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3 Recent Advances in Pathogenic Human Viruses. H. Smithand Charles E. Samuel

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Inhibitors of Histone Deacetylases. CORRELATIONBETWEENISOFORMSPECIFICITYANDREACTIVATIONOFHIVTYPE1(HIV-1)FROMLATENTLYINFECTEDCELLS. KellyHuber,Genevie`veDoyon,JosephPlaks,ElizabethFyne,JohnW.Mellors,andNicolasSluis-Cremer

15 HostProteinKu70BindsandProtectsHIV-1 IntegrasefromProteasomalDegradationandIsRequiredforHIVReplication.YingfengZheng, ZhujunAo, BinchenWang, KalleshDanappa Jayappa,andXiaojianYao

29S

Impaired Infectivity of Ritonavir-resistant HIV Is Rescued byHeat Shock Protein 90AB1. Pheroze Joshi and Cheryl A. Stoddart

41 A Chimeric HIV-1 Envelope Glycoprotein Trimer with anEmbedded Granulocyte-Macrophage Colony-stimulatingFactor (GM-CSF) Domain Induces Enhanced Antibody and TCell Responses. Thijs vanMontfort, Mark Melchers, Gozde Isik,SergeyMenis, Po-Ssu Huang, Katie Matthews, ElizabethMichael,Ben Berkhout, William R. Schief, John P. Moore, and Rogier W. Sanders

53 Identification of Interactions in the E1E2 Heterodimer ofHepatitis C Virus Important for Cell Entry. Guillemette Maurin,Judith Fresquet, Ophelia Granio, Czeslaw Wychowski,Francois-Loc Cosset, and Dimitri Lavillette

65S

Identification of Cis-Acting Elements in the 3-UntranslatedRegion of the Dengue Virus Type 2 RNA That ModulateTranslation and Replication. Mark Manzano, Erin D. Reichert,Stephanie Polo, Barry Falgout, Wojciech Kasprzak, Bruce A. Shapiro,and Radhakrishnan Padmanabhan

79S

Structural Characterization of the Crimean-CongoHemorrhagic Fever Virus Gn Tail Provides Insight into VirusAssembly. D. Fernando Estrada and Roberto N. De Guzman

The Journal of Biological ChemistryTABLE OF CONTENTS

2011 COMPENDIA COLLECTION: Recent Advances in PathogenicHuman Viruses

S Online version of this article contains supplemental material.

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Recent Advances in Pathogenic Human Viruses*H. SmithFrom the American Society for Biochemistry andMolecular Biology, Rockville, Maryland 20852

Edited and curated by Charles E. Samuel1

From the Department of Molecular, Cellular, and Developmental Biology and the Bimolecular Sciences and Engineering Program, University of California,Santa Barbara, California 93106

Written histories of virology research frequently start withthe year 1796, when English physician Edward Jenner used vac-cinia-laden pus, collected from the cowpox lesions of a milk-maid, to inoculate a young patient against smallpox. Mostaccounts are quick to acknowledge that prophylactic inocula-tion did not begin with Jenner but had in fact been practicedcenturies beforehand, in China and other regions, and that theuse of cowpox lesions had been used even in Jenners day inEurope. However, the details of Jenners experiments were bothspectacular and well told. For example, after treating an 8-year-old patient with the cowpox inoculum, Jenner challenged thepatient by intentionally inoculating himwith smallpox. By care-fully recording his work, Jenner resonated with the Enlighten-ment ideals of the scientific method. Ever since, Jennersrecords have provided a convenient milestone in the develop-ment of modern antiviral therapies.Virologys historical importance to basic molecular biology

and the disciplines crucial role in health care around the globehave been very much on the minds of the organizers of RecentAdvances in Pathogenic Human Viruses, a meeting sponsoredin part by the American Society for Biochemistry and Molecu-lar Biology, thismonth inGuangzhou, China. Attendance at themeeting by virologists from around the world not only reflectsthe unprecedented therapeutic opportunities to be developedfrom studies of viruses but also attests to the worldwide risksposed by viral pathogens in our global age. A single case of adeadly viral disease appearing in the English countryside today,or in a small Chinese village, or anywhere else in the world, hasramifications for global health that were unimaginable in Jen-ners day. Virology today must be considered along dimensionsthat cross previously recognized borders, including thosedrawn between countries, scientific disciplines, political andeconomic ideologies, and even animal species.Organization of the meeting on Recent Advances in Patho-

genic Human Viruses in Guangzhou also reflects a deliberatesentiment that the benefits and risks of virology-relatedresearch are met best through collaborations that are also builtto global proportions. The American Society for Biochemistryand Molecular Biology, founded through a shared sense ofexcitement for scientific discovery more than a hundred yearsago, is proud to support the collegial spirit that has broughtinternational scientists together in Guangzhou. We are partic-

ularly delighted to offer meeting delegates the following com-pendium of recent research findings in virology, presented byauthors from around the world within the pages of The Journalof Biological Chemistry (JBC). These current research paperstouch on a variety of topics related to viral disease in humansand promise to provide avenues for better understanding andcombating viral pathogens. Beyond their clinical implications,these papers also represent the light that virology inevitablycasts on processes essential to the wide spectrum of intereststhat define modern biochemistry and molecular biology.

Its All Very Retro

Groundbreaking investigation into the molecular biology ofthe retroviruses, culminating in the early 1970s, establishedthat RNA could be reverse-transcribed into DNA. This find-ing, made in laboratories focusing on the nucleic acid metabo-lism of avian viruses, broke the central dogma of molecularbiology that had been pronounced by Francis Crick in the1950s. Cricks declaration that the expression of genetic infor-mation within all cellular organisms flows in a single-directionsynthetic pathway, from DNA to RNA to protein, reflected thebasic conceptual framework by which biologists had begun tounlock mechanisms of gene regulation. This framework hadbeen built in large part through the work of basic virologists.Similarly, it was a framework that was recast through the workof virologists such as David Baltimore and Howard Temin.2The discovery of reverse transcriptase, which revolutionized

molecular biology, originated from studies of basic retroviralbiology. In the field of retrovirology, the discovery broughtcredibility particularly to Howard Temins thesis for the exist-ence of a proviral intermediate for Rous sarcoma: that is, for theexistence of a DNA form of the viral genome that becamespliced into the host genome as a normal phase of viral infec-tion. Temins proposal thus echoedDulbeccos identification ofthe proviral intermediate in the replication cycle of the polyoma(DNA) virus.2 The elucidation of reverse transcriptase, alongwith other elements of the HIV-1 molecular machinery thatdrive the retroviral life cycle, has paved the way for therapeuticapproaches that have alleviated greatly the worldwide burdenof illness caused by HIV-1. In addition, whereas drugs thatinhibit the HIV-1 reverse transcriptase, integrase, and proteasehave become important in the clinic, host factors that affect

* Translated into Chinese by Xing Guo, University of California, San Diego.To cite articles in this collection, use the citation information that appears in

the upper right-hand corner of the first page of the article.1 To whom correspondence should be addressed. Tel.: 805-893-3097; Fax:

805-893-5780; E-mail: [email protected].

2 The Nobel Prize in Physiology or Medicine 1975 was awarded jointly toDavid Baltimore, Renato Dulbecco, and Howard Martin Temin for theirdiscoveries concerning the interaction between tumour viruses and thegenetic material of the cell. See http://nobelprize.org/nobel_prizes/med-icine/laureates/1975/. Accessed June 14, 2011.

2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.PROLOGUE This paper is available online at www.jbc.org

3

retroviral infection have also entered center stage in the effortto understand and combat HIV-1 infection. Four of the papersin this compendium attest to the diversity of HIV-1 targets.Going Pro-ViralThe proviral stage of the HIV-1 life cycle

poses a great challenge in treating infection because the viralgenome, once integrated into host DNA, can remain quiescentthroughout therapeutic regimens that otherwise clear theblood of virus. Latent infection is thus a constant threat despitethe success of combinatorial drug strategies. HIV-1 latency isthe issue at hand in the JBC paper from Huber and colleagues(1), who explore the therapeutic potential of pharmacologicallymanipulating epigenetic regulation ofHIV-1 proviral elements.Specifically, the recruitment of histone deacetylase (HDAC) tothe long terminal repeats (LTRs) of theHIV-1 genome has beenlinked experimentally to the induction of HIV-1 latency, lead-ing Huber et al. (1) to question whether specific isoforms ofhistone deacetylase might function differentially to maintainproviral DNA in the form of quiescent chromatin. Indeed, in Tcells isolated from aviremic HIV-1-infected individuals under-going combination antiretroviral therapy, HDAC inhibitorshave been able to induce chromatin relaxation as well as theactivation of viral genes. Huber et al. (1) use a number of knowninhibitors of HDAC activity to characterize nine distinctHDAC isoforms in Jurkat cells with regard to HDAC inhibitionkinetics, association of HDAC isoforms with provirus-contain-ing chromatin, and the effectiveness of distinct HDAC inhibi-tors in activating latent virus in vitro. In this way, the authorsreport that the inhibition of HDAC3 is essential for activatingthe provirus but that HDAC1, although amenable to inhibition,is not a suitable target in terms of therapeutic HDAC inhibitionas reflected by activity in Jurkat T cells. The investigators thusestablish the importance of isoform-specific targeting in anyattempt to add HDAC inhibitors to combination antiretroviraltherapy. Moreover, as there are distinct cellular reservoirs thatmay support latent virus, the authors offer their pharmacolog-ical method of profiling HDAC activities as a relatively directmeans of surveying HDAC isoform-specific activities in othercell types.Binding and Hijacking: HIV-1 IntegraseAlthough inhibi-

tors of theHIV-1 integrase have reached clinical trials, with oneinhibitor having been approved for prescription, the roles ofcellular proteins in regulating the viral integrase are not fullyunderstood. Researchers have thus not yet tapped the thera-peutic potential of inhibiting viral replication by blocking any ofthe dozens of interactions that occur between the viral enzymeintegrase and cellular proteins. One such cellular protein isKu70, which participates in nonhomologous end-joining DNArepair (a process implicated in retroviral infection) and hasbeen identified, in the form of a p70/p80 dimer, as an autoanti-gen in systemic lupus erythematosus. The current JBC paperfrom Zheng et al. (2) adds a new dimension to Ku70 function-ality in HIV-1 replication, capitalizing on the recent identifica-tion of Ku70 as a deubiquitinating enzyme. The authors estab-lish that the C terminus of the integrase binds to N-terminalsequenceswithinKu70 and that Ku70 promotes the deubiquiti-nation of the integrase.Moreover, integrasemediates the incor-poration of Ku70 into progeny virus. The Ku70 that is thushijacked upon virion assembly ultimately promotes viral repli-

cation in distinct ways, protecting the integrase from degrada-tion and mediating the genesis of viral nucleic acid intermedi-ates upon cell entry.Aiding and Abetting: Cell Proteins That Enable HIV Drug

