Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies.
References
[1]
de Visser JA, Hermisson J, Wagner GP, Ancel Meyers L, Bagheri-Chaichian H, et al. (2003) Perspective: Evolution and detection of genetic robustness. Evolution 57: 1959–1972. doi: 10.1111/j.0014-3820.2003.tb00377.x
[2]
Wagner A (2005) Robustness, evolvability, and neutrality. FEBS Lett 579: 1772–1778. doi: 10.1016/j.febslet.2005.01.063
[3]
Elena SF (2012) RNA virus genetic robustness: possible causes and some consequences. Curr Opin Virol 2: 525–530. doi: 10.1016/j.coviro.2012.06.008
[4]
Montville R, Froissart R, Remold SK, Tenaillon O, Turner PE (2005) Evolution of mutational robustness in an RNA virus. PLoS Biol 3: e381. doi: 10.1371/journal.pbio.0030381
[5]
Codoner FM, Daros JA, Sole RV, Elena SF (2006) The fittest versus the flattest: experimental confirmation of the quasispecies effect with subviral pathogens. PLoS Pathog 2: e136. doi: 10.1371/journal.ppat.0020136
[6]
Sanjuan R, Cuevas JM, Furio V, Holmes EC, Moya A (2007) Selection for robustness in mutagenized RNA viruses. PLoS Genet 3: e93. doi: 10.1371/journal.pgen.0030093.eor
[7]
Domingo-Calap P, Cuevas JM, Sanjuan R (2009) The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet 5: e1000742. doi: 10.1371/journal.pgen.1000742
[8]
Sanjuan R, Moya A, Elena SF (2004) The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc Natl Acad Sci U S A 101: 8396–8401. doi: 10.1073/pnas.0400146101
[9]
Carrasco P, de la Iglesia F, Elena SF (2007) Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco Etch virus. J Virol 81: 12979–12984. doi: 10.1128/jvi.00524-07
[10]
Bieniasz PD (2009) The cell biology of HIV-1 virion genesis. Cell Host Microbe 5: 550–558. doi: 10.1016/j.chom.2009.05.015
[11]
Freed EO (1998) HIV-1 gag proteins: diverse functions in the virus life cycle. Virology 251: 1–15. doi: 10.1006/viro.1998.9398
[12]
Ganser-Pornillos BK, Yeager M, Sundquist WI (2008) The structural biology of HIV assembly. Curr Opin Struct Biol 18: 203–217. doi: 10.1016/j.sbi.2008.02.001
[13]
Sundquist WI, Krausslich HG (2012) HIV-1 Assembly, Budding, and Maturation. Cold Spring Harb Perspect Med 2: a006924. doi: 10.1101/cshperspect.a006924
[14]
Briggs JA, Krausslich HG (2011) The molecular architecture of HIV. J Mol Biol 410: 491–500. doi: 10.1016/j.jmb.2011.04.021
[15]
Briggs JA, Riches JD, Glass B, Bartonova V, Zanetti G, et al. (2009) Structure and assembly of immature HIV. Proc Natl Acad Sci U S A 106: 11090–11095. doi: 10.1073/pnas.0903535106
[16]
Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI (1999) Assembly and analysis of conical models for the HIV-1 core. Science 283: 80–83. doi: 10.1126/science.283.5398.80
[17]
Ganser-Pornillos BK, von Schwedler UK, Stray KM, Aiken C, Sundquist WI (2004) Assembly properties of the human immunodeficiency virus type 1 CA protein. J Virol 78: 2545–2552. doi: 10.1128/jvi.78.5.2545-2552.2004
[18]
Gamble TR, Yoo S, Vajdos FF, von Schwedler UK, Worthylake DK, et al. (1997) Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science 278: 849–853. doi: 10.1126/science.278.5339.849
[19]
Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, et al. (1996) Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell 87: 1285–1294. doi: 10.1016/s0092-8674(00)81823-1
[20]
Gitti RK, Lee BM, Walker J, Summers MF, Yoo S, et al. (1996) Structure of the amino-terminal core domain of the HIV-1 capsid protein. Science 273: 231–235. doi: 10.1126/science.273.5272.231
[21]
Chang YF, Wang SM, Huang KJ, Wang CT (2007) Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag. J Mol Biol 370: 585–597. doi: 10.1016/j.jmb.2007.05.020
[22]
Zimmerman C, Klein KC, Kiser PK, Singh AR, Firestein BL, et al. (2002) Identification of a host protein essential for assembly of immature HIV-1 capsids. Nature 415: 88–92. doi: 10.1038/415088a
[23]
Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP (1993) Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell 73: 1067–1078. doi: 10.1016/0092-8674(93)90637-6
[24]
Pornillos O, Ganser-Pornillos BK, Yeager M (2011) Atomic-level modelling of the HIV capsid. Nature 469: 424–427. doi: 10.1038/nature09640
[25]
Li S, Hill CP, Sundquist WI, Finch JT (2000) Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 407: 409–413. doi: 10.1038/35030177
[26]
Pornillos O, Ganser-Pornillos BK, Kelly BN, Hua Y, Whitby FG, et al. (2009) X-ray structures of the hexameric building block of the HIV capsid. Cell 137: 1282–1292. doi: 10.1016/j.cell.2009.04.063
[27]
Lanman J, Lam TT, Barnes S, Sakalian M, Emmett MR, et al. (2003) Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry. J Mol Biol 325: 759–772. doi: 10.1016/j.jmb.2003.10.046
[28]
Lanman J, Lam TT, Emmett MR, Marshall AG, Sakalian M, et al. (2004) Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange. Nat Struct Mol Biol 11: 676–677. doi: 10.1038/nsmb790
[29]
Mateu MG (2009) The capsid protein of human immunodeficiency virus: intersubunit interactions during virus assembly. FEBS J 276: 6098–6109. doi: 10.1111/j.1742-4658.2009.07313.x
[30]
Yamashita M, Perez O, Hope TJ, Emerman M (2007) Evidence for direct involvement of the capsid protein in HIV infection of nondividing cells. PLoS Pathog 3: 1502–1510. doi: 10.1371/journal.ppat.0030156
[31]
Yamashita M, Emerman M (2004) Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol 78: 5670–5678. doi: 10.1128/jvi.78.11.5670-5678.2004
[32]
Krishnan L, Matreyek KA, Oztop I, Lee K, Tipper CH, et al. (2010) The requirement for cellular transportin 3 (TNPO3 or TRN-SR2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase. J Virol 84: 397–406. doi: 10.1128/jvi.01899-09
[33]
Matreyek KA, Engelman A (2011) The requirement for nucleoporin NUP153 during human immunodeficiency virus type 1 infection is determined by the viral capsid. J Virol 85: 7818–7827. doi: 10.1128/jvi.00325-11
[34]
Lee K, Mulky A, Yuen W, Martin TD, Meyerson NR, et al. (2012) HIV-1 capsid-targeting domain of cleavage and polyadenylation specificity factor 6. J Virol 86: 3851–3860. doi: 10.1128/jvi.06607-11
[35]
Lee K, Ambrose Z, Martin TD, Oztop I, Mulky A, et al. (2010) Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 7: 221–233. doi: 10.1016/j.chom.2010.02.007
[36]
Price AJ, Fletcher AJ, Schaller T, Elliott T, Lee K, et al. (2012) CPSF6 defines a conserved capsid interface that modulates HIV-1 replication. PLoS Pathog 8: e1002896. doi: 10.1371/journal.ppat.1002896
Hatziioannou T, Perez-Caballero D, Cowan S, Bieniasz PD (2005) Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells. J Virol 79: 176–183. doi: 10.1128/jvi.79.1.176-183.2005
[39]
Sokolskaja E, Sayah DM, Luban J (2004) Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity. J Virol 78: 12800–12808. doi: 10.1128/jvi.78.23.12800-12808.2004
[40]
Qi M, Yang R, Aiken C (2008) Cyclophilin A-dependent restriction of human immunodeficiency virus type 1 capsid mutants for infection of nondividing cells. J Virol 82: 12001–12008. doi: 10.1128/jvi.01518-08
[41]
Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, et al. (2004) The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427: 848–853. doi: 10.1038/nature02343
[42]
Manel N, Hogstad B, Wang Y, Levy DE, Unutmaz D, et al. (2010) A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells. Nature 467: 214–217. doi: 10.1038/nature09337
[43]
Crawford H, Matthews PC, Schaefer M, Carlson JM, Leslie A, et al. (2011) The hypervariable HIV-1 capsid protein residues comprise HLA-driven CD8+ T-cell escape mutations and covarying HLA-independent polymorphisms. J Virol 85: 1384–1390. doi: 10.1128/jvi.01879-10
[44]
Dahirel V, Shekhar K, Pereyra F, Miura T, Artyomov M, et al. (2011) Coordinate linkage of HIV evolution reveals regions of immunological vulnerability. Proc Natl Acad Sci U S A 108: 11530–11535. doi: 10.1073/pnas.1105315108
