Real-Time Traffic Report: How HIV-1 Travels from Cell to Cell

Although person-to-person transmission of HIV has been well undersood for years, researchers are still trying to figure out how the virus moves from one cell to another.  To shed light on this matter, Eric O. Freed, Ph.D., and Karine Gousset, Ph.D., both of the HIV Drug Resistance Program in the Center for Cancer Research (CCR), conducted a study with collaborators from the University of Michigan and NCI's AIDS Vaccine and Advanced Technology programs at SAIC-Frederick, Inc.  The results of their work were selected as the CCR Science Advance of the year in HIV/AIDS research.

To visualize trafficking of Gag proteins in real time, Freed and colleagues inserted a small molecular tag—tetracysteine (TC)—into the HIV-1 Gag protein.  A cellular stain that becomes fluorescent upon binding to the TC tag was applied so that Gag's position could be tracked by fluorescence microscopy.  It was determined that Gag accumulates at the cell membrane and in compartments in the interior of infected macrophages.  Remarkably, when uninfected macrophages or T cells were added, the researchers were able to observe migration of fluorescent Gag to the points of cell-cell contact, so-called "virological synapses."  Electron microscopy analysis demonstrated budding of viral particles at the synapse.

This study demonstrates that HIV-1 particles are retained in internal reservoirs from which they can be rapidly released at opportune times, such as when contact is established with uninfected cells.  The TC system provides an efficient way of observing Gag trafficking in living cells without disrupting the normal virus assembly and release and is likely to become an important tool in identifying molecular clues for HIV-1 trafficking within and between cells.  The results of this study may set the groundwork for the development of new HIV treatments based on interruption of intracellular viral trafficking.  [Excerpted from the CCR In the Journals article "Revealing the Gag Itinerary: How HIV Is Transmitted One Cell at a Time."  To read more, click here (PDF - 104 K).]

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 (PDF - 667 K).  PLoS Pathog. 4: e1000015.

Retroviral Gymnastics: Slip Sliding Away

HIV reverse transcriptase (RT) catalyzes a series of intricate reactions through which it converts single-stranded viral RNA of the invading virus into integration-competent double-stranded DNA.  This process requires a variety of enzymatic activities encoded within its p66 subunit, including DNA synthesis, RNase H-mediated cleavage of the RNA/DNA replication intermediate, strand transfer, and strand displacement synthesis.  Using single-molecule fluorescence resonance energy transfer (FRET), we have probed interactions between HIV-1 RT and nucleic acid substrates in real time.  RT was observed to slide on nucleic acid duplexes, rapidly shuttling between opposite termini of the duplex.  Upon reaching the DNA 3' terminus, the enzyme can spontaneously “flip” into a polymerization orientation.  Sliding kinetics were also regulated by cognate nucleotides and anti-HIV drugs, which stabilized and destabilized the polymerization mode, respectively.  These long-range translocation activities facilitate multiple stages of the reverse transcription pathway and possibly provide insights into a unique mechanism of action of nonnucleoside RT inhibitors.  To read more, click here (PDF - 470 K).

Liu, S., Abbondanzieri, E., Rausch, J.W., Le Grice, S.F.J., and Zhuang, X.  (2008)  Slide into action: Dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates (PDF - 470 K).  Science 322: 1092-1097.

Science online supporting material related to this article (PDF - 2432 K)

Science Perspectives feature related to this article:  Sarafianos, S.G., and Arnold, E.  (2008)  Biochemistry: RT Slides Home… (PDF - 280 K).  Science 322: 1059-1060.

HHMS News November 2008 feature about this article:  An HIV enzyme with a flair for the acrobatic.

See also the related article by Abbondanzieri et al.:  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 - 546 K).  Nature 453: 184-189.

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.   To read more, click here (PDF - 958 K).

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 (PDF - 958 K).  Proc. Natl. Acad. Sci. USA 105: 14365-14370.

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.  To read more, click here (PDF - 1450 K).

Wendeler, M., Lee, H.-F., Bermingham, A., Miller, J.T., Chertov, O., Bona, M.K., Baichoo, N.S., Ehteshami, M., Beutler, J., O'Keefe, B.R., Götte, M., Kvaratskhelia, M., and Le Grice, S.  (2008)  Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity (PDF - 1450 K).  ACS Chem. Biol. 3: 635-644.

ACS Chemical Biology online supporting material related to this article (PDF - 336 K)

ACS Chemical Biology feature about Michaela Wendeler:  Introducing our Authors.  Chem. Biol. 3: 593.

ACS Chemical Biology podcast featuring Stuart Le Grice:  October 2008 ACS Chemical Biology Podcast.

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.  [Excerpted from the CCR In the Journals article "HIV and Drug Resistance: Hitting a Moving Target."  To read more, click here (PDF - 284 K).]

