nature biotechnology • VOLUME 21 • MARCH 2003 • www.nature.com/naturebiotechnology

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Choosing CCR5 or Rev siRNA in HIV-1

To the editor:In a report in Nature Biotechnology1 andanother in Nature2, it was demonstratedthat small inhibitory RNAs (siRNAs) effi-ciently target and degrade human immun-odeficiency virus (HIV)-1 RNA sequencesand inhibit viral replication. Lee et al.1 usedsiRNA against Rev generated by indepen-dent transcription of sense and antisensestrands, whereas Jacque et al.2 targeted sev-eral sequences of the HIV-1 RNA tran-scripts using both synthetic siRNA andplasmid-mediated delivery of siRNA. In amore recent report, Novina et al.3 haveused synthetic siRNA to inhibit HIV-1infection by impairing CD4 expression.Thus, siRNA technology can be used todegrade efficiently both endogenous andvirus-derived RNA.

Although both approaches seem worth-while, we believe that treatments limited tothe inhibition of viral products by siRNAwill not be clinically feasible. Thus, it seemshighly likely that inhibition of Rev willresult in the selection of escape mutantsencoding Rev, but with silent mutationsimpairing siRNA recognition, and recent

experimental evidence supports thisnotion4.

To avoid escape mutants, we suggest tar-geting RNA encoding cellular proteins.However, when selecting a cellular target, itis important to identify a protein that doesnot cause deleterious effects when down-regulated. As Novina et al.3 themselvespoint out, silencing of CD4 may becomelimited by the role of this receptor in nor-mal host immune function, and co-recep-tors, in particular CC-motif receptor-5(CCR5), may be the preferred target in thathomozygous mutations in CCR5 (ref. 5)effectively confer protection from HIV-1 inthe absence of any known adverse effectson the immune system. Finally, the recentdevelopment of plasmid-mediated expres-sion of siRNA in a single transcript6, allow-ing long-term expression, represents fur-ther improvement toward clinical applica-tion of siRNA.

We have used plasmid-mediated deliveryof siRNA and successfully impaired HIV-1infection as well as replication by usingsiRNAs directed against the 3′ ends of theCCR5 and HIV-1 Rev transcripts, respec-tively. U937 cells (a human promonocyticcell line expressing CCR5) transientlytransfected with the plasmid pRS-siCCR5,showed ∼ 40% reduction of CCR5 expres-sion 48 hours post-transfection, as detect-ed by FACS analysis (Fig. 1A). This treat-ment inhibited infection with a primaryCCR5-dependent, M-tropic, HIV-1 isolate.Quantification of the HIV-1 p24 antigen insupernatants of the cell cultures by ELISAassay, 3 days post-infection, demonstrated68% inhibition (Fig. 1B; left panel) com-pared with cells transfected with a plasmidexpressing a hairpin against green fluores-cent protein (GFP). A comparison of thedecrease in CCR5 expression and the mag-nitude of protection against infection sug-gests that even partial reduction of CCR5co-receptors on the surface of target cellscould result in considerable protectionagainst viral infection.

Following submission of this correspon-dence, two articles have appeared using asimilar approach, but with differentsequences of the siRNA for CCR5 (refs.7,8), demonstrating the versatility ofchemokine receptor downregulation bythis method. Furthermore, the vector pSR-siRev, which expresses a hairpin directedagainst the 3′ end of the HIV-1 Rev tran-scripts, blocked >90% of expression of areporter plasmid expressing a Rev–GFPfusion protein, in transiently co-transfect-ed 293 cells (Fig. 1C,D; right panel).Moreover, transient transfection of HeLa-CD4 with the siRev vector, followed byinfection with the CXC chemokine recep-

to 5-bromo-2-deoxyuridine incorporatedsubsequent to a 48-hour labeling period),and most showed a typical senescent mor-phology at one week after replating.Interestingly, sorted cells had, on average,much shorter telomeres, whereas the sort-ing process itself did not influence telom-ere length (Fig. 1). The wide spread intelomere length of the sorted populationmost probably reflects the imprecision ofthe FACS sorting, rather than intracellularvariation. However, the data clearly indi-cate that human fibroblasts that acquire asenescent phenotype at an early stage do sowith short telomeres. It should be men-tioned that lipofuscin accumulation infibroblast cultures is an indicator of oxida-tive stress5, again supporting the associa-tion between oxidative stress and telomereshortening.

It seems highly likely that there is a spec-trum of stress-induced growth arrests.Intense stress causes a variety of cell dam-age, which will activate cell cycle check-points. Most of the damage caused by mildstress will be repaired before it causesarrest, but telomere shortening will beaccelerated, resulting in a cumulative indexof cell damage3. We anticipate that stress-independent arrest driven solely by telom-ere reduction resulting from the end-repli-cation problem may be the exception,which will tend to be seen only in cells thathave uncommonly good defense mecha-nisms, such as some BJ foreskin fibrob-lasts6.

There is growing evidence, from a combi-nation of empirical and theoretical7,8 stud-ies, that cellular ageing is a network process.Thus, it is not a case of this or that alterna-tive but rather of synergy and interactionbetween different mechanisms actingtogether. Stress-induced telomere reduc-tion is a strong candidate to reconcile theviews expressed by Rubin and by Wrightand Shay.

