A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus.
BACKGROUND
The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.


METHODS
Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.


RESULTS
Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.


CONCLUSIONS
The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.
Medical Research 151MV, Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, Georgia 30033, USA. plsharm@emory.edu
Himabindu
H
Chunduri
David
D
Rimland
Viktoria
V
Nurpeisov
Clyde S
CS
Crumpacker
Prem L
PL
Sharma
D019943:Amino Acid Substitution / Q000235:genetics*; D002478:Cells, Cultured; D024882:Drug Resistance, Viral; D016655:HIV Core Protein p24 / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011533:Proviruses / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D014779:Virus Replication / Q000187:drug effects*
D016655:HIV Core Protein p24; D050505:Mutant Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
J Clin Microbiol. 2003 Jul;41(7):3306-11
12843079
J Virol. 1994 May;68(5):3421-4
7512165
EMBO J. 1996 Aug 1;15(15):4040-9
8670908
AIDS. 2009 Jan 2;23(1):95-9
19050391
J Biol Chem. 2004 Jun 11;279(24):25489-96
15044478
AIDS. 2007 Jul 11;21(11):1405-14
17589186
J Virol. 1986 Aug;59(2):284-91
3016298
J Virol. 1997 Nov;71(11):8846-51
9343245
Antimicrob Agents Chemother. 1998 Feb;42(2):447-52
9527805
J Biol Chem. 2004 Jan 2;279(1):509-16
14551187
J Biol Chem. 1999 Apr 16;274(16):11283-8
10196217
Clin Infect Dis. 2008 Jan 1;46(1):96-100
18171220
J Biol Chem. 2008 Apr 4;283(14):9196-205
18218634
AIDS. 2005 Mar 24;19(6):630-2
15802984
Biochemistry. 1998 Jul 7;37(27):9630-40
9657675
Oncogene. 2001 Nov 15;20(52):7635-44
11753641
Science. 1998 Nov 27;282(5394):1669-75
9831551
Antivir Ther. 2008;13(2):189-97
18505170
Antimicrob Agents Chemother. 2002 Nov;46(11):3437-46
12384348
J Biol Chem. 2009 Mar 20;284(12):7931-9
19150986
AIDS Rev. 2004 Jan-Mar;6(1):22-33
15168738
J Biol Chem. 2008 Apr 4;283(14):9206-16
18218633
AIDS. 2007 Mar 30;21(6):665-75
17413687
Nucleic Acids Res. 1998 Apr 1;26(7):1713-7
9512543
J Virol. 1999 Aug;73(8):6293-8
10400720
Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5694-8
7539924
Retrovirology. 2009 Feb 11;6:14
19210791
J Virol. 2006 May;80(10):4971-7
16641288
Science. 1995 Aug 4;269(5224):696-9
7542804
Antimicrob Agents Chemother. 2000 Jul;44(7):1783-8
10858331
J Virol. 1999 Oct;73(10):8448-56
10482597
Virology. 1996 Sep 15;223(2):365-9
8806572
PLoS Comput Biol. 2007 May;3(5):e87
17500586
Virology. 2004 Apr 10;321(2):222-34
15051383
J Acquir Immune Defic Syndr. 2008 Aug 1;48(4):428-36
18614922
J Mol Biol. 2007 Jan 12;365(2):310-24
17070544
J Virol. 1992 Apr;66(4):2232-9
1548759
J Clin Virol. 2006 Nov;37(3):227-30
16931138
J Acquir Immune Defic Syndr. 2007 Oct 1;46(2):174-80
17667333
J Virol. 2004 May;78(10):5056-67
15113887
Antimicrob Agents Chemother. 1995 Jul;39(7):1624-8
7492119
J Virol. 1996 Aug;70(8):4996-5004
8764006
Antimicrob Agents Chemother. 1997 Nov;41(11):2484-91
9371354
J Mol Biol. 1999 Feb 12;286(1):15-31
9931246
Adv Exp Med Biol. 1996;394:383-95
8815703
Antiviral Res. 2009 Sep;83(3):317-23
19555722