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.

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.  Nature 453: 184-189.

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

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.

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.  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.  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.  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.

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.  Proc. Natl. Acad. Sci. USA 104: 317-322.

Recent HIV DRP Publications (February – May 2008)

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

Adamson, C.S., and Freed, E.O.  (2008)  Recent progress in antiretrovirals – lessons from resistance.  Drug Discovery Today 13: 424-432.
[Abstract]     [Full-text PDF article]

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 SIV vaginal transmission in rhesus macaques by a topical antiviral agent revealed by repeat challenges.  J. Virol., in press (April 23 Epub ahead of print).
[Abstract]     [Full-text PDF article]

Chen, H., Lee, J.S., Liang, X., Zhang, H., Zhu, T., Zhang, Z., Taylor, M.E., Zahnow, C., Feigenbaum, L., Rein, A., and Sukumar, S.  (2008)  Hoxb7 inhibits transgenic HER-2/neu-induced mouse mammary tumor onset but promotes progression and lung metastasis.  Cancer Res. 68: 3637-3644.      [Abstract]     [Full-text PDF article]

Chin, M.P., Lee, S.-K., Chen, J., Nikolaitchik, O.A., Powell, D.A., Fivash, M.J., Jr., and Hu, W.-S.  (2008)  Long-range recombination gradient between HIV-1 subtypes B and C variants caused by sequence differences in the dimerization initiation signal region.  J. Mol. Biol. 377: 1324-1333.     [Abstract]     [Full-text PDF article]

Chukkapalli, V., Hogue, I.B., Boyko, V., Hu, W.-S., and Ono, A.  (2008)  Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient Gag-membrane binding.  J. Virol. 82: 2405-2417.
[Abstract]     [Full-text PDF article]

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, in press (April 9 Epub ahead of print).
[Abstract]     [Full-text PDF article]

Das, K., Bauman, J.D., Clark, A.D., Jr., Frenkel, Y.V., Lewi, P.J., Shatkin, A.J., Hughes, S.H., and Arnold, E.  (2008)  High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: Strategic flexibility explains potency against resistance mutations.  Proc. Natl. Acad. Sci. USA 105: 1466-1471.     [Abstract]     [Full-text PDF article]

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.

Derse, D.  (2008)  Role of mouse APOBEC3 in the restriction of mouse mammary tumor virus replication in vivo.  Future HIV Ther., in press.

Efroni, S., Duttagupta, R., Cheng, J., Deghnani, H., Hoeppner, D.J., Dash, C., Bazett-Jones, D.P., Le Grice, S.F.J., McKay, R.D.G., Buetow, K.H., Gingeras, T.R., Misteli, T., and Meshorer, E.  (2008)  Global transcription in pluripotent embryonic stem cells.  Cell Stem Cell 2: 437-447.
[Abstract]     [Full-text PDF article]

Gao, L., Hanson, M.N., Balakrishnan, M., Boyer, P.L., Roques, B.P., Hughes, S.H., Kim, B., and Bambara, R.A.  (2008)  Apparent defects in processive DNA synthesis, strand transfer, and primer elongation of Met-184 mutants of HIV-1 reverse transcriptase derive solely from a dNTP utilization defect.  J. Biol. Chem. 283: 9196-9205.
[Abstract]     [Full-text PDF article]

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.

Gousset, K., Ablan, S.D., Coren, L.V., Ono, A., Soheilian, F., Nagashima, K., Ott, D.E., and Freed, E.O.  (2008)  Real-time visualization of HIV-1 Gag trafficking in infected macrophages.  PLoS Pathog. 4: e1000015.     [Abstract]     [Full-text PDF article]

Joshi, A., Garg, H., Nagashima, K., Bonifacino, J.S., and Freed, E.O.  (2008)  GGA and Arf proteins modulate retrovirus assembly and release.  Mol. Cell 30: 227-238.
[Abstract]     [Full-text PDF article]

Kearney, M., Palmer, S., Maldarelli, F., Shao, W., Polis, M.A., Mican, J., Rock-Kress, D., Margolick, J.B., Coffin, J.M., and Mellors, J.W.  (2008)  Frequent polymorphism at drug resistance sites in HIV-1 protease and reverse transcriptase.  AIDS 22: 497-501.
[Abstract]     [Full-text PDF article]

Kutty, G., Maldarelli, F., Achaz, G., and Kovacs, J.A.  (2008)  Variation in the major surface glycoprotein genes in Pneumocystis jirovecii.  J. Infect. Dis., in press.

Kvaratskhelia, M., and Le Grice, S.F.J.  (2008)  Structural analysis of protein-RNA interactions with mass spectrometry.  Methods Mol. Biol., in press.

