Drug Resistance in HIV: The SHAPE of Things to Come

Nuclear export of certain HIV-1 mRNAs requires an interaction between the retroviral Rev protein and the Rev response element (RRE), a structured element located in the Env region of its RNA genome.  Disrupting this interaction has been an attractive target for drug design and gene therapy, exemplified by RevM10, a transdominant negative protein that, when introduced into host cells, disrupts viral mRNA export and inhibits virus replication.  However, two silent G->A mutations in the RRE (designated RRE61) conferred RevM10 resistance.  This observation prompted Legiewicz et al. to examine RRE evolution at the structural level using SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) chemistry, a novel footprinting approach that interrogates the base pairing status of all RNA nucleotides in a single reaction.  Structural variations in region III/IV/V of mutant RNAs suggest a stepwise rearrangement of the RRE to RevM10 resistance.  Using high-resolution mass spectrometry, these authors could also demonstrate that the stoichiometry of Rev "loading" onto RRE61 is unaffected by these structural changes, while chemical footprinting highlighted subtle differences between wild-type and mutant Rev and the RRE variants.

Legiewicz, M., Badorrek, C.S., Turner, K.B., Fabris, D., Hamm, T.E., Rekosh, D., Hammarskjold, M.-L., and Le Grice, S.F.J.  (2008)  Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element.  Proc. Natl. Acad. Sci. USA, in press.

Dirt Under the Thumbs: Allosteric Inhibition of HIV-1 RNase H by Vinylogous Ureas

High-throughput screening of NCI libraries of synthetic and natural compounds, totaling ~230,000, has identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT).  Synergy studies demonstrated that NSC727447 and the active site hydroxytropolone RNase H inhibitor b-thujaplicinol were mutually exclusive in their interaction with the RNase H domain of RT.  Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the p51 thumb subdomain were affected by inhibitor binding.  Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb.  Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity.  The study by Wendeler et al. shows that NSC727447 represents a novel class of allosteric RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.

Wendeler, M., Lee, H.-F., Bermingham, A., Miller, J.T., Chertov, O., Bona, M.K., Baichoo, N.S., Ehteshami, M., Beutler, J.A., O'Keefe, B.R., Götte, M., Kvaratskhelia, M., and Le Grice, S.F.J.  (2008)  Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity.  ACS Chem. Biol., in press.

An Unusual Turn of Events in Reverse Transcription

HIV catalyzes a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration.  This task requires the multifunctional reverse transcriptase (RT) to bind and discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support RNA-directed DNA synthesis, DNA-directed DNA synthesis, and DNA-directed RNA hydrolysis.  However, the mechanism by which substrates regulate the activity of the enzyme remains unclear.  In their recent publication, Abbondanzieri et al. have reported distinct orientational dynamics of the RT observed on different substrates using a single-molecule assay.  The enzyme adopted opposite binding orientations on duplexes containing generic DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively.  On duplexes containing the HIV polypurine tracts, which function as unique primers for plus-strand DNA synthesis, RT binds in both orientations and rapidly switches between the two states.  Switching kinetics were regulated by cognate nucleotides and non-nucleoside RT inhibitors, a major class of anti-HIV drugs.  These results indicate that the enzymatic activities of the RT are determined by its binding orientation on the substrate.   To read more, click here (PDF - 546KB).

Abbondanzieri, E.A., Bokinsky, G., Rausch, J.W., Zhang, J.X., Le Grice, S.F.J., and Zhuang, X.  (2008)  Dynamic binding orientations direct activity of HIV reverse transcriptase (PDF - 546KB).  Nature 453: 184-189.

Nature News and Views feature related to this article:  Arnold, E., and Sarafianos, S.G.  (2008)  Molecular biology: An HIV secret uncovered (PDF - 451KB).  Nature 453: 169-170.

Harvard University Gazette Online feature about this article:  Bradt, S.  (2008)  Research reveals workings of anti-HIV drugs.

CCR Connections feature about this article:  Reverse transcriptase: When function follows direction.  CCR Connections 2 (1): 4.