ResistanceThe integrase is not the only viral enzyme to col-lude with specific cellular proteins in the service of HIV repli-cation. As the JBC paper from Joshi and Stoddart (3) shows,mutant forms of the HIV protease that fail to promote full mat-uration of the capsid protein (CA) can nevertheless attain enzy-mic functionality and restore viral replication by associatingwith HSP90AB1, a cellular heat shock protein. This cell pro-tein-virus protein collusion, moreover, depends on the activa-tion status of the infected T cell: rescue of protease function byHSP90AB1 occurs only in T cells that have been activated.These results have fascinating ramifications for drug develop-ment, as pharmacological inhibition ofHSP90AB1 prevents therescue of impaired virus, and most significantly, the mutantproteases at the center of Joshi and Stoddarts experiments (3)are typical ofHIV that has become resistant to theHIV antipro-tease drug ritonavir (Norvir). Admittedly, the actual mecha-nism of viral rescue (e.g. chaperone activity) by HSP90AB1 isnot yet clear, but CA conformation and interaction with otherhost factors have been implicated in the postentry stage of HIVinfectivity. In any event, the authors illustrate the consummatemolecular behavior of HIV in usurping cellular functions.Engineering of Anti-HIV Vaccines That Carry Cytokine

SignalsThe development of antiretroviral drugs that targetintracellular viral enzyme activity has been particularly crucialto clinical efforts as effective HIV vaccines have remained elu-sive. Indeed, one of the hallmark challenges in combating AIDShas come from attempts to marshal viral immunogenicity byexploiting the envelope glycoprotein complex (Env). In theirprovocative report, vanMontfort et al. (4) directly address HIVimmunogenicity, hypothesizing that a vaccine component thatcould carry a direct signal of immune activationthat is, a sortof chemokinemight better alert host defense systems againstthe invading virus. The authors hypothesize that if the overallantigenic message can be amplified, the host might mount abetter defense. To investigate, the authors have constructed achimericmolecule that consists of the Envprotein (in triplicate)along with the immunostimulatory domain of the granulocyte-macrophage colony-stimulating factor (GM-CSF). Upon injec-tion into mice, the chimeric construct enhances both humoraland cellular responses, compared with injection of Env alone.The improved immunogenicity appears to reflect cytokine sig-naling, as the chimera retains GM-CSF activity in vitro.Whether other cytokinesmay prove effective as immunostimu-latory components when chimerically partnered with Envremains to be seen.

Flaviviridae: Hepatitis C Virus and Dengue Virus

The Flaviviridae family includes scores of viruses, several ofwhich are important pathogens in humans. The genomes ofFlaviviridae viruses consist of single-stranded RNA of positivepolarity but, unlike the case of the retroviruses, engender noDNA intermediate. Two of the JBC papers included in thiscompendium concern the biology of Flaviviridae. The firstdealswith envelope glycoproteins of the hepatitis C virus (of the

PROLOGUE: Recent Advances in Pathogenic Human Viruses

4

Hepacivirus genus), which remains a pathogen of global impor-tance. The second focuses on the RNA genome of the denguevirus (a Flavivirus), which is endemic to tropical and subtropi-cal regions, causing tens of millions of infections per year.HepatitisCVirus: Cell Entry andEnvelopeProteins E1andE2

Since discovery of the hepatitis C virus, in 1989, investigationsinto the two HCV envelope glycoproteins (E1 and E2) havebeen hampered by technological difficulties associated withcarrying the virus in culture and by the high degree of sequencevariabilitymanifest in both E1 and E2. Each of the two envelopeproteins contains a large glycosylated N-terminal ectodomainand a C-terminal transmembrane anchor, and despite the chal-lenges of assessing discrete infective stages of HCV in cell cul-ture, specific roles for each protein in cell receptor binding andcell fusion have been suggested (5). One of the characteristics ofthe E1 and E2 proteins, which was quite remarkable whenreported in JBC well over a decade ago, is that discrete muta-tions that are confined to the transmembrane domain of eitherprotein can affect heterodimerization and virus replication (6).In their current JBC paper, Maurin et al. (5) exploit the nat-

urally occurring variability of E1 and E2 sequences to elucidatethe structural basis of intra- and intersubunit interactions thatenable E1E2 heterodimers to subserve cell entry in the infectiv-ity cycle. In particular, the authors have identified combina-tions of E1 and E2 variants that can be coexpressed to producestable heterodimers that are nevertheless nonfunctional. Byapplying site-directed mutagenesis to such inactive het-erodimers, moreover, the authors have determined sequencesand residues, from both envelope proteins, that function con-certedly to culminate in viral infectivity. In this way, the authorsestablish structure-function relationships that go beyondmerely descriptive terms such as transmembrane sequence orectodomain. Indeed, discrete interactions within the E1 sub-unit of the heterodimer, as well as interprotomeric interactionsthat emanate from discrete residues within the E1 transmem-brane sequence, prove essential to entry of the virus into thecell. Ultimately, the authors indicate, functionalmechanisms ofviral replication and infection are effected through subtle facetsof the E1E2 interaction related to cell entry.Dengue Virus: Structure and Function of the Viral Genome

Manzano et al. (7) focus their attention on the structure of thessRNA genome of the dengue virus. Typical of Flaviviridaegenomes, the dengue virus RNA is of positive polarity (()-RNA), meaning that it can serve directly as message for thetranslation of viral proteins. The genome also serves as a tem-plate for the RNA-dependent RNA polymerase activity of theviral nonstructural protein 5 (NS5), whereby a negative RNAstrand is synthesized to serve as the template for production ofviral progeny genomes. In this way, replication of the viralgenome is intimately tied to the translation of viral proteins,with both processes regulated by means of structural elementsthat arise through intramolecular associations within thessRNA genome. One important element, or group of elements,is the 3-untranslated region of the genome.Manzano et al. (7) have employed a high-performance com-

puter algorithm that duly considers the stabilities and probabil-ities of RNA folding intermediates as related to the core regionof the 3-untranslated region. In addition, the authors use in

vitro assays and immunofluorescence to compare the muta-tional effects of RNA elements upon viral replication and trans-lation. The authors markedly refine previous speculations con-cerning many secondary structures of the viral genome, andthey offer stringent evidence for regulatory roles of at least fiveoligonucleotide sequences that cooperate differentially,depending upon whether the ()-ssRNA genome is directingtranslation or RNA-dependent RNA synthesis. The latter proc-ess, moreover, appears to utilize certain genomic RNA ele-ments during ()-RNA synthesis but not ()-RNA synthesis,whereas the former process (i.e. translation) may utilize ele-ments in a context-dependent manner, depending on whethertranslation occurs by canonical (i.e. 5-cap-dependent transla-tion) or noncanonical mechanisms. The implications for con-trolling dengue viral infections and basic molecular biology areunclear, but the virology of the system is compelling whenviewed in terms of mechanistic intricacies that are both elegantand complex.

The Gn Protein of the Crimean Congo Hemorrhagic FeverVirus

Twomembers of the Bunyaviridae provide focus for the JBCpaper contributed by Estrada and De Guzman (8), who provideatomic resolution, in solution, for the cytoplasmic portion ofenvelope glycoprotein Gn of the Crimean Congo hemorrhagicfever (CCHF) virus (of the genusNairovirus). By comparison totheir previous assessment of the similar protein in a member oftheHantavirus genus (also in JBC (9)), the authors offer insightsinto the role of the Gn protein in CCHF virion assembly. The100-residue cytoplasmic tail (in the respective proteins fromboth viruses) contains two zinc finger-like CCHC sequences,and the investigators establish by NMR that the four definingresidues of the twoCys-Cys-His-Cysmotifs participate in func-tional zinc coordination. The two CCHC motifs, moreover,interact to form a very stable compact structure, such that thetwo zinc fingers appear, according to solution dynamics, tobehave as a single entity. Results for the Gn protein, moreover,indicate that the zinc finger functionality is distinctive in fur-ther respects. First, the -fold that typifies the CCHC motifis supplemented, to the N-terminal side of the second suchsequence, by a third (3) helix, so that the structure ultimatelyconsists of four -folds and three helices. The distribution ofcharged residues in the Gn 43 structure, furthermore, is highlysuggestive of a function, typically associated with zinc fingers, ininteracting with RNA: multiple basic residues define one face ofthe globular structure, ideal for interactions with nucleic acid,whereas acidic residues tend to cluster on the opposing hemi-sphere of the structure. Indeed, the researchers confirm that theelectrophoretic mobility of CCHF viral RNA is significantlyretarded through interactionswith the cytoplasmic tail ofGn.Theauthors thereby offer amodel inwhich the native integral Gn pro-tein is envisaged to orchestrate the assembly of lipid envelope andnucleoprotein components into progeny virions.

