Retrovirology

BioMed Central

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Optimal design and validation of antiviral siRNA for targeting HIV-1
Yuki Naito*1, Kyoko Nohtomi2, Toshinari Onogi2, Rie Uenishi2, Kumiko UiTei1, Kaoru Saigo1 and Yutaka Takebe*2
Address: 1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 1130033, Japan and 2Laboratory of Molecular Virology and Epidemiology, AIDS Research Center, National Institute of Infectious Diseases, 1-23-1
Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan
Email: Yuki Naito* - y-naito@RNAi.jp; Kyoko Nohtomi - notomi@nih.go.jp; Toshinari Onogi - onogit@nih.go.jp;
Rie Uenishi - uenishir@nih.go.jp; Kumiko Ui-Tei - ktei@biochem.s.u-tokyo.ac.jp; Kaoru Saigo - saigo@biochem.s.u-tokyo.ac.jp;
Yutaka Takebe* - takebe@nih.go.jp* Corresponding authors

Published: 8 November 2007
Retrovirology 2007, 4:80

doi:10.1186/1742-4690-4-80

Received: 6 August 2007
Accepted: 8 November 2007

This article is available from: http://www.retrovirology.com/content/4/1/80© 2007 Naito et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent
HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an
exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible
conserved regions was validated. We present several promising antiviral siRNA candidates that
effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic
HIV-1 group M strains.

Findings
RNA interference (RNAi) is now widely used to knockdown gene expression in a sequence-specific manner,
making it a powerful tool not only for studying gene function, but also for therapeutic applications including antiviral treatments [1,2]. The replication of a wide range ofviruses can be successfully inhibited using RNAi with both
short interfering RNA (siRNA) and siRNA expression vectors [3,4]. However, for RNA viruses such as HIV-1,
designing functional siRNAs that target viral sequences isproblematic because of their extraordinarily high genetic
diversity. We analyzed 495 entries of near full-length HIV1 group M sequences available in the Los Alamos HIV
Sequence Database, and selected the highest-possible
conserved target sites for designing optimal antiviral siRNAs. It is known that RNAi-resistant viral mutants emerge
rapidly when targeting viral sequences due to their highmutation rate [5-7]. Since highly conserved sequences arelikely to contain structurally or functionally constrained

elements, our approach is anticipated to resist viral mutational escape.
First, we performed a detailed analysis on the HIV-1
genome to identify highly conserved targets by using 495
near full-length genome sequences of HIV-1 group M(listed in Additional file 1). Every possible 21-mer was
generated from all of the HIV-1 group M sequences, andtheir conservations among the 495 HIV-1 sequences were
exhaustively determined using siVirus engine [8]. We
defined 'conservation' as the percentage of sequence
entries out of the 495 HIV-1 sequences that showed perfect identity (i.e., 21/21 matches) with the cognate 21mer. Since many of the HIV-1 sequence entries lack 5'
untranslated region (5' UTR), the 3' LTR sequence wasused to compensate for the lack of 5' LTR sequences inorder to avoid underestimating conservation in suchregions. For the regions that cannot be compensated for inthis way (depicted in Figure 1A and 1B left panel, colored

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Retrovirology 2007, 4:80

A

Position :1

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1000

2000

3000

4000

5000

6000

7000

8000

9000

9699

100 %

0%

Subtype A

Conservation among
HIV-1 group M

Subtype B
Subtype C
Subtype D
Subtype F,G,H,J,K
CRF01_AE
CRF02_AG
CRF03 -16

URFs
R U5
U3

p2 p1
p17

5′ LTR

p24

tat

vif

p7 p6

gag

vpu
prot

p51 RT

p15

vpr

p31 int

nef

R U5

100%

U3

rev
gp120

gp41

3′ LTR

env

pol

50%

B

300

400

500

600

700

800

900

4600

4700

4800

4900

5000

5100

9000

9100
0%

100 %

0%

Subtype A

Conservation anomg
HIV-1 group M

Subtype B
Subtype C
Subtype D
Subtype F,G,H,J,K
CRF01_AE
CRF02_AG
CRF03 -16

URFs
5′ LTR U3

R

U5

gag

p31 int

3′ LTR U33′ PPTPAS
TCF-1α
Sp1
TAR
NFκB
TATA
poly ACPBS DIS SD

cPPT

CTS
vif

nef

0.5% 0.8%
0.3%
1.5%
2.1%
Conservation among
HIV-1 group M
90 - 100%
80 - 90%
70 - 80%
94.8%