HIV and Drug Resistance: Hitting a Moving Target

HIV can take many roads to evade the effects of drug therapy.  Investigators at CCR [the National Cancer Institute's Center for Cancer Research] and Rutgers University recently identified a novel mechanism by which HIV can circumvent the antiviral activity of a compound called amphotericin B methyl ester (AME), providing new insights into how the virus replicates and evolves into more resistant strains.

Prior research revealed how HIV-1 makes its destructive entry into the target cell by fusing together the cholesterol-rich lipid bilayer of the viral envelope—made with key glycoproteins gp120 and gp41—and the host cell's plasma membrane.  Cell-viral interactions begin with the binding of gp120 to the CD4 receptor molecule on the target cell, followed by gp120 binding to coreceptors.  These coreceptors likely reside in structures called lipid rafts—areas in the cell plasma membrane that are rich in cholesterol, saturated fatty acids, and certain proteins—that facilitate the entry of viruses into host cells.  Finally, sequences in gp41 trigger the fusion of the viral and cellular lipid bilayers.  The lipid rafts are then involved in the production of new viral particles.

Drugs that hone in on the close interaction between cell and virus by disrupting lipid rafts would likely slow the virus's spread because they would hinder its ability to enter and leave host cells.  AME is such an agent; it acts by binding to cholesterol in the viral membrane, which itself is lipid raft like, potently blocking the virus’s entry into immune cells.  Eric O. Freed, Ph.D., and first author Abdul A. Waheed, Ph.D., both of CCR's HIV Drug Resistance Program, along with other researchers at CCR and Rutgers University, used AME in experimental systems to learn more about how HIV attaches to and infects cells.  They found that continual HIV exposure to low levels of AME induced the virus to mutate and become resistant to AME.  To read more, click here (284KB).

Waheed, A.A., Ablan, S.D., Roser, J.D., Sowder, R.C., Schaffner, C.P., Chertova, E., and Freed, E.O.  (2007)  HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease (PDF - 284KB).  Proc. Natl. Acad. Sci. USA 104: 8467–8471.

Probing the Building Block of HIV-1 and Other Retroviruses

A single viral protein, termed "Gag," is sufficient for efficient assembly and release of retrovirus-like particles from mammalian cells.  Furthermore, purified HIV-1 Gag protein can be induced to assemble into virus-like particles in a defined system in vitro by the addition of nucleic acid.  Thus, the Gag protein is the fundamental building block of retrovirus particles.  As reported in a pair of recent publications, research conducted principally in Alan Rein's laboratory has studied the properties of assembly-competent HIV-1 Gag in solution; this is the first published analysis of this type for any retroviral Gag protein.  In order to more fully characterize this key building block, they have analyzed both its conformation in solution and its intermolecular interactions.  This approach to probing the intricacies of Gag should advance the understanding of molecular mechanisms involved in formation of infectious retrovirus particles, and could ultimately reveal new clinical approaches to inhibiting the replication of viruses such as HIV-1.  To read more, click on the titles shown below.

Datta, S.A.K., Zhao, Z., Clark, P.K., Tarasov, S., Alexandratos, J.N., Campbell, S.J., Kvaratskhelia, M., Lebowitz, J., and Rein, A.  (2007)  Interactions between HIV-1 Gag molecules in solution: An inositol phosphate-mediated switch (PDF - 726KB).  J. Mol. Biol. 365: 799-811.

Datta, S.A.K., Curtis, J.E., Ratcliff, W., Clark, P.K., Crist, R.M., Lebowitz, J., Krueger, S., and Rein, A.  (2007)  Conformation of the HIV-1 Gag protein in solution (PDF - 1376KB).  J. Mol. Biol. 365: 812-824.

