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Publications 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 SIVmac 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 | ... |