnas.0307953101
[36]
Donzella GA, Schols D, Lin SW, Este JA, Nagashima KA, et al. (1998) AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat Med 4: 72–77. doi: 10.1038/nm0198-072
[37]
Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, et al. (1999) A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A 96: 5698–5703. doi: 10.1073/pnas.96.10.5698
[38]
Deng Y, Zheng Q, Ketas TJ, Moore JP, Lu M (2007) Protein design of a bacterially expressed HIV-1 gp41 fusion inhibitor. Biochemistry 46: 4360–4369. doi: 10.1021/bi7001289
[39]
Jha NK, Latinovic O, Martin E, Novitskiy G, Marin M, et al. (2011) Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion. PLoS Pathog 7: e1001260. doi: 10.1371/journal.ppat.1001260
[40]
Miyauchi K, Marin M, Melikyan GB (2011) Visualization of retrovirus uptake and delivery into acidic endosomes. Biochem J 434: 559–569. doi: 10.1042/bj20101588
[41]
Henderson HI, Hope TJ (2006) The temperature arrested intermediate of virus-cell fusion is a functional step in HIV infection. Virol J 3: 36.
[42]
Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, et al. (2009) Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326: 285–289. doi: 10.1126/science.1178746
[43]
Pancera M, McLellan JS, Wu X, Zhu J, Changela A, et al. (2010) Crystal structure of PG16 and chimeric dissection with somatically related PG9: structure-function analysis of two quaternary-specific antibodies that effectively neutralize HIV-1. J Virol 84: 8098–8110. doi: 10.1128/jvi.00966-10
[44]
McLellan JS, Pancera M, Carrico C, Gorman J, Julien JP, et al. (2011) Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature 480: 336–343. doi: 10.1038/nature10696
[45]
Davenport TM, Friend D, Ellingson K, Xu H, Caldwell Z, et al. (2011) Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16. J Virol 85: 7095–7107. doi: 10.1128/jvi.00411-11
[46]
Prabakaran P, Gan J, Wu YQ, Zhang MY, Dimitrov DS, et al. (2006) Structural mimicry of CD4 by a cross-reactive HIV-1 neutralizing antibody with CDR-H2 and H3 containing unique motifs. J Mol Biol 357: 82–99. doi: 10.1016/j.jmb.2005.12.062
[47]
Sullivan N, Sun Y, Sattentau Q, Thali M, Wu D, et al. (1998) CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization. J Virol 72: 4694–4703.
[48]
Thali M, Moore JP, Furman C, Charles M, Ho DD, et al. (1993) Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol 67: 3978–3988.
[49]
Zhang MY, Shu Y, Rudolph D, Prabakaran P, Labrijn AF, et al. (2004) Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J Mol Biol 335: 209–219. doi: 10.1016/j.jmb.2003.09.055
[50]
Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, et al. (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody [see comments]. Nature 393: 648–659.
[51]
Bewley CA, Louis JM, Ghirlando R, Clore GM (2002) Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41. J Biol Chem 277: 14238–14245. doi: 10.1074/jbc.m201453200
[52]
Dimitrov AS, Louis JM, Bewley CA, Clore GM, Blumenthal R (2005) Conformational changes in HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion and inactivation. Biochemistry 44: 12471–12479. doi: 10.1021/bi051092d
[53]
Miller MD, Geleziunas R, Bianchi E, Lennard S, Hrin R, et al. (2005) A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope. Proc Natl Acad Sci U S A 102: 14759–14764. doi: 10.1073/pnas.0506927102
[54]
Sabin C, Corti D, Buzon V, Seaman MS, Lutje Hulsik D, et al. (2010) Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41. PLoS Pathog 6: e1001195. doi: 10.1371/journal.ppat.1001195
[55]
Nelson JD, Kinkead H, Brunel FM, Leaman D, Jensen R, et al. (2008) Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics. Virology 377: 170–183. doi: 10.1016/j.virol.2008.04.005
[56]
Gustchina E, Louis JM, Lam SN, Bewley CA, Clore GM (2007) A monoclonal Fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains. J Virol 81: 12946–12953. doi: 10.1128/jvi.01260-07
[57]
Jiang S, Lin K, Lu M (1998) A conformation-specific monoclonal antibody reacting with fusion-active gp41 from the human immunodeficiency virus type 1 envelope glycoprotein. J Virol 72: 10213–10217.
[58]
Root MJ, Kay MS, Kim PS (2001) Protein design of an HIV-1 entry inhibitor. Science 291: 884–888. doi: 10.1126/science.1057453
[59]
Gorny MK, Gianakakos V, Sharpe S, Zolla-Pazner S (1989) Generation of human monoclonal antibodies to human immunodeficiency virus. Proc Natl Acad Sci U S A 86: 1624–1628. doi: 10.1073/pnas.86.5.1624
[60]
Xu JY, Gorny MK, Palker T, Karwowska S, Zolla-Pazner S (1991) Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies. J Virol 65: 4832–4838.