A New "Connection" Between HIV-1 Drug Resistance and RNase H Activity

Reverse transcriptase (RT), a key enzyme in the life cycle of HIV-1, possesses DNA polymerase and RNase H activities.  Because RT is essential for viral replication, it has been one of the attractive targets for antiretroviral drugs.  However, drug resistance remains a major obstacle to the effective management of HIV-1 infection and AIDS, as drug-resistance mutations arise very quickly in response to treatment.  A greater understanding of the molecular mechanisms that mediate HIV-1 drug resistance is therefore critical for developing more effective antiretroviral agents and successful therapy.  New insights into drug-resistance mechanisms have been provided by Nikolenko et al., whose recently published study revealed that mutations in the C-terminal domains of HIV-1 RT that are selected in response to antiviral therapy play a critical role in resistance to nucleoside RT inhibitors (NRTIs), a major class of clinically available antiretroviral drugs.  The authors propose that an increase in resistance to AZT (one of the NRTIs) is dependent on the balance between the RNase H activity of RT and the rate of removal of AZT from terminated DNA.   Because only the N-terminal portions of RT from clinical samples are included in standard genotypic and phenotypic drug-resistance testing, this study highlights the importance of analyzing the whole RT sequence for more effective control of HIV-1 infection and development of improved antiviral strategies.  To read more, click here (PDF - 898KB).

Nikolenko, G.N., Delviks-Frankenberry, K.A., Palmer, S., Maldarelli, F., Fivash, M.J., Jr., Coffin, J.M., and Pathak, V.K.  (2007)  Mutations in the connection domain of HIV-1 reverse transcriptase increase 3'-azido-3'-deoxythymidine resistance (PDF - 898KB).  Proc. Natl. Acad. Sci. USA 104: 317-322.

Recent HIV DRP Publications (June–September 2008)

Ambrose, Z., Compton, L., Piatak, M., Jr., Lu, D., Alvord, W.G., Lubomirski, M.S., Hildreth, J.E.K., Lifson, J.D., Miller, C.J., and KewalRamani, V.N.  (2008)  Incomplete protection against simian immunodeficiency virus vaginal transmission in rhesus macaques by a topical antiviral agent revealed by repeat challenges.  J. Virol. 82: 6591-6599.
[Abstract]     [Full-text PDF article - 680KB]

Ambrose, Z., and KewalRamani, V.N.  (2008)  Of mice and monkeys: New advances in animal models to study HIV-1 therapy and prophylaxis.  Future HIV Ther. 2: 363-373.
[Abstract]     [Full-text PDF article - 535KB]

Archin, N.M., Eron, J.J., Palmer, S., Hartmann-Duff, A., Martinson, J.A., Wiegand, A., Bandarenko, N., Schmitz, J.L., Bosch, R.J., Landay, A.L., Coffin, J.M., and Margolis, D.M.  (2008)  Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection of resting CD4+ T cells.  AIDS 22: 1131-1135.
[Abstract]     [Full-text PDF article - 96KB]

Bauman, J.D., Das, K., Ho, W.C., Baweja, M., Himmel, D.M., Clark, A.D., Jr., Oren, D.A., Boyer, P.L., Hughes, S.H., Shatkin, A.J., and Arnold, E.  (2008)  Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design.  Nucleic Acids Res. 36: 5083-5092.
[Abstract]     [Full-text PDF article - 927KB]

Chang, K.W., Oh, J., Alvord, W.G., and Hughes, S.H.  (2008)  The effects of alternate polypurine tracts (PPTs) and mutations of sequences adjacent to the PPT on viral replication and cleavage specificity of the Rous sarcoma virus reverse transcriptase.  J. Virol. 82: 8592-8604.
[Abstract]     [Full-text PDF article - 1596KB]

Chen, J., Pathak, V.K., Peng, W., and Hu, W.-S.  (2008)  Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIV_mac can coassemble into mature cores of infectious viruses.  J. Virol. 82: 8253-8261.
[Abstract]     [Full-text PDF article 472KB]

Comin, M.J., Vu, B.C., Boyer, P.L., Liao, C., Hughes, S.H., and Marquez, V.E.  (2008)
D-(+)-iso-methanocarbathymidine, a high-affinity substrate for herpes simplex virus 1 thymidine kinase.  ChemMedChem 3: 1129-1134.     [Abstract]     [Full-text PDF article - 417KB]

Das, K., Clark, A.D., Lewi, P., Hughes, S.H., and Arnold, E.  (2008)  Detection of non-nucleoside inhibitor binding to HIV-1 reverse transcriptase by dynamic light scattering.  Biophys. J., in press.

Datta, S.A.K., and Rein, A.  (2008)  Preparation of recombinant HIV-1 Gag protein and assembly of virus-like particles in vitro.  In Prasad, V., and Kalpana, G. (eds.), HIV Protocols, 2nd Ed., Humana Press, Inc., in press.