Conclusion

The seven papers presented in this compendium touch ona variety of issues related to the basic biology of distinctviruses. Many of the observations can be related to potential


5

clinical opportunities, whereas others shed light not only onviral processes of metabolism and replication, but also onfundamental cell functions. The Journal of Biological Chem-istry is proud to be a part of the unfolding history of virology,and we continue to welcome important research findingsfrom virologists from around the world. We hope that par-ticipants of the Recent Advances in Pathogenic HumanViruses will enjoy reading the stories told in the papers ofthis compendium, assembled especially for the internationalmeeting in China.REFERENCES1. Huber, K., Doyon, G., Plaks, J., Fyne, E., Mellors, J. W., and Sluis-Cremer,

N. (2011) J. Biol. Chem. 286, 22211222182. Zheng, Y., Ao, Z., Wang, B., Jayappa, K. D., and Yao, X. (2011) J. Biol.

Chem. 286, 17722177353. Joshi, P., and Stoddart, C. A. (2011) J. Biol. Chem. 286, 24581245924. van Montfort, T., Melchers, M., Isik, G., Menis, S., Huang, P.-S., Mat-

thews, K., Michael, E., Berkhout, B., Schief, W. R., Moore, J. P., and Sand-ers, R. W. (2011) J. Biol. Chem. 286, 2225022261

5. Maurin, G., Fresquet, J., Granio, O., Wychowski, C., Cosset, F.-L., andLavillette, D. (2011) J. Biol. Chem. 286, 2386523876

6. Op De Beeck, A., Montserret, R., Duvet, S., Cocquerel, L., Cacan, R., Bar-berot, B., Le Maire, M., Penin, F., and Dubuisson, J. (2000) J. Biol. Chem.275, 3142831437

7. Manzano, M., Reichert, E. D., Polo, S., Falgout, B., Kasprzak, W., Shapiro,B. A., and Padmanabhan, R. (2011) J. Biol. Chem. 286, 2252122534

8. Estrada, D. F., and De Guzman, R. N. (2011) J. Biol. Chem. 286,2167821686

9. Estrada,D. F., Boudreaux,D.M., Zhong,D., St. Jeor, S. C., andDeGuzman,R. N. (2009) J. Biol. Chem. 284, 86548660


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Inhibitors of Histone DeacetylasesCORRELATION BETWEEN ISOFORM SPECIFICITY AND REACTIVATIONOFHIV TYPE 1(HIV-1) FROM LATENTLY INFECTED CELLSSReceived for publication,August 30, 2010, and in revised form, April 29, 2011 Published, JBC Papers in Press, April 29, 2011, DOI 10.1074/jbc.M110.180224

Kelly Huber1, Genevie`ve Doyon1, Joseph Plaks, Elizabeth Fyne, John W. Mellors, and Nicolas Sluis-Cremer2

From the Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania 15261

Deacetylation of histone proteins at the HIV type 1 (HIV-1)long terminal repeat (LTR) by histone deactylases (HDACs) canpromote transcriptional repression and virus latency. As such,HDAC inhibitors (HDACI) could be used to deplete reservoirsof persistent, quiescent HIV-1 proviral infection. However, thedevelopment of HDACI to purge latent HIV-1 requires knowl-edge of theHDAC isoforms contributing to viral latency and thedevelopment of inhibitors specific to these isoforms. In thisstudy, we identify the HDACs responsible for HIV-1 latency inJurkat J89GFP cells using a chemical approach that correlatesHDACI isoform specificity with their ability to reactivate latentHIV-1 expression. We demonstrate that potent inhibition orknockdown of HDAC1, an HDAC isoform reported to driveHIV-1 into latency, was not sufficient to de-repress the viralLTR. Instead, we found that inhibition ofHDAC3was necessaryto activate latent HIV-1. Consistent with this finding, we iden-tified HDAC3 at the HIV-1 LTR by chromatin immunoprecipi-tation. Interestingly, we show that valproic acid is a weak inhib-itor of HDAC3 (IC50 5.5 mM) relative to HDAC1 (IC50 170M). Because the total therapeutic concentration of valproicacid ranges from275 to 700M in adults, these datamay explainwhy this inhibitor has no effect on the decay of latent HIV res-ervoirs inpatients. Taken together, our study suggests an impor-tant role for HDAC3 in HIV-1 latency and, importantly,describes a chemical approach that can readily be used to iden-tify the HDAC isoforms that contribute to HIV-1 latency inother cell types.

Combination antiretroviral therapy (cART)3 can effectivelyreduce plasmaHIV-1 to undetectable levels. However, upon itsinterruption, there is usually a rapid rebound of viremia (1).This viremia is thought to arise from latently infected reservoirssuch as memory CD4() T cells or CD34() multipotenthematopoietic progenitor cells (25). Therefore, any long termtherapeutic strategy targeted toward eliminating HIV-1 infec-

tion must include compounds that purge the latent viral reser-voirs thereby rendering them susceptible to cART.HIV-1 can be maintained in a latent state by multiple differ-

ent mechanisms that inhibit virus gene expression after inte-gration into the cellular DNA (68). For example, epigeneticmodifications at or near the HIV-1 5-long terminal repeat(LTR) can induce chromatin condensation that diminishes theaccessibility of the HIV-1 promoter to transcription factors. Inthis regard, it has been well documented that different tran-scription factors can recruit histone deacetylase (HDAC)enzymes to the HIV-1 LTR where they promote chromatincondensation by deacetylating the -amino groups of lysine res-idues in histone tails (914). Eleven distinct zinc-dependentHDAC isoforms have been identified in humans. These can beclassified into four families, namely class I (HDAC13 and -8),IIa (HDAC4, -5, -7, and -9), IIb (HDAC6 and -10), and IV(HDAC11), which differ in structure, enzymatic function, sub-cellular localization, and expression patterns (15). To date,multiple studies have demonstrated that recruitment ofHDAC1 to the HIV-1 LTR by different DNA-binding com-plexes is sufficient to induce viral latency (914). However,HDAC2 and HDAC3 can also bind to the HIV-1 LTR and mayalso play an important role in viral latency (12, 16, 17).Treatment of latently infected HIV-1 cell lines and/or

CD4() T cells from aviremic HIV-1-infected individuals oncARTwithHDACI can lead to chromatin relaxation and induc-tion of viral transcription (reviewed in Ref. 6). Therefore, HDA-CIs are considered as potential therapeutic agents for purgingthe latent viral reservoir in HIV-1-infected individuals. How-ever, the active site structures of the HDAC family are largelyconserved, and many HDACIs exhibit activity against multipleHDAC isoforms. For example, suberoylanilide hydroxamic acid(SAHA, vorinostat), an activator of latent HIV-1 expression(1820), is a nonselective HDACI that inhibits both class I andclass II HDAC isoforms (21). Because HDACs exert crucialroles in numerous biological processes, including cell cycle, celldifferentiation, and survival (15), simultaneous inhibition ofmultiple HDAC isoforms will likely reduce their therapeuticwindow by promoting undesirable side effects and/or toxicity.Accordingly, the development of HDACI for anHIV-1 curativestrategy requires knowledge of the HDAC isoforms contribut-ing to viral latency and the development of inhibitors targetingthese isoforms. In this study, we use a chemical approach thatcorrelates the isoform specificities of HDACI with their abili-ties to reactivate latent HIV-1 expression to identify the HDACisoforms responsible for HIV-1 latency in Jurkat J89GFP cells.

S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1 and Tables 1 and 2.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: S817 Scaife Hall, 3550 Ter-

race St., Pittsburgh, PA 15261. Tel.: 412-648-8457; Fax: 412-648-8521;E-mail: [email protected].

3 The abbreviations used are: cART, combination antiretroviral therapy;HDAC, histone deacetylase; HDACI, HDAC inhibitor; SAHA, suberoylanilidehydroxamic acid; EGFP, enhanced green fluorescent protein; FW, forward;REV, reverse; HIV-1, HIV type 1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 25, pp. 2221122218, June 24, 2011 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

7

The results from this study suggest that potent inhibition ofHDAC3 may be important for reactivation of latent HIV-1.

EXPERIMENTAL PROCEDURES

MaterialsThe HDACI 4,5:8,9-dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol (depudecin),suberoyl bis-hydroxamic acid, cyclo[(2S)-2-amino-8-oxo-decanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperi-dinecarbonyl] (apicidin), cyclo-(D-Pro-L-Ala-D-Ala-L-2-amino-8-oxo-9,10-epoxydecanoic acid) (HC toxin), (2E)-5-[3-(phenylsulfonylamino)phenyl]-pent-2-en-4-ynohydro-xamic acid (oxamflatin), 6-(1,3-dioxo-1H,3H-benzo[de]iso-quinolin-2-yl)-hexanoic acid hydroxyamide (scriptaid), sodiumbutyrate, sodium 4-phenylbutyrate, SAHA, valproic acid, and[(R)-(E,E)]-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-di-methyl-7-oxo-2,4-heptadienamide] (trichostatin A) were ob-tained from Enzo Life Sciences (Plymouth Meeting, PA). 4-Di-methylamino-N-(6-hydroxyamino)-6-(oxohexyl]-benzamide(CAY10398) and N-phenyl-N-(2-aminophenyl) hexamethyl-enediamide (CAY10433) were obtained from Cayman Chemi-cal Co. (Ann Arbor, MI). Sodium 1-naphthoate was obtainedfrom TCI America (Portland, OR). Droxinostat was pur-chased from Sigma. Wortmannin was obtained from Sigma.The AKT inhibitor IV was obtained from EMD Biosciences(Gibbstown, NJ). The phospho-AKT antibody and AKT an-tibody were obtained from Cell Signaling Technology (Bos-ton). The -actin antibody was obtained from Abcam (Cam-bridge,MA). DNA oligonucleotide primers were synthesizedby Integrated DNA Technologies (San Diego). The recombi-nant purified HDAC isoforms, the Fluorogenic HDAC assaykit, and the HDAC assay substrates were purchased fromBPS Bioscience (San Diego). The J89GFP cells were a kindgift from Dr David Levy.HDAC Activity AssaysThe lysine deacetylase activity of

HDAC19 was assessed using the fluorogenic HDAC assay(BPS Bioscience) according to the manufacturers instructions.The HDAC3 used in this assay was complexed with humannuclear receptor co-repressor 2 (NCOR2; amino acids 395489), which is an activating co-factor of this HDAC isoform(31). All assays were carried out under steady-state conditions,and the assay read-out was optimized for linearity both as afunction of time and enzyme concentration. Inhibition assayswere carried out in 384-well plates. The assay volume was 25land contained 0.1 mg/ml BSA, 20 M substrate, and varyingconcentrations of the HDACI. All HDACI were dissolved inDMSO. The final concentration of DMSO did not exceed 5.0%(v/v). The formation of the fluorescent product was measuredusing a SpectraMax M2 plate reader (Molecular Devices). Theexcitation and emission wavelengths were 360 and 450 nm,respectively. The concentrations of HDACI required to inhibit50% of the deacetylase activity of an HDAC isoform (i.e. IC50)were calculated by regression analysis using SigmaPlot software(Systat Software, Inc., San Jose, CA).HDACI CytotoxicityJurkat cells were maintained in RMPI

1640 medium supplemented with 10% FBS (Atlanta Biologi-cals), 0.3 mg/ml L-glutamine, 100 units/ml penicillin, and 100g/ml streptomycin. HeLa and 293T cells were maintained inDMEMmedium supplementedwith 10% FBS, 0.3mg/ml L-glu-

tamine, 100 units/ml penicillin, and 100 g/ml streptomycin.CD8()-depleted peripheral bloodmononuclear cellswere iso-lated from fresh whole blood (100 ml) of HIV-negative individ-uals, as described previously (38). To determine HDACI cyto-toxicity, 1 104 cells were plated in 96-well plates with varyingconcentrations of drug. Following a 24-h incubation period, cellviability was measured using either the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Roche Applied Sci-ence) or CellTiter 96 proliferation (Promega, Madison, WI)assay. The concentration ofHDACI that decreased cell viabilityby 50% (i.e. 50% cytotoxic concentration (CC50)) was calculatedby regression analysis using SigmaPlot software.Reactivation of Latent HIV-1 by HDACIJ89GFP cells are a