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 as an approach towards new anti-HIV-1 therapeutics.  ChemBioChem, in press.

Luttge, B.G., Shehu-Xhilaga, M., Demirov, D.G., Adamson, C.S., Soheilian, F., Nagashima, K., Stephen, A.G., Fisher, R.J., and Freed, E.O..  (2008)  Molecular characterization of feline immunodeficiency virus budding.  J. Virol. 82: 2106-2109.
[Abstract]     [Full-text PDF article]

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.

McWilliams, M.J., Julias, J.G., and Hughes, S.H.  (2008)  Mutations in the human immunodeficiency virus type 1 polypurine tract (PPT) reduce the rate of PPT cleavage and plus-strand DNA synthesis.  J. Virol. 82: 5104-5108.     [Abstract]     [Full-text PDF article]

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.

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

Motomura, K., Chen, J., and Hu, W.-H.  (2008)  Genetic recombination between human immunodeficiency virus type 1 (HIV-1) and HIV-2, two distinct human lentiviruses.  J. Virol. 82: 1923-1933.     [Abstract]     [Full-text PDF article]

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 HIV-1 and MLV infection.  J. Virol., in press (April 9 Epub ahead of print).
[Abstract]     [Full-text PDF article]

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, in press (Mar 17 Epub ahead of print).
[Abstract]     [Full-text PDF article]

Oh, J., McWilliams, M.J., Julias, J.G., and Hughes, S.H.  (2008)  Mutations in the U5 region adjacent to the primer binding site affect tRNA cleavage by human immunodeficiency virus type 1 reverse transcriptase in vivo.  J. Virol. 82: 719-727.     [Abstract]     [Full-text PDF article]

Palmer, S., Maldarelli, F., Wiegand, A., Bernstein, B., Hanna, G.J., Brun, S.C., Kempf, D.J., Mellors, J.W., Coffin, J.M., and King, M.S.  (2008)  Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy.  Proc. Natl. Acad. Sci. USA 105: 3879-3884.     [Abstract]     [Full-text PDF article]

Ricci, E.P., Herbreteau, C.H., Decimo, C., 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 3 distinct IRESes which are regulated by the HIV protease and the Gag polyprotein.  RNA, in press.

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., in press (April 30 Epub ahead of print).
[Abstract]     [Full-text PDF article]

Santos, A.F., Lengruber, R.B., Soares, E.A., Jere, A., Sprinz, E., Martinez, A.M., Silveira, J., Sion, F.S., Pathak, V.K., and Soares, M.A.  (2008)  Conservation patterns of HIV-1 RT connection and RNase H domains: Identification of new mutations in NRTI-treated patients.  PLoS ONE 3: e1781.     [Abstract]     [Full-text PDF article]

Sheng, L., Cai, F., Zhu, Y., Pal, A., Athanasiou, M., Orrison, B., Blair, D.G., Hughes, S.H., Coffin, J.M., Lewis, A.M., and Peden, K.  (2008)  Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H-ras and c-myc.  Biologicals 36: 184-197.
[Abstract]     [Full-text PDF article]

Turner, K.B., Brinson, R.G., Yi-Brunozzi, H.Y., Rausch, J.W., Miller, J.T., Le Grice, S.F.J., Marino, J.P., and Fabris, D.  (2008)  Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.  Nucleic Acids Res. 36: 2799-2810.
[Abstract]     [Full-text PDF article]

Viard, M., Ablan, S.D., Zhou, M., Veenstra, T.D., Freed, E.O., Raviv, Y., and Blumenthal, R.  (2008)  Photoinduced reactivity of the HIV-1 envelope glycoprotein with a membrane-embedded probe reveals insertion of portions of the HIV-1 gp41 cytoplasmic tail into the viral membrane.  Biochemistry 47: 1977-1983.     [Abstract]     [Full-text PDF article]

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., 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.

Wilkinson, K.A., Gorelick, R.J., Vasa, S.M., Guex, N., Rein, A., Mathews, D.H., Giddings, M.C., and Weeks, K.M.  (2008)  High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.  PLoS Biol. 6: e96.
[Abstract]     [Full-text PDF article]

Yi-Brunozzi, H.Y., Brinson, R.G., Brabazon, D.M., Lener, D., Le Grice, S.F.J., and Marino, J.P.  (2008)  High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers.  Chem. Biol. 15: 254-262.
[Abstract]     [Full-text PDF article]

Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A.A., Tong, X., Hong, A., Heck, S., Curreli, F., Goger, M., Cowburn, D., Freed, E.O., and Debnath, A.K.  (2008)  A cell-penetrating helical peptide as a potential HIV-1 inhibitor.  J. Mol. Biol. 378: 565-580.
[Abstract]     [Full-text PDF article]