Delviks-Frankenberry, K.A., Nikolenko, G.N., Boyer, P.L., Hughes, S.H., Coffin, J.M., Jere, A., and Pathak, V.K.  (2008)  HIV-1 reverse transcriptase connection subdomain mutations reduce template RNA degradation and enhance AZT excision.  Proc. Natl. Acad. Sci. USA 105: 10943-10948.     [Abstract]     [Full-text PDF article - 536KB]

Ehteshami, M., Scarth, B.J., Tchesnokov, E.P., Dash, C., Le Grice, S.F.J., Hallenberger, S., Jochmans, D., and Goette, M.  (2008)  Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against nucleotide-competing reverse transcriptase inhibitors.  J. Biol. Chem., in press (Aug 25 Epub ahead of print).
[Abstract]     [Full-text PDF article - 883K]

Gorelick, R.J., Thomas, J.A., Coren, L.V., Bosche, W.J., Gagliardi, T.D., Shulenin, S., and Oroszlan, S.  (2008)  Characterization of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein mutants with alterations at a putative HIV-1 protease cleavage site.  Virology, in press.

Greene, W.C., Debyser, Z., Ikeda, Y., Freed, E.O., Stephens, E., Yonemoto, W., Buckheit, R., Este, J., and Cihlar, T.  (2008)  Novel targets for HIV therapy.  Antiviral Res., in press.

Hu, J., Renaud, G., Golmes, T., Ferris, A., Hendrie, P.C., Donahue, R.E., Hughes, S.H., Wolfsberg, T.G., Russell, D.W., and Dunbar, C.E.  (2008)  Reduced genotoxicity of avian sarcoma leukosis virus vectors in rhesus long-term repopulating cells compared to standard murine retrovirus vectors.  Mol. Ther. 16: 1617-1623.
[Abstract]     [Full-text PDF article - 367KB]

Joshi, A., Munshi, U., Ablan, S.D., Nagashima, K., and Freed, E.O.  (2008)  Functional replacement of a retroviral late domain by ubiquitin fusion.  Traffic, in press.

Kutty, G., Maldarelli, F., Achaz, G., and Kovacs, J.A.  (2008)  Variation in the major surface glycoprotein genes in Pneumocystis jirovecii.  J. Infect. Dis. 198: 741-749.
Abstract]     [Full-text PDF article - 523KB]

Kvaratskhelia, M., and Le Grice, S.F.J.  (2008)  Structural analysis of protein-RNA interactions with mass spectrometry.  In Lin, R.J., (ed.), RNA-Protein Interaction Protocols, Methods in Molecular Biology, Vol. 488, Humana Press, Totowa, NJ, pp. 213-220.

Legiewicz, M., Badorrek, C.S., Turner, K.B., Fabris, D., Hamm, T.E., Rekosh, D., Hammarskjold, M.-L., and Le Grice, S.F.J.  (2008)  Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element.  Proc. Natl. Acad. Sci. USA, in press.

Li, K., Zhang, S., Kronqvist, M., Wallin, M., Ekström, M., Derse, D., and Garoff, H.  (2008)  Intersubunit disulfide isomerization controls membrane fusion of human T-cell leukemia virus Env.  J. Virol. 82: 7135-7143.     [Abstract]     [Full-text PDF article - 1112KB]

Liu, F., Stephen, A.G., Waheed, A.A., Aman, M.J., Freed, E.O., Fisher, R.J., and Burke, T.R., Jr.  (2008)  SAR by oxime-containing peptide libraries: Application to Tsg101 ligand optimization.  ChemBioChem 9: 2000-2004.
[Abstract]     [Full-text PDF article - 313KB]

Mbisa, J., K.A. Delviks-Frankenberry, J.A. Thomas, R.J. Gorelick, and Pathak, V.K.  (2008)  Real-time PCR analysis of HIV-1 replication post-entry events.  In Prasad, V., and Kalpana, G. (eds.), HIV Protocols, 2nd Ed., Humana Press, Inc., in press.

Moore, M.D., and Hu, W.-S.  (2008)  HIV-1 RNA dimerization: It takes two to tango.  AIDS Rev., in press.