Jurkat T-cell line that contains a stably integrated, full-lengthHIV-1 provirus (strain 89.6) with an enhanced green fluores-cent protein (EFGP) reporter incorporated into the viralgenome (22). The viral genome in these cells is transcriptionallysilent. However, upon stimulation with tumor necrosis factor or HDACI, viral transcription was activated, and viral expres-sion can be measured by EGFP production. We chose this celllinemodel ofHIV-1 latency because the absence of viral expres-sion was not due to mutations in either the Tat-TAR axis (e.g.the ACH2 cell line (34) and the U1 promonocytic cell line (36))or in the 5-LTR (e.g. the JK cell line (35)). The J89GFP cellswere maintained in RMPI 1640 medium supplemented with10% FBS, 0.3 mg/ml L-glutamine, 100 units/ml penicillin, and100 g/ml streptomycin. 5 105 cells/ml cells were plated in6-well plateswith varying concentrations ofHDACI for 672h.The PI3K and Akt inhibitors wortmannin and Akt inhibitor IV(AI4) were used at concentrations of 100 nM and 10M, respec-tively. The cells were thenwashed in PBS, fixed in 4%paraform-aldehyde, and stored at 4 Cuntil analysis. Reactivation of latentHIV-1 was determined by quantifying the percentage of EGFP-positive cells using a FACScan flow cytometer with FACSDivasoftware (BD Biosciences).DNA Microarray Analyses5 105 J89GFP cells were

treated with 200 nM SAHA, oxamflatin, scriptaid, and apicidinfor 24 h. Control experiments included J89GFP cells grown inthe absence of HDACI and Jurkat cells infected with HIV-1(multiplicity of infection of 1) for 24 h. Total cellular RNA wasextracted from these cells using the RNeasy Plus RNA extrac-tion kit (Qiagen Inc.) according to themanufacturers protocol.RNA quantification, quality assessment, and DNA microarrayanalyses were carried out by PhalanxBio, Inc. (Palo Alto, CA),using the Human Whole Genome OneArrayTM microarray.Each treatment condition and control were assessed in dupli-cate biological replicates, and all samples were run in duplicatetechnical replicates on the arrays. Data analysis was performedby PhalanxBio, Inc., using Rosetta Resolver software.siRNAKnockdownsiRNAs targetingHDAC1,HDAC2, and

HDAC3, as well as a control scrambled sequence controlsiRNA, were purchased from Qiagen (SA Biosciences). TheJ89GFP cells were transfected with 60 nM siRNA using theNeon Transfection System from Invitrogen, according tothemanufacturers protocol. The efficiency of gene knockdownwas assessed by determining mRNA levels (described below)and by Western blot analyses of protein expression.

Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

8

Quantitative Analysis of Gene TranscriptsRNA wasextracted from treated cells using RNeasy Plus RNA extractionkit (Qiagen Inc., Valencia, CA) according to themanufacturersprotocol. RNA was quantified using a Nanodrop 2000, and200400 ng of total RNA was used in each reaction. RNA wasamplified using the QuantiTect SYBR Green RT-PCR kit(Qiagen Inc., Valencia, CA) and the DNA Engine Opticonsystem (Bio-Rad). Initiated HIV-1 transcripts were detectedusing primers TAR-FW (5-GTTAGACCAGATCTGAGCCT-3) and TAR-Rev (5-GTGGGTTCCCTAGTTAGCCA-3).Elongated HIV-1 transcripts were detected using primersTAT-FW (5-ACTCGACAGAGGAGAGCAAG-3) and TAT-REV (5-GAGTCTGACTGTTCTGATGA-3). HDAC1 wasquantified using primers HDAC1-FW (5-CCAGTATTC-GATGGCCTGTT-3) andHDAC1-REV (5-TGTACAGCAC-CCTCTGGTGA-3). HDAC2 was quantified using primersHDAC2-FW (5-ATAAAGCCACTGCCGAAGAA-3) andHDAC2-REV (5-TCCTCCAGCCCAATTAACAG-3). HDAC3was quantified using primers HDAC3-FW (5-TGGCTTCT-GCTATGTCAACG-3) and HDAC3-REV (5-TCTCTGC-CCCGACTTCATAC-3). -Actin mRNA copies were used asthe normalization control (10) and were quantified using theprimers -actin-FW (5-GTCGACAACGGCTCCGGC-3)and -actin-REV (5-GGTGTGGTGCCAGATTTTCT-3).Relative gene expression levels were calculated using theC(T) method (23).Chromatin Immunoprecipitation (ChIP) Assays2 106

J89GFP cells were fixed with 1% formaldehyde for 10 min atroom temperature. ChIP assays were carried out using the EZ-ChIP assay kit (Millipore Billerica, MA). Immunoprecipitationwas performed using 5 g of antibody (Invitrogen) againstHDAC13 or -8. Rabbit immunoglobulin G serum (5g, SantaCruz Biotechnology Inc., Santa Cruz, CA) was used to controlfor nonspecific immunoprecipitation of DNA. Forward(LTRB primer 5, 5-AGGTTTGACAGCCGCCTA-3) andreverse (LTRB primer 3, 5-AGAGACCCAGTACAG-GCAAAA-3) primers specific for a 203-bp region in theHIV-1LTR that encompasses the NF-B-binding site LTR were usedto detect a specific interaction between an HDAC isoform andHIV-1 DNA, as described previously (10).

RESULTS ANDDISCUSSION

Potent Inhibition of HDAC1 Is Not Sufficient to ReactivateLatent HIV-1Previous studies have demonstrated thatrecruitment of HDAC1 to the HIV-1 LTR by different DNA-binding complexes is sufficient to induce viral latency (911,13, 14). Accordingly, we hypothesized that a potent inhibitor ofHDAC1 would reactivate HIV-1 expression in the J89GFP cellline model of viral latency. Initially, we screened 16 structurallydiverse HDACI (Fig. 1) for the following: (i) their inhibitoryactivity against recombinant purified HDAC1; (ii) their cyto-toxicity (CC50) in Jurkat cells; and (iii) their ability to reactivateHIV-1 expression in J89GFP cells (Table 1). For point iii, thehighest possible sub-cytotoxic concentration ofHDACI (deter-mined from the cytotoxicity assessments) was used. Of the 16HDACI tested, 6 (apicidin, HC toxin, scriptaid, oxamflatin,SAHA, and trichostatinA) exhibited potent activity (IC50100nM) against purified HDAC1. Of these, only three (oxamflatin,

apicidin, and trichostatin A) were able to stimulate HIV-1expression bymore than 5% in the J89GFP cells, asmeasured byflow cytometry analysis of EGFP expression. In this regard, itshould be noted that apicidin, scriptaid, oxamflatin, and SAHAexhibited similar CC50 values and were tested in the J89GFPcells at identical concentrations, therefore allowing for a directcomparison of their ability to reactivate latent HIV-1 expres-sion. Interestingly, valproic acid and sodium butyrate werefound to be relatively weak inhibitors of HDAC1 (IC50 175M), but each elicited a different effect in the J89GFP cells asfollows: 1 mM valproic acid reactivated HIV-1 expression inonly 4.3% of cells; 1 mM sodium butyrate reactivated HIV-1expression in 66.4% of cells. Taken together, these HDACIscreening studies show that inhibition of HDAC1 is not suffi-cient to reactivate HIV-1 expression in J89GFP cells.Inhibition of HDAC3 Correlates with the Reactivation of

Latent HIV-1Based on the data described above, we next car-ried out in-depth analyses on theHDACIs apicidin, oxamflatin,scriptaid, and SAHA (Fig. 2). Each of these HDACIs exhibitsimilar potency against purified HDAC1 and similar cytotoxic-ity (CC50) values in Jurkat cells (Table 1). However, at concen-trations of inhibitor ranging from 0 to 500 nM, apicidin andoxamflatin reactivated HIV-1 expression in the J89GFP cells ina dose-dependent manner, whereas SAHA and scriptaid elic-ited no effect (Fig. 2A). A time course experiment demonstratedthat this lack of activity was not due to an early or late EGFPpeak that was missed at the 24-h time point used in the dose-response experiments (Fig. 2B). Because the EGFP expressionquantitated in Fig. 2,A and B, only provides information on thetranslated protein, we also used quantitative RT-PCR to assessthe formation of HIV-1 RNA transcripts (Fig. 2C). Consistentwith the flow cytometry analyses, oxamflatin and apicidin sig-nificantly increased the abundance of elongated HIV-1 tran-scripts, but not initiated transcripts, compared with untreatedJ89GFP cells. By contrast, scriptaid and SAHA did not signifi-cantly increase the formation of either initiated or elongatedHIV-1 transcripts.We also carried out cDNAmicroarray analyses to determine

themagnitude and the extent of global gene expression changesobserved in the J89GFP cells after 24 h of treatmentwith 200 nMoxamflatin, scriptaid, SAHA, or apicidin. Control experimentsincluded untreated J89GFP cells and Jurkat cells infected withHIV-1 for 24 h. The cDNA microarray data are provided insupplemental Tables 1 and 2 and Fig. 1. SAHA and scriptaidwere found to significantly (p 0.01) up- or down-regulate 3and 1% of all genes compared with untreated cells, respectively.One study reported that SAHA altered regulation in at least22% of genes in CEM cells; however, a much higher concentra-tion of drug (2.5M) was used which caused50% of cell deathafter 24 h (30).Oxamflatin and apicidin resulted in gene expres-sion changes in 11 and 8% of all genes compared withuntreated J89GFP, respectively. These higher gene expressionlevels compared with scriptaid and SAHA could be due to inhibi-tionofdifferentHDACisoforms(seebelow)and/or theexpressionof HIV-1 proteins in the J89GFP cells. Microarray DNA analysesrevealed that 2.7% of all genes displayed altered expressionchanges in HIV-1-infected Jurkat cells compared with uninfectedcells. Nevertheless, these data indicate that SAHA and scriptaid


9

were taken up into the J89GFP cells and induced gene expressionchanges. However, at the concentrations tested, they lacked theability to induce expression of latent HIV-1.

To gain insight into the mechanisms by which oxamflatinand apicidin, but not scriptaid and SAHA, reactivated latentHIV-1, we determined the in vitro inhibitory activity of each of

FIGURE 1. Chemical structures of HDACI used in this study.