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 - 284 K).  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 - 726 K).  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 - 1376 K).  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 - 898 K).

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 - 898 K).  Proc. Natl. Acad. Sci. USA 104: 317-322.

Recent HIV DRP Publications (November 2008–February 2009)

Crist, R.M., Datta, S.A.K., Stephen, A.G., Soheilian, F., Mirro, J., Fisher, R.J., Nagashima, K., and Rein, A.  (2009)  Assembly properties of HIV-1 Gag-leucine zipper chimeras: Implications for retroviral assembly.  J. Virol., in press (Dec 10 Epub ahead of print).
[Abstract]     [Full-text PDF article]

Dash, C., Scarth, B.J., Badorrek, C., Götte, M., and Le Grice, S.F.J.  (2008)  Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids.  Nucleic Acids Res. 36: 6363-6371.
[Abstract]     [Full-text PDF article - 8700 K]

Datta, S.A.K., and Rein, A.  (2009)  Preparation of recombinant HIV-1 Gag protein and assembly of virus-like particles in vitro.  In Prasad, V.R., and Kalpana, G.V. (eds.), HIV Protocols, 2nd Ed., Methods in Molecular Biology, Vol. 485, Humana Press, pp. 197-208.     [Abstract]

Gorelick, R.J., Thomas, J.A., Coren, L.V., Bosche, W.J., Gagliardi, T.D., Shulenin, S., and Oroszlan, S.  (2009)  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.W., Este, J.A., and Cihlar, T.  (2008)  Novel targets for HIV therapy.  Antiviral Res. 80: 251-265.     [Abstract]     [Full-text PDF article - 1737 K]

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 9: 1972-1983.
[Abstract]     [Full-text PDF article - 796 K]

Kearney, M., Maldarelli, F., Shao, W., Margolick, J.B., Daar, E.S., Mellors, J.W., Rao, V., Coffin, J.M., and Palmer, S.  (2009)  HIV-1 population genetics and adaptation in newly infected individuals.  J. Virol. , in press (Dec 30 Epub ahead of print).
[Abstract]     [Full-text PDF article - 293 K]

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.

LaRue, R.S., Andrésdóttir, V., Blanchard, Y., Conticello, S.G., Derse, D., Emerman, M., Greene, W.C., Jónsson, S.R., Landau, N.R., Löchelt, M., Malik, H.S., Malim, M.H., Münk, C., O'Brien, S.J., Pathak, V.K., Strebel, K., Wain-Hobson, S., Yu, X.F., Yuhki, N., and Harris, R.S.  (2009)  Guidelines for naming nonprimate APOBEC3 genes and proteins.  J. Virol. 83: 494-497.     [PubMed citation]     [Full-text PDF article - 148 K]

Liu, S., Abbondanzieri, E., Rausch, J.W., Le Grice, S.F.J., and Zhuang, X.  (2008)  Slide into action: Dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates.  Science 322: 1092-1097.     [Abstract]     [Full-text PDF article - 470 K]
[Science online supporting material - 2432 K PDF]
[HHMI News: An HIV Enzyme with a Flair for the Acrobatic]
[Science Perspectives feature: Biochemistry: RT Slides Home… (PDF - 470 K)]

Mbisa, J., K.A. Delviks-Frankenberry, J.A. Thomas, R.J. Gorelick, and Pathak, V.K.  (2009)  Real-time PCR analysis of HIV-1 replication post-entry events.  In Prasad, V.R., and Kalpana, G.V. (eds.), HIV Protocols, 2nd Ed., Methods in Molecular Biology, Vol. 485, Humana Press, pp. 55-72.     [Abstract]

Michel, N., Goffinet, C., Ganter, K., Allespach, I., KewalRamani, V.N.,Saifuddin, M., Littman, D.R., Greene, W.C., Goldsmith, M.A., and Keppler, O.T.  (2009)  Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo.  Retrovirology 6:2.
[Abstract]     [Full-text PDF article - 856 K]

Migueles, S.A., Osborne, C.M., Royce, C., Compton, A.A., Joshi, R.P., Weeks, K.A., Rood, J.E., Berkley, A.M., Sacha ,J.B., Cogliano-Shutta, N.A., Lloyd, M., Roby, G., Kwan, R., McLaughlin, M., Stallings, S., Rehm, C., O'Shea, M.A., Mican, J., Packard, B.Z., Komoriya, A., Palmer, S., Wiegand, A.P., Maldarelli, F., Coffin, J.M., Mellors, J.W., Hallahan, C.W., Follman, D.A., and Connors, M.  (2008)  Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control.  Immunity 29: 1009-1021.
[Abstract]     [Full-text PDF article - 1649 K]

Moore, M.D., and Hu, W.-S.  (2009)  HIV-1 RNA dimerization: It takes two to tango.  AIDS Rev., in press.