[45]
Brockman MA, Brumme ZL, Brumme CJ, Miura T, Sela J, et al. (2010) Early selection in Gag by protective HLA alleles contributes to reduced HIV-1 replication capacity that may be largely compensated for in chronic infection. J Virol 84: 11937–11949. doi: 10.1128/jvi.01086-10
[46]
Carlson JM, Brumme CJ, Martin E, Listgarten J, Brockman MA, et al. (2012) Correlates of Protective Cellular Immunity Revealed by Analysis of Population-Level Immune Escape Pathways in HIV-1. J Virol 86: 13202–13216. doi: 10.1128/jvi.01998-12
[47]
Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB (1994) Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol 68: 6103–6110.
[48]
Pereyra F, Jia X, McLaren PJ, Telenti A, de Bakker PI, et al. (2010) The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Science 330: 1551–1557. doi: 10.1126/science.1195271
[49]
Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, et al. (2007) CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 13: 46–53. doi: 10.1038/nm1520
[50]
Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, et al. (2001) Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J Exp Med 193: 375–386. doi: 10.1084/jem.193.3.375
[51]
Troyer RM, McNevin J, Liu Y, Zhang SC, Krizan RW, et al. (2009) Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response. PLoS Pathog 5: e1000365. doi: 10.1371/journal.ppat.1000365
[52]
Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, et al. (2004) HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 10: 282–289. doi: 10.1038/nm992
[53]
Reicin AS, Paik S, Berkowitz RD, Luban J, Lowy I, et al. (1995) Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity. J Virol 69: 642–650.
[54]
Srinivasakumar N, Hammarskjold ML, Rekosh D (1995) Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation. J Virol 69: 6106–6114.
[55]
Fitzon T, Leschonsky B, Bieler K, Paulus C, Schroder J, et al. (2000) Proline residues in the HIV-1 NH2-terminal capsid domain: structure determinants for proper core assembly and subsequent steps of early replication. Virology 268: 294–307. doi: 10.1006/viro.1999.0178
[56]
von Schwedler UK, Stray KM, Garrus JE, Sundquist WI (2003) Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol 77: 5439–5450. doi: 10.1128/jvi.77.9.5439-5450.2003
[57]
Holm L, Koivula AK, Lehtovaara PM, Hemminki A, Knowles JK (1990) Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase. Protein Eng 3: 181–191. doi: 10.1093/protein/3.3.181
[58]
McNatt MW, Zang T, Hatziioannou T, Bartlett M, Fofana IB, et al. (2009) Species-specific activity of HIV-1 Vpu and positive selection of tetherin transmembrane domain variants. PLoS Pathog 5: e1000300. doi: 10.1371/journal.ppat.1000300
[59]
Hatziioannou T, Cowan S, Von Schwedler UK, Sundquist WI, Bieniasz PD (2004) Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid. J Virol 78: 6005–6012. doi: 10.1128/jvi.78.11.6005-6012.2004
[60]
Ambrose Z, Lee K, Ndjomou J, Xu H, Oztop I, et al. (2012) Human immunodeficiency virus type 1 capsid mutation N74D alters cyclophilin A dependence and impairs macrophage infection. J Virol 86: 4708–4714. doi: 10.1128/jvi.05887-11
[61]
Sanjuan R, Nebot MR, Chirico N, Mansky LM, Belshaw R (2010) Viral mutation rates. J Virol 84: 9733–9748. doi: 10.1128/jvi.00694-10
[62]
Sanjuan R (2010) Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies. Philos Trans R Soc Lond B Biol Sci 365: 1975–1982. doi: 10.1098/rstb.2010.0063
[63]
Forshey BM, von Schwedler U, Sundquist WI, Aiken C (2002) Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol 76: 5667–5677. doi: 10.1128/jvi.76.11.5667-5677.2002
[64]
Jouvenet N, Bieniasz PD, Simon SM (2008) Imaging the biogenesis of individual HIV-1 virions in live cells. Nature 454: 236–240. doi: 10.1038/nature06998
