Search tips
Search criteria 


Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. 1992 November; 66(11): 6806–6812.
PMCID: PMC240183

In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome.


Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Araya A, Sarih L, Litvak S. Reverse transcriptase mediated binding of primer tRNA to the viral genome. Nucleic Acids Res. 1979 Aug 24;6(12):3831–3843. [PMC free article] [PubMed]
  • Argos P. A sequence motif in many polymerases. Nucleic Acids Res. 1988 Nov 11;16(21):9909–9916. [PMC free article] [PubMed]
  • Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. [PubMed]
  • Barat C, Lullien V, Schatz O, Keith G, Nugeyre MT, Grüninger-Leitch F, Barré-Sinoussi F, LeGrice SF, Darlix JL. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. [PubMed]
  • Barber AM, Hizi A, Maizel JV, Jr, Hughes SH. HIV-1 reverse transcriptase: structure predictions for the polymerase domain. AIDS Res Hum Retroviruses. 1990 Sep;6(9):1061–1072. [PubMed]
  • Beese LS, Steitz TA. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991 Jan;10(1):25–33. [PubMed]
  • Bernad A, Lázaro JM, Salas M, Blanco L. The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4610–4614. [PubMed]
  • Cann AJ, Karn J. Molecular biology of HIV: new insights into the virus life-cycle. AIDS. 1989;3 (Suppl 1):S19–S34. [PubMed]
  • Cobrinik D, Soskey L, Leis J. A retroviral RNA secondary structure required for efficient initiation of reverse transcription. J Virol. 1988 Oct;62(10):3622–3630. [PMC free article] [PubMed]
  • Delarue M, Poch O, Tordo N, Moras D, Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. [PubMed]
  • Donahue PR, Hoover EA, Beltz GA, Riedel N, Hirsch VM, Overbaugh J, Mullins JI. Strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses. J Virol. 1988 Mar;62(3):722–731. [PMC free article] [PubMed]
  • Dorsky DI, Crumpacker CS. Site-specific mutagenesis of a highly conserved region of the herpes simplex virus type 1 DNA polymerase gene. J Virol. 1990 Mar;64(3):1394–1397. [PMC free article] [PubMed]
  • Farmerie WG, Loeb DD, Casavant NC, Hutchison CA, 3rd, Edgell MH, Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science. 1987 Apr 17;236(4799):305–308. [PubMed]
  • Gilboa E, Mitra SW, Goff S, Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. [PubMed]
  • Goff SP. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3(8):817–831. [PubMed]
  • Haffey ML, Novotny J, Bruccoleri RE, Carroll RD, Stevens JT, Matthews JT. Structure-function studies of the herpes simplex virus type 1 DNA polymerase. J Virol. 1990 Oct;64(10):5008–5018. [PMC free article] [PubMed]
  • Hu WS, Temin HM. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. [PubMed]
  • Inokuchi Y, Hirashima A. Interference with viral infection by defective RNA replicase. J Virol. 1987 Dec;61(12):3946–3949. [PMC free article] [PubMed]
  • Inouye S, Hsu MY, Eagle S, Inouye M. Reverse transcriptase associated with the biosynthesis of the branched RNA-linked msDNA in Myxococcus xanthus. Cell. 1989 Feb 24;56(4):709–717. [PubMed]
  • Jablonski SA, Luo M, Morrow CD. Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol. 1991 Sep;65(9):4565–4572. [PMC free article] [PubMed]
  • Johnson MS, McClure MA, Feng DF, Gray J, Doolittle RF. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. [PubMed]
  • Joung I, Horwitz MS, Engler JA. Mutagenesis of conserved region I in the DNA polymerase from human adenovirus serotype 2. Virology. 1991 Sep;184(1):235–241. [PubMed]
  • Kamer G, Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 1984 Sep 25;12(18):7269–7282. [PMC free article] [PubMed]
  • Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Lampson BC, Inouye M, Inouye S. Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus. Cell. 1989 Feb 24;56(4):701–707. [PubMed]
  • Lampson BC, Sun J, Hsu MY, Vallejo-Ramirez J, Inouye S, Inouye M. Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA. Science. 1989 Feb 24;243(4894 Pt 1):1033–1038. [PubMed]
  • Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. [PubMed]
  • Larder BA, Kemp SD, Purifoy DJ. Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4803–4807. [PubMed]
  • Larder BA, Purifoy DJ, Powell KL, Darby G. Site-specific mutagenesis of AIDS virus reverse transcriptase. Nature. 327(6124):716–717. [PubMed]
  • Le Grice SF, Naas T, Wohlgensinger B, Schatz O. Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. EMBO J. 1991 Dec;10(12):3905–3911. [PubMed]
  • Lightfoote MM, Coligan JE, Folks TM, Fauci AS, Martin MA, Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. [PMC free article] [PubMed]
  • Lopata MA, Cleveland DW, Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. [PMC free article] [PubMed]
  • Lowe DM, Parmar V, Kemp SD, Larder BA. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 1991 May 6;282(2):231–234. [PubMed]
  • Marcy AI, Hwang CB, Ruffner KL, Coen DM. Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition. J Virol. 1990 Dec;64(12):5883–5890. [PMC free article] [PubMed]
  • Morrow CD, Warren B, Lentz MR. Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6050–6054. [PubMed]
  • Ollis DL, Brick P, Hamlin R, Xuong NG, Steitz TA. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature. 313(6005):762–766. [PubMed]
  • Panganiban AT, Fiore D. Ordered interstrand and intrastrand DNA transfer during reverse transcription. Science. 1988 Aug 26;241(4869):1064–1069. [PubMed]
  • Poch O, Sauvaget I, Delarue M, Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. [PubMed]
  • Ratner L, Fisher A, Jagodzinski LL, Mitsuya H, Liou RS, Gallo RC, Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. [PubMed]
  • Rey MA, Spire B, Dormont D, Barre-Sinoussi F, Montagnier L, Chermann JC. Characterization of the RNA dependent DNA polymerase of a new human T-lymphotropic retrovirus (lymphadenopathy associated virus). Biochem Biophys Res Commun. 1984 May 31;121(1):126–133. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Schulte U, Lambowitz AM. The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase. Mol Cell Biol. 1991 Mar;11(3):1696–1706. [PMC free article] [PubMed]
  • Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC, Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. Science. 1984 Dec 7;226(4679):1165–1171. [PubMed]
  • Shinnick TM, Lerner RA, Sutcliffe JG. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. [PubMed]
  • Taylor JM, Illmensee R. Site on the RNA of an avian sarcoma virus at which primer is bound. J Virol. 1975 Sep;16(3):553–558. [PMC free article] [PubMed]
  • Temin HM. Structure, variation and synthesis of retrovirus long terminal repeat. Cell. 1981 Nov;27(1 Pt 2):1–3. [PubMed]
  • Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. [PubMed]
  • Varmus HE. Form and function of retroviral proviruses. Science. 1982 May 21;216(4548):812–820. [PubMed]
  • Wong SW, Wahl AF, Yuan PM, Arai N, Pearson BE, Arai K, Korn D, Hunkapiller MW, Wang TS. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. [PubMed]
  • Xiong Y, Eickbush TH. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 1990 Oct;9(10):3353–3362. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. [PubMed]
  • Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)