[61]
Golding H, Zaitseva M, de Rosny E, King LR, Manischewitz J, et al. (2002) Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates. J Virol 76: 6780–6790. doi: 10.1128/jvi.76.13.6780-6790.2002
[62]
Gorny MK, Zolla-Pazner S (2000) Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41. J Virol 74: 6186–6192. doi: 10.1128/jvi.74.13.6186-6192.2000
[63]
Yuan W, Li X, Kasterka M, Gorny MK, Zolla-Pazner S, et al. (2009) Oligomer-specific conformations of the human immunodeficiency virus (HIV-1) gp41 envelope glycoprotein ectodomain recognized by human monoclonal antibodies. AIDS Res Hum Retroviruses 25: 319–328. doi: 10.1089/aid.2008.0213
[64]
Frey G, Chen J, Rits-Volloch S, Freeman MM, Zolla-Pazner S, et al. (2010) Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies. Nat Struct Mol Biol 17: 1486–1491. doi: 10.1038/nsmb.1950
[65]
Dennison SM, Anasti K, Scearce RM, Sutherland L, Parks R, et al. (2011) Nonneutralizing HIV-1 gp41 envelope cluster II human monoclonal antibodies show polyreactivity for binding to phospholipids and protein autoantigens. J Virol 85: 1340–1347. doi: 10.1128/jvi.01680-10
[66]
Buchacher A, Predl R, Strutzenberger K, Steinfellner W, Trkola A, et al. (1994) Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res Hum Retroviruses 10: 359–369. doi: 10.1089/aid.1994.10.359
[67]
Stiegler G, Kunert R, Purtscher M, Wolbank S, Voglauer R, et al. (2001) A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 17: 1757–1765. doi: 10.1089/08892220152741450
[68]
Dimitrov AS, Jacobs A, Finnegan CM, Stiegler G, Katinger H, et al. (2007) Exposure of the Membrane-Proximal External Region of HIV-1 gp41 in the Course of HIV-1 Envelope Glycoprotein-Mediated Fusion. Biochemistry 46: 1398–1401. doi: 10.1021/bi062245f
[69]
de Rosny E, Vassell R, Jiang S, Kunert R, Weiss CD (2004) Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes. J Virol 78: 2627–2631. doi: 10.1128/jvi.78.5.2627-2631.2004
[70]
Finnegan CM, Berg W, Lewis GK, DeVico AL (2002) Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion. J Virol 76: 12123–12134. doi: 10.1128/jvi.76.23.12123-12134.2002
[71]
Alam SM, Scearce RM, Parks RJ, Plonk K, Plonk SG, et al. (2008) Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection. J Virol 82: 115–125. doi: 10.1128/jvi.00927-07
[72]
Sattentau QJ, Moore JP (1991) Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding. J Exp Med 174: 407–415. doi: 10.1084/jem.174.2.407
[73]
Abrahamyan LG, Mkrtchyan SR, Binley J, Lu M, Melikyan GB, et al. (2005) The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle. J Virol 79: 106–115. doi: 10.1128/jvi.79.1.106-115.2005
[74]
Chakrabarti BK, Walker LM, Guenaga JF, Ghobbeh A, Poignard P, et al. (2011) Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity. J Virol 85: 8217–8226. doi: 10.1128/jvi.00756-11
[75]
Gallo SA, Wang W, Rawat SS, Jung G, Waring AJ, 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. doi: 10.1074/jbc.m602422200
[76]
Panyutich AV, Panyutich EA, Krapivin VA, Baturevich EA, Ganz T (1993) Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis. J Lab Clin Med 122: 202–207.
[77]
Shiomi K, Nakazato M, Ihi T, Kangawa K, Matsuo H, et al. (1993) Establishment of radioimmunoassay for human neutrophil peptides and their increases in plasma and neutrophil in infection. Biochem Biophys Res Commun 195: 1336–1344. doi: 10.1006/bbrc.1993.2190
[78]
Lahm HW, Stein S (1985) Characterization of recombinant human interleukin-2 with micromethods. J Chromatogr 326: 357–361. doi: 10.1016/s0021-9673(01)87461-6
[79]
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: 1896–1905. doi: 10.1128/aac.46.6.1896-1905.2002
[80]
Vujcic LK, Quinnan GV Jr (1995) Preparation and characterization of human HIV type 1 neutralizing reference sera. AIDS Res Hum Retroviruses 11: 783–787. doi: 10.1089/aid.1995.11.783
[81]
Li Y, Svehla K, Mathy NL, Voss G, Mascola JR, et al. (2006) Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants. J Virol 80: 1414–1426. doi: 10.1128/jvi.80.3.1414-1426.2006
[82]
Schnolzer M, Alewood P, Jones A, Alewood D, Kent SB (1992) In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int J Pept Protein Res 40: 180–193. doi: 10.1111/j.1399-3011.1992.tb00291.x
[83]
Wu Z, Powell R, Lu W (2003) Productive folding of human neutrophil alpha-defensins in vitro without the pro-peptide. J Am Chem Soc 125: 2402–2403. doi: 10.1021/ja0294257
[84]
Wei G, de Leeuw E, Pazgier M, Yuan W, Zou G, et al. (2009) Through the looking glass, mechanistic insights from enantiomeric human defensins. J Biol Chem 284: 29180–29192. doi: 10.1074/jbc.m109.018085
[85]
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4: 2411–2423. doi: 10.1002/pro.5560041120
[86]
Platt EJ, Kozak SL, Durnin JP, Hope TJ, Kabat D (2010) Rapid dissociation of HIV-1 from cultured cells severely limits infectivity assays, causes the inactivation ascribed to entry inhibitors, and masks the inherently high level of infectivity of virions. J Virol 84: 3106–3110. doi: 10.1128/jvi.01958-09