TABLE 1In vitro activity against HDAC1, cytotoxicity in Jurkat cells, and reactivation of latent HIV-1 in J89GFP cells by structurally diverse HDACI

HDACI IC50 against HDAC1 Cytotoxicity CC50Reactivation

dosea% inhibition of HDAC1 at

reactivation doseb% EGFP-positive J89GFP

cells (after 24 h)M M M

HC toxin 0.000154 0.05 0.005 99.7 1.3Apicidin 0.000299 10.0 0.5 99.7 40.6Oxamflatin 0.003959 6.0 0.5 99.2 31.2Scriptaid 0.006421 6.0 0.5 98.7 3.2SAHA 0.0137 10.0 0.5 97.3 2.5TSA 0.0169 0.10 0.05 74.7 17.2M344 0.0941 0.5 0.1 51.5 3.1CAY10398 1.7780 1.0 0.5 21.9 0.9MC1293 4.245 10.0 5 54.1 2.7CAY10433 9.36 1000 10 51.6 5.2SBHA 4.54 100 100 95.6 57.5Depudecin 25.33 5.0 1 3.8 1.3Sodium 1-naphthoate 200.6 10 10 4.75 1Valproic acid 171 10,000 1000 85.4 4.3Sodium butyrate 175 10,000 1000 85.1 66.4Sodium 4-phenylbutyrate 162 10,000 1000 86.1 2.5

a The highest nontoxic concentration of HDACI (determined from the cytotoxicity assays in Jurkat cells) was administered to the J89GFP cells to determine the inhibitorsability to reactivate latent HIV-1.

b Maximum possible inhibition of HDAC1 at the concentration of inhibitor used in the reactivation experiments in J89GFP cells is shown (the actual inhibition in theJ89GFP cells is likely to be significantly less due to inefficient cellular uptake and nonspecific protein binding).


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the HDACI against recombinant purified HDAC1 and -2, the3-NCOR2 complex, and -49 (Table 2). In general, each of theHDACI exhibited little or no activity against the class II HDAC

isoforms, although scriptaid exhibited excellent activity againstHDAC6 (IC50 34 nM) and oxamflatin activity againstHDAC6(IC50 390 nM) and HDAC7 (IC50 840 nM). By contrast, allfour of the HDACI were found to be very potent inhibitors ofthe class I HDAC isoforms 1, 2, and 8 (IC5020 nM). Interest-ingly, oxamflatin and apicidin, both of which induced HIV-1outgrowth in the J89GFP cells, also potently inhibited theHDAC3-NCOR2complex (IC5010nM). By contrast, scriptaid(IC50 320 nM) and SAHA (IC50 600 nM) were 100-foldless active against the HDAC3-NCOR2 complex. Based on ourIC50 calculations, a 200 nM dose of apicidin and oxamflatinwould inhibit 95% of the deacetylase activity of HDAC13and -8. (In the J89GFP cells, these values would likely be signif-icantly less due to inefficient inhibitor uptake and nonspecificprotein binding.) By contrast, a 200 nM dose of scriptaid andSAHA would inhibit 95% of HDAC1, -2, and -8 but wouldonly inhibit 25 and 18% of the deacetylase activity of HDAC3,respectively. These values for HDAC3would increase to 45 and36%, respectively, at a dose of 500 nM. Taken together, thesedata provide strong evidence that potent inhibition of HDAC3is required to reactivate the expression of HIV-1 in the J89GFPcells. The data also suggested that increasing the concentra-tions of scriptaid and SAHA to allow inhibition of HDAC3would result in the activation of latent HIV-1 in the J89GFPcells. Indeed, in Fig. 2Dwe show that 2 M scriptaid and SAHApromote activation of latent HIV-1 in the J89GFP. At this con-centration, the total inhibition of HDAC3 approaches 76 and70% for scriptaid and SAHA, respectively. To further assess theimportance of HDAC3 inhibition in the reactivation of latentHIV-1 infection, we assessed the ability of droxinostat to reac-tivate latent HIV-1 expression in the J89GFP cells. Previousstudies reported that this HDACI selectively inhibited HDAC3and -8 but notHDAC1 and -2 (37). Indeed, we found that droxi-nostat is a reasonably potent inhibitor of recombinantHDAC3-NCOR2 complex and HDAC8 (IC50 2.0 and 3.0 M, respec-tively) but shows only weak activity against HDAC1 and -2(IC50 63 and 250 M, respectively) (Table 2). Of note, droxi-nostat was found to reactivate latent HIV-1 expression in adose-dependent manner (Fig. 3). At a 40 M concentration ofdroxinostat, HDAC3 and -8 would be inhibited by 95%,whereas therewould be only partial inhibition ofHDAC1 (38%)andHDAC2 (14%). Taken together, these studies provide addi-

FIGURE 2. Reactivation of latent HIV-1 in J89GFP cells by oxamflatin, api-cidin, scriptaid, and SAHA. A, J89GFP cells were treated with varying con-centrationsofHDACI (0500nM), and thepercentageof cells expressingEGFPwasquantitated after 24hbyFACs. Error bars represent S.E. fromat least threeindependent experiments. B, J89GFP cells were treated with 200 nM HDACI,and thepercentageof cells expressingEGFPwasquantitatedatdifferent timeintervals (075 h) by FACs. C, increase in HIV-1 RNA transcripts in cells treatedwith 200 nM HDACI for 24 h as measured by quantitative RT-PCR. Error barsrepresent S.E. fromat least three independent experiments. The fold increasein transcript relative to untreated cells is indicated above each bar for allfour HDACI. D, J89GFP cells were treated with varying concentrations ofHDACI (0 2 M), and the percentage of cells expressing EGFP was quan-titated after 24 h by FACs. Error bars represent S.E. from at least threeindependent experiments.

TABLE 2In vitro activity of HDACI against class I and class II HDAC isoforms

HDAC isoformIC50 against HDAC isoforms (nM )

Apicidin Oxamflatin Scriptaid SAHA Droxinostat Valproic acidClass IHDAC1 0.30 0.15a1 3.96 0.87 0.64 0.09 13.7 0.15 63,000 171,000HDAC2 1.2 0.80 0.16 0.11 1.4 0.74 62.0 0.15 250,000 634,000HDAC3 0.98 0.22 10.3 1.2 607 93 869 0.15 2000 5,500,000HDAC8 0.26 0.09 0.37 0.15 14.5 1.1 6.8 0.15 5000 756,000

Class IIHDAC4 50,000 3800 1100 14,000 1500 50,000 NDb NDHDAC5 50,000 50,000 50,000 50,000 ND NDHDAC6 50,000 390 73 34 9 5500 760 ND NDHDAC7 50,000 840 39 2200 350 50,000 ND NDHDAC9 50,000 50,000 50,000 50,000 ND ND

a Data represent the mean S.D. from three replicate experiments.b ND, not determined.


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tional evidence that HDACI with specificity toward HDAC3can reactivate latent HIV-1 expression in J89GFP cells.Several studies have recently demonstrated that oxamflatin,

apicidin, scriptaid, and SAHA can reactivate latent HIV-1 indifferent cell lines and/or in resting CD4() T cells from avire-mic patients (18, 19, 20, 32). In each of these studies relativelyhigh concentrations (500 nM) of inhibitor were used. Of note,each of these HDACI display significant toxicity in cell lines(CC50 values range from 5 to 10 M) and in CD8()-depletedperipheral blood mononuclear cells (CC50 values range from0.1 to 7.5 M) (Table 3). In this regard, the small therapeuticwindow of these HDACI highlights one potential limitation fortheir inclusion in therapeutic combinations targeted towardthe eradication of HIV-1.HDAC3 Resides at the HIV-1 LTR in J89GFP CellsThe data

described above provide strong evidence that inhibition ofHDAC3 is important for the activation of latent HIV-1. How-ever, only two studies have identified this HDAC isoform at theHIV-1 LTR (16, 17). Accordingly, we performed ChIP assays inthe J89GFP cells using antibodies specific for the class I HDACisoforms (Fig. 4A).We detected a strong signal for HDAC1 and-3 indicating that both of these HDAC isoforms resided at theHIV-1 LTR. We also detected a weak signal for HDAC2 sug-gesting that this isoformmay also be present at the HIV-1 LTR

in J89GFP cells. HDAC8 did not associate with the HIV-1 LTR.These findings are consistent with a recent study byKeedy et al.(17), who also reported that HDAC13 resided at the HIV-1LTR in J89GFP cells. Of note, this study also reported thatHDAC8 is primarily sequestered in the cytoplasm and not thenucleus in J89GFP cells (17). Quantitative real time PCR exper-iments confirmed the presence of HDAC1 and -3, but notHDAC2, at the HIV-1 LTR (Fig. 4B). Importantly, the occu-pancy of HDAC1 and -3 at theHIV-1 LTR in the J89GFP cells islost upon treatment with apicidin (Fig. 4B) or oxamflatin (datanot shown). To further assess the role of HDAC13 in main-taining HIV-1 latency, we knocked down their gene expression

FIGURE 3. Reactivation of latent HIV-1 in J89GFP cells by theHDAC3-spe-cific inhibitor droxinostat. The chemical structure of droxinostat is shown.The number of J89GFP cells expressing EGFP was quantitated after 24 h byFACs. Error bars represent S.E. from two independent experiments.

TABLE 3Cytotoxicity of HDACI in different cells

HDACI

CC50 (M)

Jurkata HeLaa 293TaCD8()-depleted

PBMCb

Apicidin 10.0 0.1 11.3 1.7 12.2 1.4 0.1Oxamflatin 5.9 0.1 9.4 0.8 6.0 1.0 0.3SAHA 10.0 0.1 11.3 0.3 14.1 0.2 7.5Scriptaid 6.1 0.3 8.9 1.8 5.8 0.2 0.7

a Data represent the mean S.D. from three replicate experiments.b Data represent the mean from two independent replicate experiments.

FIGURE 4. Role of HDAC13 in maintaining HIV-1 latency. A, ChIP assaysidentify HDAC13, but not HDAC8, at the HIV-1 LTR in J89GFP cells. Two dif-ferent HDAC1 antibodies (a and b) were used in the ChIP assays. B, quantita-tive real timePCRexperiments showenrichment (versus rabbit IgG) of HDAC1and -3 at the HIV-1 LTR in J89GFP cells that is lost upon treatment with apici-din. PCR data were normalized by quantification of the GAPDH promoter inthe input samples. C, mRNA expression of HDAC13 after knockdown bysiRNA. HDAC1* and HDAC2* reports on the mRNA levels in experiments inwhichbothHDAC1and -2were knockeddownsimultaneously. An siRNAwitha scrambled sequencewas used as a control. D, reactivation of latent HIV-1 inJ89GFPcells after knockdownofHDAC1or -2orHDAC1and -2. ThenumberofJ89GFP cells expressing EGFP was quantitated after 24 h by FACs. Error barsrepresent S.E. from three independent experiments.