60 - 70%
50 - 60%
0 - 50%Figure 1
Conservationsof siRNA target sequences among HIV-1 group M
Conservations of siRNA target sequences among HIV-1 group M. (A) A total of 4,417,157 siRNA targets were generated from the 495 HIV-1 sequences, and their conservations within the HIV-1 genomes are represented using a color density
plot. The line plot above the color chart represents the highest value in each position. (B) A detailed view of the three conserved regions; 5' LTR, the cPPT/CTS in the integrase gene, and 3' PPT. 'Position' indicates the 5'-most position of each 21mer. The landmarks of the HIV-1 genome are adjusted to align at the center of the siRNAs by shifting 10 bp to the left. (C) Pie
chart indicating the percentage of the 4,417,157 siRNA target sites at each conservation level.

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Retrovirology 2007, 4:80

black), conservation was calculated by considering only
the HIV-1 sequences that contain the corresponding
regions. The result revealed that HIV-1 genomes are notconserved for consecutive 21 bp for the most part, resulting in the poor conservation of many of the 21-mers overthe HIV-1 sequences (Figure 1A, colored blue). As shownin Figure 1C, only 5.2% of the possible 21-mers are >50%
conserved. Furthermore, highly (>70%) conserved 21mers constitute only 1.6% of all 21-mers. It is of note thatmany of the published anti-HIV-1 siRNA sequences do
not fall into this 'highly conserved' category (Additional
file 2 and [9]). From these results, we anticipate that mostof the possible siRNAs are not suitable for the efficient targeting of HIV-1.
However, our analysis has identified several distinct
regions that are highly conserved in the HIV-1 genome(Figure 1B). Such regions include the regulatory domains
responsible for the viral gene expression, such as the TATA
sequence and polyadenylation signal (AAUAAA). In addition, several regions essential for the regulation of viral
replication were also highly conserved, including theprimer activation signal (PAS)[10], primer binding site(PBS), packaging signal (Ψ), central polypurine tract(cPPT), central termination sequence (CTS), and 3' polypurine tract (3' PPT). All of these highly conserved
sequences are constrained at the nucleotide sequence level
or by their RNA secondary structure in order to execute
their functions. In contrast, regions constrained by aminoacid sequences were not necessarily conserved at thenucleotide sequence level due to the wobbling of the thirdbase in the codon (data not shown). siRNAs targeting thehighly conserved regions are expected to overwhelm thehigh level of sequence diversity of the HIV-1 genome, andalso to reduce the chances of viral mutational escapes.
Total of 216 highly conserved (>70%) siRNA targets identified in this study are listed in Additional file 3. In mammalian RNAi, the efficacy of each siRNA varies markedly
depending on its sequence. According to our guidelines
for the selection of effective siRNAs [11,12], 31 out of 216
siRNAs were predicted to be functional. Similarly, 30 and44 siRNAs are functional according to the algorithms
reported by Reynolds et al. [13], and Amarzguioui et al.
[14], respectively (Additional file 3). This suggests that
only a limited fraction of 21-mers is best suited for use asfunctional antiviral siRNAs.
For the functional validation, 23 siRNAs from Additional
file 3, and 18 additional siRNAs targeting moderatelyconserved regions were selected based on the following
criteria: (I) predicted to be functional by the algorithm of
Ui-Tei et al. [11,12], and (II) the sequence has perfectidentity with pNL4-3 (GenBank M19921). The 41 siRNAsequences selected and their target sites are detailed in