Thomas von Zglinicki,Joanne Petrie,

and Thomas B.L. KirkwoodDepartment of Gerontology,

Institute for Ageing and Health,

Newcastle University,Newcastle upon Tyne

NE4 6BE, UK(t.vonzglinicki@ncl.ac.uk)

1. von Zglinicki, T. Trends Biochem. Sci. 27, 339–344(2002).

2. Serra, V. et al. J. Biol. Chem., in press (2003).3. von Zglinicki, T. et al. Lab. Invest. 80, 1739–1747

(2000).4. Henle, E.S. et al. J. Biol. Chem. 274, 962–971

(1999).5. Sitte, N., Merker, K., Grune, T. & von Zglinicki, T.

Exp. Gerontol. 36, 475–486 (2001).6. Lorenz, M., Saretzki, G., Sitte, N., Metzkow, S. &

von Zglinicki, T. Free Radic. Biol. Med. 31, 824–831(2001).

7. Proctor, C.J. & Kirkwood, T.B.L. Mech. Ageing Dev.123, 351–363 (2002).

8. Sozou, P.D. & Kirkwood, T.B.L. J. Theor. Biol. 213,573–586 (2001).

Figure 1. Telomere Southern blot of MRC-5fibroblast cells. Lanes 1 and 6, λ-Hind marker;lane 2, unsorted cells; lane 3, cells sorted forlarge size and high autofluorescence; lane 4,cells sorted for small size and lowautofluorescence; lane 5, cells collected in thewaste from the sort for large size and highautofluorescence.

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www.nature.com/naturebiotechnology • MARCH 2003 • VOLUME 21 • nature biotechnology

tor 4 (CXCR4)-dependent, T-tropic HIV-1LAI strain inhibited viral replication in>90% of cells, 3 days post-infection (Fig.1B; right panel).

In summary, we argue that it is importantto avoid treatment approaches limited to thetargeting of single sequences for whichescape mutants may develop and that theuse of siRNA directed against suitable cellu-lar products such as CCR5 is preferable.However, it is not unlikely that the com-bined targeting of cellular and viral RNAtranscripts could have a synergistic effect.

H. Jose Arteaga,Department of Medicine and Department of Immunology,Microbiology and Pathology,

Huddinge University Hospital,Karolinska Institutet,

SE- 141 86 Huddinge,(jose.arteaga@crc.ki.se)

Jorma Hinkula,Institute for Infectious

Disease Control,Department of Virology,

Stockholm and Malmö University,Department of Health and Society,

Malmö, Sweden,Iris van Dijk-Härd,

Microbiology and Tumor Biology Center,Karolinska Institutet,

Stockholm, Sweden,M. Sirac Dilber,

Department of Medicine,Huddinge University Hospital,

Britta Wahren,Institute for Infectious

Disease Control,Department of Virology,

Stockholm, Sweden,Birger Christensson,

Department of Immunology,Microbiology and Pathology

Huddinge University Hospital,Abdalla J. Mohamed,

University College of South Stockholm,Sweden, and

C. I. Edvard Smith,Clinical Research Center at Novum,

Karolinska Institutet,SE- 141 86 Huddinge,

Sweden (edvard.smith@crc.ki.se)

1. Lee, N.S. et al. Nat. Biotechnol. 20, 500–505(2002).

2. Jacque, J.M., Triques, K. & Stevenson, M. Nature418, 435–438 (2002).

3. Novina, C.D. et al. Nat. Med. 8, 681–686 (2002).4. Gitlin, L., Karelsky. S. & Andino, R. Nature 418,

430–434 (2002).5. Huang, Y. et al. Nat. Med. 2, 1240–1243 (1996).6. Brummelkamp, T.R., Bernards, R. & Agami, R.

Science 296, 550–553 (2002).7. Martinez, M.A. et al. AIDS 16, 2385–2390 (2002).8. Qin X.-F., An D.S., Chen I.S.Y. & Baltimore D.

Proc. Natl. Acad. Sci. USA 100, 183–188 (2003).

231

Figure 1. Analysis of siRNA-mediated inhibitionof HIV-1. (A) Downregulation of chemokinereceptor CCR5 expression with siRNA. FACSanalysis of GFP-gated U937 cells, aftertransfection with (right panel) or without (leftpanel) a plasmid encoding a CCR5-specificsiRNA (siCCR5; 5′-caggttggaccaagctatg-3′)together with a plasmid encoding GFP. (B)Inhibition of HIV-1 infection and replication intissue culture cells. U937 cells expressing aCCR5-specific siRNA were challenged with aprimary macrophage-tropic isolate of HIV-1.HeLa-CD4 cells expressing Rev-specific siRNA(siRev; 5′-acttactcttgattgtaac-3′) were infectedwith a T-cell-tropic strain of HIV-1. In both cases,a GFP-specific siRNA (siGFP; 5′-gaacggcatcaaggtgaac-3′) was used as control.Virus replication was monitored by detection ofthe HIV-1 p24 antigen. (C) siRNA blocks theexpression of HIV-1 Rev. Plasmids expressingsiRNAs directed against GFP or Rev were co-transfected with constructs encoding GFP orRev-GFP fusion protein into 293 cells.Determination of protein steady-state levels bywestern blot analysis 48 h post-transfectionshows that expression of the Rev-GFP fusionprotein was substantially downregulated by bothsiRNAs. (D) Imaging of cells treated asdescribed in (C) 72 h post-transfection. Mocktransfection (upper panel). The siRNA cloningvector6 was provided by R. Agami, TheNetherlands Cancer Institute.

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