[65]
Krausslich HG (1991) Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. Proc Natl Acad Sci U S A 88: 3213–3217. doi: 10.1073/pnas.88.8.3213
[66]
Ott DE, Coren LV, Chertova EN, Gagliardi TD, Nagashima K, et al. (2003) Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant. J Virol 77: 5547–5556. doi: 10.1128/jvi.77.10.5547-5556.2003
[67]
Ott DE, Coren LV, Shatzer T (2009) The nucleocapsid region of human immunodeficiency virus type 1 Gag assists in the coordination of assembly and Gag processing: role for RNA-Gag binding in the early stages of assembly. J Virol 83: 7718–7727. doi: 10.1128/jvi.00099-09
[68]
Borsetti A, Ohagen A, Gottlinger HG (1998) The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly. J Virol 72: 9313–9317.
[69]
Auerbach MR, Shu C, Kaplan A, Singh IR (2003) Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting. Proc Natl Acad Sci U S A 100: 11678–11683. doi: 10.1073/pnas.2034020100
[70]
Auerbach MR, Brown KR, Singh IR (2007) Mutational analysis of the N-terminal domain of Moloney murine leukemia virus capsid protein. J Virol 81: 12337–12347. doi: 10.1128/jvi.01286-07
[71]
Alin K, Goff SP (1996) Amino acid substitutions in the CA protein of Moloney murine leukemia virus that block early events in infection. Virology 222: 339–351. doi: 10.1006/viro.1996.0431
[72]
Allen TM, Altfeld M, Geer SC, Kalife ET, Moore C, et al. (2005) Selective escape from CD8+ T-cell responses represents a major driving force of human immunodeficiency virus type 1 (HIV-1) sequence diversity and reveals constraints on HIV-1 evolution. J Virol 79: 13239–13249. doi: 10.1128/jvi.79.21.13239-13249.2005
[73]
Martinez-Picado J, Prado JG, Fry EE, Pfafferott K, Leslie A, et al. (2006) Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1. J Virol 80: 3617–3623. doi: 10.1128/jvi.80.7.3617-3623.2006
[74]
Mansky LM, Temin HM (1995) Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol 69: 5087–5094.
[75]
Abram ME, Ferris AL, Shao W, Alvord WG, Hughes SH (2010) Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication. J Virol 84: 9864–9878. doi: 10.1128/jvi.00915-10
[76]
Perelson AS, Neumann AU, Markowitz M, Leonard JM, Ho DD (1996) HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271: 1582–1586. doi: 10.1126/science.271.5255.1582
[77]
Rolland M, Manocheewa S, Swain JV, Lanxon-Cookson EC, Kim M, et al. (2013) HIV-1 Conserved-Element Vaccines: Relationship between Sequence Conservation and Replicative Capacity. J Virol 87: 5461–5467. doi: 10.1128/jvi.03033-12
[78]
Tang C, Loeliger E, Kinde I, Kyere S, Mayo K, et al. (2003) Antiviral inhibition of the HIV-1 capsid protein. J Mol Biol 327: 1013–1020. doi: 10.1016/s0022-2836(03)00289-4
[79]
Blair WS, Pickford C, Irving SL, Brown DG, Anderson M, et al. (2010) HIV capsid is a tractable target for small molecule therapeutic intervention. PLoS Pathog 6: e1001220. doi: 10.1371/journal.ppat.1001220
[80]
Sticht J, Humbert M, Findlow S, Bodem J, Muller B, et al. (2005) A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol 12: 671–677. doi: 10.1038/nsmb964
[81]
Zhang H, Zhao Q, Bhattacharya S, Waheed AA, Tong X, et al. (2008) A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J Mol Biol 378: 565–580. doi: 10.1016/j.jmb.2008.02.066
[82]
Lemke CT, Titolo S, von Schwedler U, Goudreau N, Mercier JF, et al. (2012) Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein. J Virol 86: 6643–6655. doi: 10.1128/jvi.00493-12
[83]
Zhang F, Zang T, Wilson SJ, Johnson MC, Bieniasz PD (2011) Clathrin facilitates the morphogenesis of retrovirus particles. PLoS Pathog 7: e1002119. doi: 10.1371/journal.ppat.1002119
[84]
Varthakavi V, Browning PJ, Spearman P (1999) Human immunodeficiency virus replication in a primary effusion lymphoma cell line stimulates lytic-phase replication of Kaposi's sarcoma-associated herpesvirus. J Virol 73: 10329–10338.