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by siRNA (Fig. 4C). The magnitude of the siRNA-mediatedgene silencing was confirmed by quantitative PCR analyses ofmRNA levels (Fig. 4C) and by Western blot analysis (data notshown). Knockdown ofHDAC3 resulted in significant and sub-stantial cell death that prevented subsequent analyses of HIV-1latency. Interestingly, the knockdown of either HDAC1 or -2,or a combination of HDAC1 and -2, did not result in the reac-tivation of latentHIV-1 expression in the J89GFP cells (Fig. 4D).These data provide strong supporting evidence that inhibitionof HDAC1 and -2 is insufficient to reactivate latent HIV-1expression.Reactivation of Latent HIV-1 Expression by Apicidin and

Oxamflatin Is Partially Dependent on the PI3K/Akt SignalingPathwayPeterlin and co-workers (20) have shown that SAHAactivates HIV-1 expression in latently infected cells via thePI3K/Akt pathway. To determine whether this pathway is alsoactivated by apicidin and oxamflatin, we first assessed Aktphosphorylation levels in J89GFP cells treated with TNF- (apositive control) or with 500 nM apicidin, oxamflatin, SAHA, orscriptaid (Fig. 5A). Increased Akt phosporylation was observedfollowing treatmentwith apicidin but not oxamflatin, SAHA, orscriptaid. Because the concentration of SAHA used in thisexperiment (500 nM) is not sufficient to reactivate latent HIV-1expression in the J89GFP cells, it was not unexpected that Aktwas not activated. To further determine the impact of apicidinand oxamflatin on activation of the PI3K/Akt signaling path-way, we used PI3K (wortmannin) and Akt (Akt inhibitor IV(AI4)) inhibitors. Indeed, Akt and PI3K inhibitors decreasedbut did not completely eliminate both apicidin and oxamflatin-induced viral replication (Fig. 5B). Importantly, these inhibitorshad no significant effect on the basal levels ofHIV-1 production(Fig. 5B). These results suggest that the latent HIV-1 reactiva-tion activity of both apicidin and oxamflatin is intertwinedwithactivation of the PI3K/Akt signaling pathway.Valproic Acid Is a Weak Inhibitor of HDAC3In 2004,

Ylisastigui et al. (24) demonstrated that treatment of restingCD4() T cells of aviremic patients with valproic acid inducedhistone acetylation and promoted virus outgrowth. An initialproof-of-concept study in which four volunteers infected withHIV added oral valproic acid to their cART regimen for 3months reported a significant decline in the frequency of rest-ing CD() T-cell infection (25). However, several follow-upstudies found that valproic acid does not reduce the size oflatent HIV reservoir (2629). Interestingly, we find that val-proic acid is a weak inhibitor of the HDAC3-NCOR2 complex(IC50 5.5mM, Table 2) and that high concentrations of inhib-itor (1 mM) are required in J89GFP cells to activate HIV-1expression (Fig. 6). The total and free therapeutic concentra-tions of valproic acid in adults range from 275 to 700 M andfrom 27 to 100 M, respectively. These therapeutic concentra-tions would be insufficient to inhibit HDAC3 in vivo. Accord-ingly, our data may explain why valproic acid has no effect onthe decay of latent HIV reservoirs in patients.ConclusionsThis study demonstrates that HDAC3 resides

at the HIV-1 LTR in J89GFP cells and that inhibition of thisHDAC isoform is required for the activation of latent HIV-1expression by HDACI. Interestingly, Archin et al. (33) recentlydemonstrated that an HDACI (MRK12) specific for HDAC1

and -2was unable to reactivate latentHIV-1 in J89GFP cells andin resting CD4() T-cells from aviremic patients. By contrast,an inhibitor (MRK13) specific for HDAC13 promoted virusoutgrowth in both assay systems (33). Taken together, thesestudies suggest that potent inhibition of HDAC3 should be animportant criterion in the development of HDACI for HIV-1curative strategies. Unfortunately, neither our study nor that ofArchin et al. (33) could address whether inhibition of HDAC3alone is sufficient to induce virus outgrowth or if inhibition ofHDAC1 and/or -2 is also required. The identification anddevelopment of inhibitors specific forHDAC3may address thisquestion.Finally, it is likely that there are several reservoirs of latent

HIV-1 infection in aviremic patients on cART. For example,resting CD4() T-cells and CD34() multipotent hematopoi-etic progenitor cells have both been identified as reservoirs oflatent HIV-1 infection (25). The HDAC isoforms recruited totheHIV-1 LTRs in these different cell typesmay be different. Inthis regard, the chemical approach described in this study canreadily be used to identify the HDAC isoforms that contributeto HIV-1 latency in other cell types. The primary advantages ofthis approach include the ability to rapidly conduct studies incell types that cannot be easily transfected with siRNA (or

FIGURE 5. Activation of the PI3K/Akt signaling pathway by apicidin andoxamflatin. A, Western blot analysis of Akt phosphorylation in J89GFP cellstreated with TNF- (positive control) or with 500 nM apicidin, oxamflatin,SAHA, or scriptaid. Phosphorylated Akt and -actin were detected usingmonoclonal antibodies specific for these proteins. B, J89GFP cells weretreated with 500 nM HDACI with or without wortmannin (100 nM) or the Aktinhibitor IV (AI 4, 10 M). The number of J89GFP cells expressing EGFP wasquantitated after 24 h by FACs. Error bars represent S.E. from three indepen-dent experiments.


13

shRNA) molecules or in patient-derived tissues or cells wherethere may be insufficient material to carry out genetic studies.Importantly, our chemical approach also provides an immedi-ate assessment of the therapeutic potential of HDACI to reac-tivate latent HIV-1 expression in different cell types and/ortissues.REFERENCES1. Davey, R. T., Jr., Bhat, N., Yoder, C., Chun, T.W., Metcalf, J. A., Dewar, R.,

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FIGURE 6. Reactivation of latent HIV-1 in J89GFP cells by varying concen-trations (010 mM) of valproic acid. The percentage of J89GFP cellsexpressing EGFP was quantitated after 24 h by FACs.


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Host Protein Ku70 Binds and Protects HIV-1 Integrase fromProteasomal Degradation and Is Required for HIV Replication*Received for publication, September 13, 2010, and in revised form, March 28, 2011 Published, JBC Papers in Press,March 29, 2011, DOI 10.1074/jbc.M110.184739

Yingfeng Zheng1, Zhujun Ao2, Binchen Wang, Kallesh Danappa Jayappa3, and Xiaojian Yao4

From the Laboratory of Molecular Human Retrovirology, Department of Medical Microbiology, Faculty of Medicine, University ofManitoba, Winnipeg, Manitoba R3E 0J9, Canada

HIV-1 integrase (IN) is a key viral enzymatic protein actingin several viral replication steps, including integration. INhas been shown to be an unstable protein degraded by theN-end rule pathway through the host ubiquitin-proteasomemachinery. However, it is still not fully understood how thisviral protein is protected from the host ubiquitin-proteasomesystem within cells during HIV replication. In the presentstudy, we provide evidence that the host protein Ku70 inter-acts with HIV-1 IN and protects it from the Lys48-linkedpolyubiquitination proteasomal pathway. Moreover, Ku70 isable to down-regulate the overall protein polyubiquitinationlevel within the host cells and to specifically deubiquitinateIN through their interaction. Mutagenic studies revealed thatthe C terminus of IN (residues 230288) is required for INbinding to the N-terminal part of Ku70 (Ku70(1430)), andtheir interaction is independent of Ku70/80 heterodimeriza-tion. Finally, knockdown of Ku70 expression in both virus-producing and target CD4 T cells significantly disruptedHIV-1 replication and rendered two-long terminal repeat cir-cles and integration undetectable, indicating that Ku70 isrequired for both the early and the late stages of theHIV-1 lifecycle. Interestingly, Ku70 was incorporated into the progenyvirus in an IN-dependent way. We proposed that Ku70 mayinteract with IN during viral assembly and accompany HIV-1IN upon entry into the new target cells, acting to 1) protect INfrom the host defense system and 2) assist IN integrationactivity. Overall, this report provides another example of howHIV-1 hijacks host cellular machinery to protect the virusitself and to facilitate its replication.

Integration is an obligatory step in the life cycle of all of theretroviruses and is performed by the viral enzyme integrase

(IN).5 During HIV-1 integration, IN catalyzes the insertion ofnewly reverse-transcribed 10-kb viral DNA into the hostgenome. In addition, IN plays important roles in other viralreplication steps, such as reverse transcription, the nuclearimport of preintegration complexes (PICs), and chromatin tar-geting. By interaction with the host chromatin-tethering factorLEDGF/p75, IN preferentially targets viral DNA into transcrip-tionally active sites in the host genome to optimize the tran-scription and translation of its gene products (13). Cellularproteins are recruited to assist IN to accomplish integrationfrom different pathway, including nuclear import, shielding INfrom proteasomal degradation, integration site selection, andgap repair (4). Recently, considerable interest has been focusedon the functional interaction between IN and host cellular pro-teins in the hope of disrupting their interactions, thereby block-ing HIV-1 replication. In an attempt to identify host cellularpartners for IN, several research groups have identified a num-ber of IN cofactors using the yeast two-hybrid system, coimmu-noprecipitation (co-IP) assays, or in vitro reconstitution of theenzymatic activity of salt-stripped PICs (511). A recent studyby Studamire et al. (5) found that 12 cellular proteins, includingKu70, could bind to the INs of both the Moloney murine leu-kemia virus (MMLV) andHIV-1 through screeningwith a yeasttwo-hybrid system. However, whether these cellular cofactorsare associated with HIV-1 IN during HIV replication and theirfunctional relevance remain unknown.Ku70 is an evolutionarily conserved protein; it is found ubiq-

uitously in eukaryotes and some prokaryotes, such as Archaeaand Bacteria (1214). It is well known as a DNA repair proteinand is part of the nonhomologous end-joining (NHEJ) path-way. Ku70 has also been implicated in many cellular pro-cesses, including antigen-receptor gene rearrangement,mobilegenetic element biology, V(D)J recombination of immunoglob-ulins, telomere maintenance, DNA replication, transcription,cell cycle control, and apoptosis (13, 15). As a DNA repair pro-tein, Ku70 can bind to any double-stranded DNA irrespectiveof sequence specificity or end configuration, including 5 over-hangs, 3 overhangs, or blunt ends (for a review, see Ref. 15).Ku70 can also bind specific DNA sequences to affect gene tran-scription (16). Formost biological functions inwhichKu70 par-ticipates, Ku functions as a heterodimer consisting of Ku70 andKu80, named according to their respectivemolecular masses of

* This work was supported by Canadian Institutes of Health Research(CIHR) Grants HOP-81180 and HBF 103212 and the Leaders OpportunityFund Award from the Canadian Foundation of Innovation (to X.-J. Y.).