Moore, M.D., Chin, M.P.S., and Hu, W.-S.  (2009)  HIV-1 recombination: An experimental assay and a phylogenetic approach.  In Prasad, V.R., and Kalpana, G.V. (eds.), HIV Protocols, 2nd Ed., Methods in Molecular Biology, Vol. 485, Humana Press, pp. 87-105.     [Abstract]

Morse, C., and Maldarelli, F.  (2009)  HIV infection.  In Strober, W., and Gottesman, S. (eds.), Immunology, Clinical Case Studies and Disease Pathophysiology, John Wiley & Sons, in press.

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. 82: 11480-11483.     [Abstract]     [Full-text PDF article - 276 K]

Perkovic, M., Schmidt, S., Marino, D., Russell, R.A., Stauch, B., Hofmann, H., Kopietz, F., Kloke, B.P., Zielonka, J., Ströver, H., Hermle, J., Lindemann, D., Pathak, V.K., Schneider, G., Löchelt, M., Cichutek, K., and Münk, C.  (2009)  Species-specific inhibition of APOBEC3C by the prototype foamy virus protein Bet.  J. Biol. Chem., in press (Dec 12 Epub ahead of print).
[Abstract]     [Full-text PDF article - 881 K]

Porter, B.O., Anthony, K.B., Shen, J., Hahn, B., Keh, C.E., Maldarelli, F., Blackwelder, W.C., Lane, H.C., Kovacs, J.A., Davey, R.T., and Sereti, I.  (2009)  Inferiority of IL-2 alone versus IL-2 with HAART in maintaining CD4 T cell counts during HAART interruption: A randomized controlled trial.  AIDS 23: 203-212.     [Abstract]     [Full-text PDF article - 252 K]

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 syndrome after epidural triamcinolone injections in an HIV type 1-infected patient receiving therapy with ritonavir-lopinavir.  Clin. Infect. Dis. 47: e97-99.     [Abstract]     [Full-text PDF article - 109 K]

Rausch, J.W., Chelico, L., Goodman, M.F., and Le Grice, S.  (2009)  Dissecting APOBEC3G substrate specificity by nucleoside analog interference.  J. Biol. Chem., in press (Jan 9 Epub ahead of print).     [Abstract]     [Full-text PDF article - 1058 K]

Robertson, S.M., Maldarelli, F., Natarajan, V., Formentini, E., Alfaro, R.M., and Penzak, S.R.  (2009)  Efavirenz induces CYP2B6-mediated hydroxylation of bupropion in healthy subjects.  J. Acquir. Immune Defic. Syndr., in press (Nov 4 Epub ahead of print).
[Abstract]     [Full-text PDF article - 119 K]

Russell, R.A., Smith, J., Barr, R., Bhattacharyya, D., and Pathak, V.K.  (2009)  Distinct domains within APOBEC3G and APOBEC3F interact with separate regions of human immunodeficiency virus type 1 Vif.  J. Virol. 83: 1992-2003.
[Abstract]     [Full-text PDF article - 1351 K]

Sarafianos, S.G., Marchand, B., Das, K., Himmel, D.M., Parniak, M.A., Hughes, S.H., and Arnold, E.  (2009)  Structure and function of HIV-1 reverse transcriptase: Molecular mechanisms of polymerization and inhibition.  J. Mol. Biol. 385: 693-713.
[Abstract]     [Full-text PDF article - 1561 K]

Waheed, A.A., and Freed, E.O.  (2008)  Peptide inhibitors of HIV-1 egress.  ACS Chem. Biol. 3: 745-747.     [Abstract]     [Full-text PDF article - 183 K]

Waheed, A.A., Ono, A., and Freed, E.O.  (2009)  Methods for the study of HIV-1 assembly.  In Prasad, V.R., and Kalpana, G.V. (eds.), HIV Protocols, 2nd Ed., Methods in Molecular Biology, Vol. 485, Humana Press, pp. 163-184.     [Abstract]

Wendeler, M., Beilhartz, G.L., Beutler, J.A., Götte, M., and Le Grice, S.F.J.  (2009)  HIV ribonuclease H: Continuing the search for small molecule antagonists.  HIV Ther. 3: 39-53.
[Full-text PDF article - 3282 K]

Wendeler, M., and Le Grice, S.F.J.  (2009)  HIV-1 reverse transcriptase.  In Gotte, M., Cameron, C.E., and Raney, K. (eds.), Viral Genome Replication, Springer Publications Company, New York, NY.

Wilkinson, T.A., Januszyk, K., Phillips, M.L., Tekeste, S.S., Zhang, M., Miller, J.T., Le Grice, S.F.J., Clubb, R.T., and Chow, S.A.  (2009)  Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase.  J. Biol. Chem., in press (Jan 16 Epub ahead of print).     [Abstract]     [Full-text PDF article - 1603 K]

Last modified: 30 January 2009