Moore, M.D., Chin, M.P.S., and Hu, W.-S.  (2008)  HIV-1 recombination: An experimental assay and a phylogenetic approach.  In Prasad, V., and Kalpana, G. (eds.), HIV Protocols, 2nd Ed., Humana Press, Inc., in press.

Moore, M.D., Fu, W., Soheilian, F., Nagashima, K., Ptak, R.G., Pathak, V.K., and Hu, W.-S.  (2008)  Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation.  Virology 379: 152-160.
[Abstract]      [Full-text PDF article - 1204KB]

Morse, C., and Maldarelli, F.  (2008)  HIV infection.  In Strober, W., and Gottesman, S. (eds.), Immunology, a Short Course, in press.

Mulky, A., Cohen, T.V., Kozlov, S.V., Korbei, B., Foisner, R., Stewart, C.L., and KewalRamani, V.N.  (2008)  The LEM domain proteins emerin and LAP2a are dispensable for human immunodeficiency virus type 1 and murine leukemia virus infections.  J. Virol. 82: 5860-5868.
[Abstract]     [Full-text PDF article - 798KB]

Nikolaitchik, O.A., Gorelick, R.J., Leavitt, M.G., Pathak, V.K., and Hu, W.-H.  (2008)  Functional complementation of nucleocapsid and late domain PTAP mutants of human immunodeficiency virus type 1 during replication.  Virology 375: 539-549.
[Abstract]     [Full-text PDF article - 824KB]

Oh, J., Chang, K.W., and Hughes, S.H.  (2008)  Integration of Rous sarcoma virus DNA: A CA dinucleotide is not required for the integration of the U3 end of viral DNA.  J. Virol., in press.

Ramanathan, R., Pau, A.K., Busse, K.H., Zemskova, M., Nieman, L., Kwan, R., Hammer, J.H., Mican, J.M., and Maldarelli, F.  (2008)  Iatrogenic Cushing's syndrome following epidural triamcinolone in an HIV-1 infected patient undergoing therapy with ritonavir-lopinavir.  Clin. Infect. Dis., in press.

Ricci, E.P., Herbreteau, C.H., Decimo, D., Schaupp, A., Datta, S.A.K., Rein, A., Darlix, J.-L., and Ohlmann, T.  (2008)  In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein.  RNA 14: 1443-1455.
[Abstract]     [Full-text PDF article - 1268KB]

Rulli, S.J., Jr., Mirro, J., Hill, S.A., Lloyd, P., Gorelick, R.J., Coffin, J.M., Derse, D., and Rein, A.  (2008)  Interactions of murine APOBEC3 and human APOBEC3G with murine leukemia viruses.  J. Virol. 82: 6566-6575.
[Abstract]     [Full-text PDF article - 429KB]

Saad, J.S., Ablan, S.D., Ghanam, R.H., Kim, A., Andrews, K., Nagashima, K., Soheilian, F., Freed, E.O., and Summers, M.F.  (2008)  Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting.  J. Mol. Biol. 382: 434-447.
[Abstract]     [Full-text PDF article - 3510KB]

Waheed, A.A., Ablan, S.D., Soheilian, F., Nagashima, K., Ono, A., Schaffner, C.P., and Freed, E.O.  (2008)  Inhibition of human immunodeficiency virus type 1 assembly and release by the cholesterol-binding compound amphotericin B methyl ester: Evidence for Vpu dependence.  J. Virol., in press (July 23 Epub ahead of print).
[Abstract]     [Full-text PDF article - 279KB]

Waheed, A.A., Ono, A., and Freed, E.O.  (2008)  Methods for the study of HIV-1 assembly.  In HIV Protocols, 2nd Ed., Humana Press, in press.

Wendeler, M., Lee, H.-F., Bermingham, A., Miller, J.T., Chertov, O., Bona, M.K., Baichoo, N.S., Ehteshami, M., Beutler, J.A., O'Keefe, B.R., Götte, M., Kvaratskhelia, M., and Le Grice, S.F.J.  (2008)  Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity.  ACS Chem. Biol., in press.

Wendeler, M., and Le Grice, S.F.J.  (2008)  HIV-1 reverse transcriptase.  In Gotte, M., Cameron, C.E., and Raney, K. (eds.), Viral Genome Replication, Springer Publications Company, New York, NY.

Last modified: 6 September 2008