1 Recipient of studentships from the Manitoba Health Research Council/Manitoba Institute of Child Health (MHRC/MICH) and the CIHR Interna-tional Infectious Disease and Global Health Training Program.

2 Recipient of a postdoctoral fellowship from the CIHR International Infec-tious Disease and Global Health Training Program.

3 Recipient of studentships from MHRC/MICH.4 Recipient of the Basic Science Career Development Research Award from

the Manitoba Medical Service Foundation. To whom correspondenceshould be addressed: Laboratory ofMolecular HumanRetrovirology, Dept.ofMedicalMicrobiology, Faculty ofMedicine,University ofManitoba, 508745 William Ave., Winnipeg R3E 0J9, Canada. Tel.: 204-977-5677; Fax: 204-789-3926; E-mail: [email protected].

5 The abbreviations used are: IN, integrase; PIC, preintegration complex; IP,immunoprecipitation;WB,Westernblot;MMLV,Moloneymurine leukemiavirus; NHEJ, nonhomologous end-joining; Ub, ubiquitin; VSV-G, vesicularstomatitis virus G; p.i., postinfection; PL, ProLabel; aa, amino acids; MOI,multiplicity of infection.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, pp. 1772217735, May 20, 2011 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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70 and 80 kDa. Two regions of Ku70 amino acids 1115 and430482 are responsible for its heterodimerization with Ku80(17). Successful HIV-1 integration requires gap repair betweenviral DNA and host genome, which is believed to be performedby host DNA repair enzymes (18). Two different host DNArepair pathways have been suggested to fill in the gap duringHIV-1 infection: theNHEJ andDNAdamage-sensing pathways(1921). TheNHEJ pathway begins with the recruitment of theKu70/80 heterodimer, followed by the catalytic subunit ofDNA-dependent protein kinase or DNA-PKcs, Xrcc4, andDNA ligase IV. Studies have shown that the NHEJ pathway isimportant for retroviral transduction or infection and for thecell survival of infected or transduced cells (20, 2225). Forexample, HIV-1-based vector transduction or infection wasmarkedly reduced in cells deficient in Ku80, DNA-PKcs, Xrcc4,or ligase IV (22, 24). Moreover, NHEJ activity is required fortwo-long terminal repeat (2-LTR) circle formation, and Ku70has been detected in MMLV PICs (24, 2628). Ku80 was alsoshown to suppress HIV transcription by specifically binding toa negative regulatory element within the LTR (29). All of theseobservations suggest that Ku70 or the K70/80 heterodimermaybe involved inHIV-1 infection by affectingmultiple steps of theviral replication cycle, such as integration. In addition, a noveldeubiquitinating enzymatic activity of Ku70 was recentlydescribed in which Ku70 has a regulatory effect on Bax-medi-ated apoptosis by decreasing the ubiquitination of Bax andblocking Bax from proteasomal degradation (30). However,whether Ku70 also exerts a deubiquitinating effect on otheridentified binding partners of Ku70 and how Ku70 interactswith the ubiquitin-proteasome pathway to deubiquitinate pro-tein substrates are still unclear.In this study, we investigated the interaction between Ku70

and HIV-1 IN and the potential roles of Ku70 during HIV-1replication using cell-based coimmunoprecipitation and shorthairpin RNA (shRNA)-mediated knockdown approaches.Interestingly, our results provide evidence that Ku70 is able toprotect HIV-1 IN from Lys48-linked polyubiquitination anddegradation by down-regulation of the overall protein poly-ubiquitination level within the host cells and by specific INdeubiquitination through its binding to IN. Moreover, ourstudy showed that Ku70 depletion in both virus-producing andtarget cells drastically inhibited HIV-1 replication and blocked2-LTR formation and integration in the real-time PCR analysis.Our data also showed that, mediated by HIV-1 IN, Ku70 wasincorporated into the progeny virus. All of these results suggestthat Ku70 may interact with IN during viral assembly andaccompany HIV-1 IN into newly infected cells to assist INintegration activity and protect IN from host-mediateddegradation.

EXPERIMENTAL PROCEDURES

Cell Lines and TransfectionHuman embryonic kidney293T and HeLa cell lines were cultured in Dulbeccos modifiedEagles medium (DMEM) supplemented with 10% fetal calfserum (FCS) and 1% penicillin/streptomycin. Human CD4C8166 T-lymphoid cells were maintained in RPMI 1640medium supplemented with 10% FCS and 1% penicillin/strep-tomycin. For the transfection of 293T cells and HeLa cells, the

standard calcium phosphate precipitation technique was used,as described previously (31).Plasmids and ReagentsTo achieve high level IN expression,

a codon-optimized IN (INopt) cDNA was synthesized andcloned into the pUC57 vector (GenScript Co., Ltd.). To con-struct pAcGFP-INopt, the INopt fragment was excised frompUC57-INopt with BamHI and cloned in frame at the 3 end ofthe pAcGFP1-C vector (Clontech) with the same restrictionenzyme. To construct pAcGFP-INwt/mut, each of the INwt/mut coding sequences, including 1230, 1250, 1270,50288, 112288, and K186A/R187A, was amplified by PCR-based mutagenesis and subcloned into the pAcGFP1-C vector(Clontech) in frame with the GFP coding sequence at the BglIIand BamHI restriction sites (32). Plasmids IN-YFP and MA-YFP were described previously (33, 34). Untagged human full-length Ku70 cDNA in the pCMV6-XL5 vector was purchasedfrom OriGene Technologies Inc. To construct SVCMV-T7-Ku70, a Ku70 cDNA without a start codon was amplified andcloned into the SVCMVin-T7 vector at the BamHI and NotIrestriction sites. The T7-Ku70 truncation mutants (1263,1430, 226609, and 430609) were obtained using the samestrategy. The nucleotide sequences of the mutagenic oligonu-cleotides are as follows: 5Ku70-BamHI, 5-TAGCCGGATCC-TCAGGGTGGGAGTCATATTA-3; 3Ku70-NotI, 5-TATAT-GCGGCCGCTCAGTCCTGGAAGTGCTT-3; Ku70263-NotI,5-AGATGCGGCCGCCTAGAGCTTCAGCTTT-3; Ku70430-NotI, 5-TATGCGGCCGCTCATGGAGGAGTCACCT-GAAT-3; Ku70226-BamHI, 5-TATGGATCCGATGAGGA-CCTCA-3; Ku70430-BamHI, 5-ATATGGATCCCCAGGC-TTCCAGCT-3. HA-tagged ubiquitin (HA-Ub) and mutantsHA-UbK48R and HA-UbK63R were described previously (35).TheHIV-1 proviruses pNL4.3-GFP,HxBru, andHxBru-IN-HAwere described earlier (32, 36). For single cycle HIV virus,RT/IN/Env gene-deleted NL4.3lucBglRI provirus andCMV-Vpr-RT-INwere described previously (34, 37, 38). CMV-Vpr-RT has two stop codons TAGTGA in place of the first sixnucleotides in IN sequences, and the sequence was confirmedby sequencing. To construct CMV-Vpr-RT-IN-ProLabel (Vpr-RT-IN-PL) plasmid, a two-step-based PCR method was used.ProLabel tag sequence was amplified from the pProLabel-Cvector from ProLabelTM Detection Kit II (Clontech) andinserted after the IN sequence in the CMV-Vpr-RT-IN plasmidwith the IN stop codon and ProLabel start codon removed. Thefollowing primers were used: RT NheI 5, 5-GCAGCTAGCA-GGGAGACTAA-3; R-RT-IN-ProLabel pstI 3, 5-GTCGACT-GCAGAATTCGAAGCTTATTC-3; IN-ProLabel 5, 5-AGA-CAGGATGAGGATAGCTCCAATTCACTG-3; IN-ProLabel3, 5-CAGTGAATTGGAGCTATCCTCATCCTGTCT-3.Antibodies and ReagentsThe rabbit anti-GFP polyclonal

antibody (Molecular Probes Inc.), the rabbit anti-HA antibody(Sigma), and mouse anti-T7 antibody (Novagen) were usedfor immunoprecipitation. The antibodies for Western blot(WB) were as follows: themousemonoclonal anti--actin anti-body (Abcam), mouse anti--tubulin (Sigma), mouse anti-Ku70 (Abcam), mouse anti-Ku80 (Abcam), horseradishperoxidase (HRP)-conjugated anti-GFP antibody (MolecularProbes), HRP-conjugated anti-HA antibody (Miltenyi Biotec),and HRP-conjugated anti-T7 antibody (Novagen). As the sec-

Ku70 Binds and Protects HIV-1 Integrase

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ondary antibodies, the ECLTM HRP-conjugated donkey anti-rabbit IgG and sheep anti-mouse IgG were purchased fromAmersham Biosciences. The WB detection ECL kit was pur-chased from PerkinElmer Life Sciences (Boston, MA). NonidetP-40 was from Roche Applied Science. Proteasome inhibitorMG-132 and puromycin were obtained fromCalbiochem. Sub-tilisin was purchased from Sigma.Transient and Stable Knockdown of Ku70 in 293T, HeLa

Cells, and C8166 T CellsTo test the effect of Ku70 levels onthe stability of IN, siRNA targeting human Ku70 (GenBankTMaccession number NM_001469) was used to transiently knockdown Ku70 expression in 293T cells and HeLa cells using theLipofectamineTM RNAiMAX transfection reagent (Invitro-gen). The sense primer for this siRNA is 5-GAUCCAGGUU-UGAUGCUCAtt-3, targeting Ku70 nucleotides 10941112.In parallel, a scrambled siRNA (Invitrogen) was used as a neg-ative control (siNC). After 5 nM siKu70 or siNC oligonucleotidewas transfected into cells for 12 h, cells were transfected againwith 5 nM siKu70 to maximize knockdown efficiency.To produce a stable Ku70-knockdown (KD) 293T and