[85]
Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113.
[86]
Shenkin PS, Erman B, Mastrandrea LD (1991) Information-theoretical entropy as a measure of sequence variability. Proteins 11: 297–313. doi: 10.1002/prot.340110408
[87]
Shao W, Everitt L, Manchester M, Loeb DD, Hutchison CA 3rd, et al. (1997) Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants. Proc Natl Acad Sci U S A 94: 2243–2248. doi: 10.1073/pnas.94.6.2243
[88]
Parera M, Fernandez G, Clotet B, Martinez MA (2007) HIV-1 protease catalytic efficiency effects caused by random single amino acid substitutions. Mol Biol Evol 24: 382–387. doi: 10.1093/molbev/msl168
[89]
Loeb DD, Swanstrom R, Everitt L, Manchester M, Stamper SE, et al. (1989) Complete mutagenesis of the HIV-1 protease. Nature 340: 397–400. doi: 10.1038/340397a0
[90]
Chao SF, Chan VL, Juranka P, Kaplan AH, Swanstrom R, et al. (1995) Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1. Nucleic Acids Res 23: 803–810. doi: 10.1093/nar/23.5.803
[91]
van den Ent FM, Vos A, Plasterk RH (1998) Mutational scan of the human immunodeficiency virus type 2 integrase protein. J Virol 72: 3916–3924.
[92]
Smith RA, Anderson DJ, Preston BD (2006) Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase. J Virol 80: 7169–7178. doi: 10.1128/jvi.00322-06
[93]
van Opijnen T, Boerlijst MC, Berkhout B (2006) Effects of random mutations in the human immunodeficiency virus type 1 transcriptional promoter on viral fitness in different host cell environments. J Virol 80: 6678–6685. doi: 10.1128/jvi.02547-05
[94]
Kim B, Hathaway TR, Loeb LA (1996) Human immunodeficiency virus reverse transcriptase. Functional mutants obtained by random mutagenesis coupled with genetic selection in Escherichia coli. J Biol Chem 271: 4872–4878.
[95]
Yasugi T, Vidal M, Sakai H, Howley PM, Benson JD (1997) Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins. J Virol 71: 5942–5951.
[96]
Morrison HG, Kirchhoff F, Desrosiers RC (1995) Effects of mutations in constant regions 3 and 4 of envelope of simian immunodeficiency virus. Virology 210: 448–455. doi: 10.1006/viro.1995.1361
[97]
Nakajima K, Nobusawa E, Tonegawa K, Nakajima S (2003) Restriction of amino acid change in influenza A virus H3HA: comparison of amino acid changes observed in nature and in vitro. J Virol 77: 10088–10098. doi: 10.1128/jvi.77.18.10088-10098.2003
[98]
Yano T, Nobusawa E, Nagy A, Nakajima S, Nakajima K (2008) Effects of single-point amino acid substitutions on the structure and function neuraminidase proteins in influenza A virus. Microbiol Immunol 52: 216–223. doi: 10.1111/j.1348-0421.2008.00034.x
[99]
Pakula AA, Young VB, Sauer RT (1986) Bacteriophage lambda cro mutations: effects on activity and intracellular degradation. Proc Natl Acad Sci U S A 83: 8829–8833. doi: 10.1073/pnas.83.23.8829
[100]
Rhee SS, Hunter E (1991) Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid. EMBO J 10: 535–546.
