title: Altered strand transfer activity of a multiple-drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutant with a dipeptide fingers domain insertion.
abstract: Prolonged highly active anti-retroviral therapy with multiple nucleoside reverse transcriptase inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1) can induce the development of an HIV-1 reverse transcriptase (RT) harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation. This mutation renders viral resistance to multiple nucleoside reverse transcriptase inhibitors. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during the pre-treatment and post-treatment of an infected patient, termed pre-drug RT without dipeptide insertion and post-drug RT with Ser-Gly insertion, respectively. First, the post-drug RT displayed elevated strand transfer activity compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate (k(on)) for template binding and a lower equilibrium binding constant K(d) for the template, leading to a template binding affinity tighter than that of the pre-drug RT. The k(off) values for the pre-drug RT and the post-drug RT were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity, in addition to the loss of azidothymidine resistance. These biochemical data suggest that the dipeptide insertion elevates strand transfer activity by increasing the interaction of the RT with the RNA donor template, promoting cleavage that generates more invasion sites for the acceptor template during DNA synthesis.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Nguyen
authors.forename: Waaqo
authors.initials: W
authors.lastname: Daddacha
authors.forename: Sean
authors.initials: S
authors.lastname: Rigby
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
mesh_terms: D019380:Anti-HIV Agents / Q000008:administration & dosage; D004279:DNA, Viral / Q000378:metabolism; D004151:Dipeptides / Q000235:genetics; D024921:Drug Resistance, Multiple, Viral; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; D004151:Dipeptides; D050505:Mutant Proteins; D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
references.citation: J Mol Biol. 2009 Apr 24;388(1):30-47
references.pmid: 19233203
references.citation: Science. 1988 Nov 25;242(4882):1168-71
references.pmid: 2460924
references.citation: J Biol Chem. 2004 Jun 4;279(23):24569-77
references.pmid: 15047690
references.citation: J Biol Chem. 2003 Mar 21;278(12):10102-11
references.pmid: 12533519
references.citation: J Biol Chem. 2005 Dec 9;280(49):40534-43
references.pmid: 16221683
references.citation: J Infect Dis. 1997 Oct;176(4):899-903
references.pmid: 9333147
references.citation: Antimicrob Agents Chemother. 1999 Aug;43(8):1961-7
references.pmid: 10428920
references.citation: J Biol Chem. 2000 Dec 1;275(48):37664-71
references.pmid: 10956669
references.citation: J Biol Chem. 2007 Apr 27;282(17):12598-609
references.pmid: 17337733
references.citation: Nucleic Acids Res. 1996 Sep 1;24(17):3407-14
references.pmid: 8811096
references.citation: Lancet. 2010 Jul 3;376(9734):49-62
references.pmid: 20609987
references.citation: Antimicrob Agents Chemother. 2004 Mar;48(3):992-1003
references.pmid: 14982794
references.citation: J Biol Chem. 2002 Jun 21;277(25):22662-9
references.pmid: 11927582
references.citation: Genetics. 2004 Aug;167(4):1573-83
references.pmid: 15342499
references.citation: EMBO J. 2000 Nov 1;19(21):5752-61
references.pmid: 11060026
references.citation: J Virol. 2002 Sep;76(18):9143-51
references.pmid: 12186898
references.citation: J Biol Chem. 2003 Aug 22;278(34):31536-46
references.pmid: 12801926
references.citation: J Virol. 1995 Aug;69(8):5087-94
references.pmid: 7541846
references.citation: Microbiol Mol Biol Rev. 2009 Sep;73(3):451-80, Table of Contents
references.pmid: 19721086
references.citation: Science. 1998 Nov 27;282(5394):1669-75
references.pmid: 9831551
references.citation: Science. 1992 Nov 13;258(5085):1112-8
references.pmid: 1279806
references.citation: J Clin Invest. 1998 Nov 15;102(10):1769-75
references.pmid: 9819361
references.citation: Antiviral Res. 2007 Aug;75(2):93-103
references.pmid: 17416429
references.citation: Virus Res. 2008 Jun;134(1-2):19-38
references.pmid: 18279992
references.citation: J Virol. 2003 Apr;77(8):4710-21
references.pmid: 12663778
references.citation: J Virol. 2003 Mar;77(6):3871-7
references.pmid: 12610164
references.citation: Virology. 2000 May 10;270(2):310-6
references.pmid: 10792990
references.citation: Antiviral Res. 1997 Dec;36(3):179-89
references.pmid: 9477118
references.citation: Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11978-83
references.pmid: 11035788
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6106-11
references.pmid: 8650227
references.citation: J Virol. 1995 Feb;69(2):669-74
references.pmid: 7529334
references.citation: Clin Microbiol Rev. 2002 Apr;15(2):247-77
references.pmid: 11932232
references.citation: J Gen Virol. 1995 Oct;76 ( Pt 10):2601-5
references.pmid: 7595365
references.citation: J Biol Chem. 2006 Oct 27;281(43):32113-21
references.pmid: 16926150
references.citation: Science. 2008 Nov 14;322(5904):1092-7
references.pmid: 19008444
references.citation: J Mol Biol. 2009 Dec 11;394(4):694-707
references.pmid: 19853618
references.citation: J Mol Biol. 2009 Jan 23;385(3):693-713
references.pmid: 19022262
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10943-8
references.pmid: 18667707
references.citation: Infect Genet Evol. 2007 Jul;7(4):476-83
references.pmid: 17369105
references.citation: J Virol. 2001 Apr;75(8):3988-92
references.pmid: 11264389
references.citation: J Infect Dis. 2000 May;181(5):1808-12
references.pmid: 10823790
references.citation: Biochemistry. 1993 Jul 13;32(27):6908-15
references.pmid: 7687463
references.citation: Science. 1988 Nov 25;242(4882):1171-3
references.pmid: 2460925
references.citation: J Biol Chem. 1998 Nov 13;273(46):30435-42
references.pmid: 9804810
references.citation: J Mol Biol. 2007 Jan 12;365(2):298-309
references.pmid: 17070543
references.citation: J Biol Chem. 2008 Apr 4;283(14):9196-205
references.pmid: 18218634
references.citation: J Gen Virol. 2006 Feb;87(Pt 2):419-428
references.pmid: 16432030