C8166 CD4 T cell line, lentivirus-like particles harboringKu70 shRNA were produced by cotransfecting the shRNApLKO.1 vector containing shRNA targeting the Ku70 mRNA(5-CCGGCGACATAAGTCGAGGGACTTTCTCGAGA-AAGTCCCTCGACTTATGTCGTTTTTG-3 (Oligo ID:TRCN0000039608; purchased from Open Biosystems)),packaging plasmid 8.2, and vesicular stomatitis virus G(VSV-G) expressor into the 293T cells. After 48 h, shRNApLKO.1 vector particles were pelleted by ultracentrifugation(32,000 rpm at 4 C for 1 h) and used to transduce cells for48 h, followed by selection with 2 g/ml puromycin for 1week. Ku70-KD efficiency was determined by WB analysiswith anti-Ku70 antibody. Endogenous -actin was used tonormalize sample loading. The pLKO.1 vector without theshRNA sequence (empty vector) was introduced into cells bythe same method as a negative control.Direct Immunofluorescence AssayTo test the effect of

Ku70-KD level on the expression of IN, HeLa cells were firsttransfected with Ku70-specific siRNA oligonucleotides or non-targeting random siRNA (siNC) for 48 h and further trans-fected with GFP-INopt for another 48 h with or withoutMG-132 (10 M) treatment. GFP fluorescence-positive cellswere imaged by microscopy under a 20 objective lens (CarlZeiss).Coimmunoprecipitation Assay in 293T Cells and in HIV-1-

infected C8166T CellsTo detect the interaction betweenGFP-INwt/mut and T7-Ku70wt/mut and to identify theirmutual binding regions, the cell-based co-IP assay was per-formed as described previously (37). Briefly, GFP orGFP-INwt/mut plasmid was cotransfected with pCMV-Ku70 or T7-Ku70,respectively, into 293T cells for 48 h. To increase GFP-IN sta-bility, 10MMG-132was added 12 h prior to cell lysis for co-IP.Then 90% of the transfected cells were lysed in 0.25% NonidetP-40 prepared in 199medium containing a mixture of proteaseinhibitors (Roche Applied Science) and clarified by centrifuga-tion at 14,000 rpm for 30 min at 4 C. Supernatant was pre-cleared with Protein G-agarose on a rotator for 2 h at 4 C andsubsequently subjected to IP with a rabbit anti-GFP antibody

and Protein A-Sepharose overnight. The IN-bound proteinswere detected by WB using anti-Ku70 or anti-T7 antibodies.The same nitrocellulose membrane was then stripped andprobed with anti-GFP antibody to detect GFP-INwt/mut orGFP expression. Meanwhile, 5% of the transfected cells werelysed in 0.5% Nonidet P-40, and the lysates were used to detectthe expression of GFP-INwt/mut and Ku70 by WB using theircorresponding antibodies.To examine the IN/Ku70 interaction inHIV-1-infected cells,

HIV-1 (HxBru or HxBru-IN-HA)-infected C8166 T cells werelysed with 0.25% Nonidet P-40 and immunoprecipitated withanti-HA antibody followed by WB with anti-Ku70 antibody todetect IN-bound Ku70.Detection ofUbiquitination of IN in theAbsence or Presence of

Ku70To determine the ubiquitination level of HIV-1 IN inthe absence and presence of Ku70, 293T cells were cotrans-fectedwithGFP-IN andHA-Ubwild type ormutants K48R andK63R with and without T7-Ku70wt or T7-Ku70(1430). After48 h, cells were lysed in 199 medium containing 0.25%NonidetP-40 and a protease/inhibitor mixture and immunoprecipi-tatedwith anti-GFP antibody. Then the precipitated complexeswere run on a 10% SDS-polyacrylamide gel and analyzed for thepresence of HA-Ub byWBwithHRP-conjugated anti-HA anti-body. Simultaneously, GFP-IN was detected by immunoblot-ting the same membrane with HRP-conjugated anti-GFP anti-body.Andprotein band intensitywas quantified usingQuantityOne 1-D analysis software (Bio-Rad).Virus Production and InfectionTo study the effect of

Ku70-KD on HIV-1 replication, equal amounts (quantified byHIV-1 p24 antigen) of pNL4.3-GFP virus were used to infectKu70-KD or empty vector-transduced C8166 T cells for 2 h;cells were then washed and cultured in a 37 C incubator. Atdifferent time points, viral replication levels weremonitored bythemeasurement of p24 levels using an HIV-1 Gag-p24 ELISA.To test the infectivity of progeny virus produced from theKu70-KD cells, empty-vector and Ku70-KDC8166 T cells wereinfected with the same amounts of pNL4.3-GFP. Progenyviruses were collected by ultracentrifugation after 4 days ofinfection, and equal amounts of viruses (quantified by HIV-1p24 antigen) were used to infect empty vector or Ku70-KDC8166 T cells. Viral infection was examined at 3 days postin-fection by monitoring HIV p24 levels in the supernatant.Quantitative Real-time PCR1.5 106 stable C8166 T cell

lines with Ku70-KD or empty vector-transduced were infectedwith the pNL4.3-GFP virus as described above. Heat-inacti-vated virus (70 C for 30min) was used as a negative control forinfection. After 4 h of infection, cells were washed and culturedin fresh RPMI medium. At 24 h postinfection, cells were har-vested and washed with PBS twice. DNA was isolated using aQIAamp blood DNA minikit (Qiagen). The total levels ofHIV-1 DNA, 2-LTR circles, and integrated DNA were quanti-fied following the same procedure in an Mx3000P real-timePCR system (Stratagene) as described (32).Virus Composition and Incorporation of Cellular Protein into

HIV-1 VirionTo examine the viral protein compositions, thepNL4.3-GFP viruses from empty vector-transduced andKu70-KD C8166 T cells were pelleted through a 20% sucrosecushion at 35,000 rpm for 1.5 h at 4 C. Then equal amounts of

Ku70 Binds and Protects HIV-1 Integrase

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viruses (normalized by p24 values) were lysedwith 4Laemmlibuffer and directly loaded onto an SDS-PAGE gel and analyzedfor IN and p24 expressions using their corresponding antibod-ies. The reverse transcription activity from the purified viruseswas analyzed by a reverse transcription assay using a commer-cial RT assay kit (RocheApplied Science) according to theman-ufacturers instructions.To detect the presence of Ku70 in the HIV-1 particles, 15

106 CD4 C8166 T cells were mock-infected or infected withpNL4.3-GFP for 3 days. Then supernatants from both cell cul-tures were ultracentrifuged at 35,000 rpm for 1.5 h through a20% sucrose cushion. The pellets were dissolved in the samevolume of radioimmune precipitation assay buffer and mixedwith 20% (v/v) TCA, followed by precipitation on ice for 30minand acetone washing. Protein precipitates were dissolved in 4Laemmli buffer and directly loaded onto a 10% SDS-polyacryl-amide gel. Virus-associated Ku70 and p24 were then examinedby WB using the corresponding antibodies.Subtilisin treatment of purified HIV-1 virions. The subtilisin

assay was performed according to the protocol as described(39). The Vpr-RT-IN or Vpr-RT expressor was cotransfectedwith VSV-G andNL4.3lucBglRI to produce single cycle INand IN virus. The viruses were first ultracentrifuged through20% sucrose at 35,000 rpm for 2 h and then mock-treated ortreated with 0.1 mg/ml of subtilisin (Sigma) for 20 h at a 37 Cincubation. Subtilisin was inactivated by phenylmethylsulfonylfluoride. Virus was then repelleted as described above, lysed inradioimmune precipitation assay buffer, and loaded onto SDS-polyacrylamide gel followed by WB. Blots were sequentiallyprobed with anti-Ku70, anti-IN, and p24 antibodies.ProLabel Detection AssayTo test the effect of Ku70 on IN

during HIV infection, VSV-G pseudotyped HIV single cyclevirus containing ProLabel tag fused to the C terminus of INwasgenerated to quantify IN expression under HIV infection.NL4.3lucBglRI was cotransfected with Vpr-RT-IN-PL andVSV-G expressor into 293T cells to generate VSV-G pseu-dotyped HIV-1 single cycle IN-PL virus. The viruses were usedto infect shKu70-KD or empty vector-transduced C8166T cellsfor 3 h. The cells were washed three times and kept in freshmedium and then lysed with lysis/complementation buffer at8 h p.i. IN-ProLabel activity in the cell lysate was measuredaccording to the manufacturers instructions from the assay kit(ProLabelTM detection kit II, Clontech).Statistical AnalysisThe statistical significance was calcu-

lated using Students t test, and a p value of0.05 was consid-ered significant.

RESULTS

Cellular Protein Ku70 Protects HIV-1 IN from ProteasomalDegradationAs a part of the NHEJ machinery, the host pro-tein Ku70 has been shown to participate inHIV integration andin the circularization of unintegrated viral DNAs (25, 27). Sur-prisingly, based on the results of a yeast two-hybrid assay, arecent study indicated that HIV-1 IN may bind to Ku70 (5),suggesting a direct association betweenHIV-1 IN andKu70. Tofurther investigate this viral/host protein interaction, we coex-pressed Ku70 (T7-tagged Ku70) and HIV-1 IN (IN-YFP) in293T cells and analyzed their interaction after 48 h of transfec-

tion. Noticeably, our results revealed that T7-Ku70 overexpres-sion significantly increased IN expression (Fig. 1A, lanes 1 and2).However, the coexpression ofKu70with anotherHIV-1 pro-tein, MA (MA-YFP), did not change the MA expression level(Fig. 1A, lanes 3 and 4). This suggests that Ku70 is able toincrease IN expression. Alternatively, Ku70 could protect theIN protein from degradation (40).To further test whether endogenous Ku70 could exert the

same activity andwhether it is due to a protective effect, we firstknocked down the Ku70 expression using specific siRNA in293T (Fig. 1B) or HeLa cells (Fig. 1C) and checked the level ofGFP-IN expression by WB or fluorescence microscopy (Fig. 1,B and C). To increase IN expression under normal conditions,we used a pAcGFP-IN with a codon-optimized IN sequence(GFP-INopt). The results showed that IN expression inKu70-KD cells was significantly decreased when comparedwith IN expression in siNC cells (Fig. 1, B (compare lanes 1 and2) and C (compare A1A3 and B1B3)). Intriguingly, in thepresence of the specific proteasome inhibitorMG-132 (10M),IN expression in Ku70-KD cells was remarkably increased,reaching levels similar to those in siNC-transfected 293T andHeLa cells (Fig. 1, B (compare lanes 4 a

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