http://www.retrovirology.com/content/4/1/80

Additional file 4. We first tested the efficacy of each siRNAusing target mRNA cleavage assay (Additional file 5 and[15]). Briefly, a vector expressing reporter mRNA that contains the siRNA target site was cotransfected into HeLa
cells with the corresponding siRNA, and the mRNA cleavage activity of the siRNA was evaluated by measuring thequantity of surviving mRNA using real-time RT-PCR. This
assay allows us to directly monitor the sequence-dependent potency of siRNA itself, without being affected by thedifferences in target gene expression level or target secondary structures. The result showed that 39 out of the 41 siRNAs gave >60% silencing at 5 nM (Figure 2, rightmost
panel). si4794 and si4888 were not functional, probably
due to the long consecutive Gs in si4794 and internal palindromes (AAAAUUUU) in si4888 [11,13].
Next, siRNAs were evaluated for their antiviral efficacy
against three evolutionary-distant groups of HIV-1: subtypes B and B' (Thailand variant of subtype B [16]); subtype C; and CRF01_AE. Each siRNA was cotransfected into
HeLa cells at 5 nM with one of the four infectious molecular clones: pNL4-3 (subtype B); 95MM-yIDU106 (subtype B'); 93IN101 (subtype C); or 93JP-NH1 (CRF01_AE).
Culture supernatants were collected 48 h after transfection
and the viral reverse transcriptase activity was measured(Additional file 5 and [17]). The results show that 26 of
the 41 siRNAs effectively inhibited viral replication of all
four strains by >80% (Figure 2, marked with red or orange
circles). Of the remaining 15 siRNAs, 13 of them (except
si4794/4888) were shown to be functional in the target
mRNA cleavage assay, and 12 of them (except si690/
4794/4888) inhibited the replication of at least one viralstrain by >80%, indicating that the designed siRNAs havethe potential to induce RNAi. In several viral strains,
nucleotide substitutions in their target sites essentiallyabolished the inhibition of viral replication (Figure 2,
blue bars with arrowheads). However, mismatches near
the ends of the target sites (see Additional file 6) did notnecessarily abolish the siRNA efficacy (Figure 2, blue bars
with asterisks). si689 and si690 did not inhibit viral replication even though these siRNAs perfectly matched totheir target sites (confirmed by DNA sequencing of theinfectious molecular clones). This is probably due to thestable secondary structure at the si689-690 target sites inboth BMH (branched multiple hairpin) conformationand LDI (long distance interaction) conformation of theHIV-1 leader RNA [18] (see Additional file 4). It should be
noted that the efficacy of si575 differed when targeting
pNL4-3 and 93IN101. One possible explanation for thisis the secondary structure differences among HIV-1 subtypes, which may alter the accessibility of the si575 target
site.
The approach described here enabled us to select highly
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DIS
TCF-1αSp1 TAR PAS
NFκB TATA poly A PBS SD

Conservation
siRNA
U3
R
U5
5′ LTR

gag

pol
vif

vpr

HIV-1 genometat

vpu rev

env

nef

3′ LTR

si505 77 %si509 84 %si510 84 %si512 85 %si515 85 %si521 94 %si554 68 %si575 68 %si689 88 %si690 69 %si764 88 %si770 88 %si1490 56 %si1817 82 %si2075 70 %si2329 77 %si2330 77 %si2333 77 %si2485 66 %si2486 66 %si3000 57 %si3005 60 %si3006 61 %si3011 47 %si4175 76 %si4373 49 %si4378 49 %si4652 64 %si4746 66 %si4750 74 %si4751 74 %si4753 71 %si4794 80 %si4806 72 %si4809 84 %si4840 60 %si4888 80 %si4960 69 %si4961 70 %si7653 67 %si7658 65 %siControl
vector only
no transfection

Overall efficacy
Inhibited all 4 strains by >90%
Inhibited all 4 strains by >80%

Relative RT activity (%)
0100

Relative RT activity (%)
0100

Relative RT activity (%)
0100

Relative RT activity (%)
0100

Relative target mRNA quantity (%)
0100

Target mRNA
cleavage assay
Subtype B
pNL4-3(M19921)Conservation among
HIV-1 group M
90 - 100%
80 - 90%
70 - 80%
60 - 70%
50 - 60%
0 - 50%

Subtype B′
95MM-yIDU106

Subtype C
93IN101(AB023804)