[101]
Terwilliger TC, Zabin HB, Horvath MP, Sandberg WS, Schlunk PM (1994) In vivo characterization of mutants of the bacteriophage f1 gene V protein isolated by saturation mutagenesis. J Mol Biol 236: 556–571. doi: 10.1006/jmbi.1994.1165
[102]
Masso M, Mathe E, Parvez N, Hijazi K, Vaisman II (2009) Modeling the functional consequences of single residue replacements in bacteriophage f1 gene V protein. Protein Eng Des Sel 22: 665–671. doi: 10.1093/protein/gzp050
[103]
Suzutani T, Lacey SF, Powell KL, Purifoy DJ, Honess RW (1992) Random mutagenesis of the thymidine kinase gene of varicella-zoster virus. J Virol 66: 2118–2124. doi: 10.1099/0022-1317-72-3-623
[104]
Eifan SA, Elliott RM (2009) Mutational analysis of the Bunyamwera orthobunyavirus nucleocapsid protein gene. J Virol 83: 11307–11317. doi: 10.1128/jvi.01460-09
[105]
Stenger DC, Young BA, French R (2006) Random mutagenesis of wheat streak mosaic virus HC-Pro: non-infectious interfering mutations in a gene dispensable for systemic infection of plants. J Gen Virol 87: 2741–2747. doi: 10.1099/vir.0.81933-0
[106]
Rennell D, Bouvier SE, Hardy LW, Poteete AR (1991) Systematic mutation of bacteriophage T4 lysozyme. J Mol Biol 222: 67–88. doi: 10.1016/0022-2836(91)90738-r
[107]
Strambio-de-Castillia C, Hunter E (1992) Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis. J Virol 66: 7021–7032.
[108]
Moyer CL, Wiethoff CM, Maier O, Smith JG, Nemerow GR (2011) Functional genetic and biophysical analyses of membrane disruption by human adenovirus. J Virol 85: 2631–2641. doi: 10.1128/jvi.02321-10
[109]
Van Der Velden A, Kaminski A, Jackson RJ, Belsham GJ (1995) Defective point mutants of the encephalomyocarditis virus internal ribosome entry site can be complemented in trans. Virology 214: 82–90. doi: 10.1006/viro.1995.9952
[110]
Peris JB, Davis P, Cuevas JM, Nebot MR, Sanjuan R (2010) Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1. Genetics 185: 603–609. doi: 10.1534/genetics.110.115162
[111]
Markiewicz P, Kleina LG, Cruz C, Ehret S, Miller JH (1994) Genetic studies of the lac repressor. XIV. Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as “spacers” which do not require a specific sequence. J Mol Biol 240: 421–433. doi: 10.1006/jmbi.1994.1458
[112]
Guo HH, Choe J, Loeb LA (2004) Protein tolerance to random amino acid change. Proc Natl Acad Sci U S A 101: 9205–9210. doi: 10.1073/pnas.0403255101
[113]
Ihssen J, Kowarik M, Wiesli L, Reiss R, Wacker M, et al. (2012) Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB. BMC Biotechnol 12: 67. doi: 10.1186/1472-6750-12-67
[114]
Olins PO, Bauer SC, Braford-Goldberg S, Sterbenz K, Polazzi JO, et al. (1995) Saturation mutagenesis of human interleukin-3. J Biol Chem 270: 23754–23760. doi: 10.1074/jbc.270.40.23754
[115]
Huang W, Petrosino J, Hirsch M, Shenkin PS, Palzkill T (1996) Amino acid sequence determinants of beta-lactamase structure and activity. J Mol Biol 258: 688–703. doi: 10.1006/jmbi.1996.0279
[116]
Chen C, Roberts VA, Rittenberg MB (1992) Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutated antibodies lose their ability to bind antigen. J Exp Med 176: 855–866. doi: 10.1084/jem.176.3.855
[117]
Axe DD, Foster NW, Fersht AR (1998) A search for single substitutions that eliminate enzymatic function in a bacterial ribonuclease. Biochemistry 37: 7157–7166. doi: 10.1021/bi9804028
[118]
Kawashima H, Yamagishi J, Yamayoshi M, Ohue M, Fukui T, et al. (1992) Structure-activity relationships in human interleukin-1 alpha: identification of key residues for expression of biological activities. Protein Eng 5: 171–176. doi: 10.1093/protein/5.2.171