CRF01_AE
93JP-NH1(AB052995)

siRNA vs. target sequence
perfect matches
imperfect matches

Figure 2 of 41 siRNAs
Validation
Validation of 41 siRNAs. The antiviral efficacy of each siRNA was tested against four HIV-1 infectious molecular clones:
pNL4-2 (subtype B); 95MM-yIDU106 (subtype B'); 93IN101 (subtype C); or 93JP-NH1 (CRF01_AE). The potency of eachsiRNA was tested using the target mRNA cleavage assay (rightmost panel). The ability of each siRNA to cleave its target wasevaluated by the target mRNA cleavage assay.

rate. The highly effective siRNAs (>90% inhibition) withmaximal conservation (>70%) identified in our study
include si521 (poly A site; 94% conservation), si764/770(Ψ; 88%), si510 (TAR/poly A; 84%), si2075 (ribosomal
slip site; 70%), si2329/2330/2333 (protease region;77%), and si4750/4751/4753 (integrase region;
71–74%). These sites are found mostly in the 5' LTR, protease, and integrase regions (Figure 2). However, theextraordinarily high genetic diversity of HIV-1 obviously
prevents us from designing a single siRNA that can nullify
all HIV-1 strains currently circulating worldwide (Additional file 7). One possible approach is to combine multiple siRNAs targeting different conserved regions [19,20].
The siRNAs selected and validated in this study have thepotential to target >99% of HIV-1 strains by combining
only two siRNAs (Additional file 7), and also considered

to resist viral mutational escape. Our approach is expected
to be highly applicable to therapeutic intervention forother pathogens of public health importance, including
HCV, influenza virus, and SARS coronavirus, that are
known to show high genetic diversity.

Competing interests
The author(s) declare that they have no competing interests.

Authors' contributions
YN performed the computational analyses and the target
mRNA cleavage assays, participated in the design of thestudy, and drafted the manuscript. KN and TO performed
the viral replication assays. RU analyzed the data. KU-T
participated in the target mRNA cleavage assays, and wasPage 4 of 5
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Retrovirology 2007, 4:80

involved in critically revising the manuscript. KS and YT
supervised the entire study and wrote the manuscript.

Additional material

http://www.retrovirology.com/content/4/1/80

Acknowledgements
This study was supported in part by grants from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (to YN, KU-T, KS, andYT), the Ministry of Health, Labour and Welfare of Japan (to YT), and theJapan Health Sciences Foundation (to YT).

Additional file 1

References

The list of 495 near full-length genome sequences of HIV-1 group M.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S1.pdf]

1.
2.
3.

Additional file 2
The list of published siRNA/shRNAs targeting HIV-1.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S2.pdf]

Additional file 3
The list of highly conserved siRNA targets identified in this study.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S3.pdf]

4.
5.
6.

7.
8.

Additional file 4
The siRNA sequences and their target sites. The sequences of 41 siRNAs
and their target sites are shown. The siRNA numbers indicate the nucleotide position in HXB2 (GenBank K03455). The conservation level ofeach siRNA in HIV-1 group M sequence is depicted in color chart at therightmost column. BMH (branched multiple hairpin) and LDI (long distance interaction) conformations of the HIV-1 leader RNA and siRNAs
targeting them are shown.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S4.pdf]

9.

10.
11.

12.

Additional file 5
Supplementary materials and methods.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S5.pdf]

13.

Additional file 6

15.

Target sites of the 41 siRNAs used in this study. Sequence alignment of
the target site from the four HIV-1 infectious molecular clones: pNL4-2(subtype B); 95MM-yIDU106 (subtype B'); 93IN101 (subtype C); or
93JP-NH1 (CRF01_AE).
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S6.pdf]

14.

16.

17.

Additional file 7
Coverage of HIV-1 group M by single siRNA or two siRNAs. (A) Coverage of HIV-1 group M by 41 siRNAs used in this study. (B) Coverage ofHIV-1 group M by combining two siRNAs from above. Coverage was calculated by considering only the HIV-1 sequences which contain the corresponding regions.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17424690-4-80-S7.pdf]

18.
19.

20.

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