HIV-1 Tat RNA silencing suppressor activity is
conserved across kingdoms and counteracts
translational repression of HIV-1
Shuiming Qiana, Xuehua Zhongb, Lianbo Yuc, Biao Dingb, Peter de Haand, and Kathleen Boris-Lawriea,1
aCenter for Retrovirus Research and Department of Veterinary Biosciences, Molecular, Cellular and Developmental Biology Graduate Program,
Comprehensive Cancer Center,bDepartment of Plant Cellular and Molecular Biology and Plant Biotechnology Center, andcCenter for Biostatistics,
Ohio State University, Columbus, OH 43210; anddPhytovation B. V., 2333 AL Leiden, The Netherlands
Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved November 24, 2008 (received for review
July 14, 2008)
The RNA silencing pathway is an intracellular innate response to
virus infections and retro-transposons. Many plant viruses counter
this host restriction by RNA silencing suppressor (RSS) activity of a
double-stranded RNA-binding protein, e.g., tomato bushy stunt
virus P19. Here, we demonstrate P19 and HIV-1 Tat function across
the plant and animal kingdoms and suppress a common step in
RNA silencing that is downstream of small RNA maturation. Our
experiments reveal that RNA silencing in HIV-1 infected human
cells severely attenuates the translational output of the unspliced
HIV-1 gag mRNA, and possibly all HIV-1 transcripts. The attenua-
tion in gag mRNA translation is exacerbated by K51A substitution
in the Tat double-stranded RNA-binding domain. Tat, plant virus
RSS, or Dicer downregulation rescues robust gag translation and
bolsters HIV-1 virion production. The reversal of HIV-1 translation
repression by plant RSS supports the recent finding in Arabidopsis
that plant miRNAs operate by translational inhibition. Our results
identify common features between RNA silencing suppression of
plant and animal viruses. We suggest that RNA silencing-mediated
set-point in a newly HIV-1-infected patient.
retrovirus gag RNA ? restriction of HIV-1 replication
tions and genetic damage by retro-transposons. Most plant
viruses encode an RNA silencing suppressor (RSS) that coun-
teracts this restriction and drives pathogenesis (reviewed in
refs.1, 2). The importance of RNA silencing suppression
in animal retrovirus infection remains controversial (reviewed in
refs. 3, 4). Antiviral RNA silencing is initiated when virus-
specific double-stranded RNA appears in the cytoplasm and is
processed by Dicer endonuclease into 21–25 nucleotide miRNA/
miRNA* (guide/passenger) duplexes (5–8). The guide strand
and complementary target mRNA is incorporated into RNA-
induced silencing complexes (RISC) (9, 10), which coalesce as
processing bodies that are sites of target mRNA degradation or
translation repression (11, 12).
Physiological expression of HIV-1-encoded miRNA remains
controversial (13–16). Evidence that RNA silencing is important
for HIV-1 includes the observation that cell-encoded miRNAs
dampen virus replication in activated T lymphocytes (17) and
contribute to viral latency in resting T lymphocytes (18). HIV-1
restriction of RNA silencing has been attributed to the viral Tat
transcriptional transactivator (16). Benasser, et al. (16) deter-
mined that Tat RSS activity was genetically separable from Tat
transcriptional activity but segregated with the arginine-rich
double-stranded RNA-binding domain (16). A similar RNA
binding domain is conserved in plant virus RSS and confers
interaction with miRNA duplexes in a sequence nonspecific
manner that blocks programming of RISC for RNA silencing
(19–21). For Tat, tomato bushy stunt virus (TBSV) P19 and
NA silencing is a eukaryotic posttranscriptional gene regu-
lation mechanism and innate defense to quell virus infec-
other Tombusvirus RSS, mutation of this domain eliminates
RNA silencing suppression (16, 22), which posits a common
mechanism of activity. The outcome of P19 mutation is reduced
TBSV RNA, which culminates in impaired virus propagation
and attenuation of disease pathogenesis (19–21). To investigate
we compared the activity of Tat and P19 in plant protoplasts and
Results demonstrate that HIV-1 Tat and TBSV P19 function
equivalently in plant protoplasts and animal cells to suppress
RNA silencing at a step downstream of the double-stranded
RNA (dsRNA) processing, most likely by sequestering mature
si/miRNAs. We present evidence that RNA silencing does not
affect HIV-1 RNA degradation and instead restricts HIV-1
mRNA translation. HIV-1 Tat and TBSV P19 function equiv-
alently to protect against RNA silencing-mediated suppression
of HIV-1 translation.
HIV-1 Tat Suppresses RNA Silencing in Plant Cells Downstream of the
Maturation Step of dsRNA Duplexes. We used Nicotiana benthami-
ana protoplasts to investigate whether or not Tat RSS activity is
maintained in the plant kingdom similar to the activity of
influenza A virus NS1 RSS (23). Tat was expressed in plant cells
downstream of the strong and constitutive 35S promoter derived
from cauliflower mosaic virus. The RSS activity of Tat was
compared to that of plant viral RSSs by coelectroporation with
GFP reporter plasmid and 700 nt GFP-specific dsRNAs that
downregulate GFP expression (24). Representative images from
5 independent triplicate transfection assays (Fig. 1A) demon-
strated that HIV-1 Tat (Tat), TBSV P19 (P19) and tobacco etch
virus helper component-protease (HC-Pro) restored GFP flu-
orescence compared to the empty vector control. Quantification
of GFP fluorescence in the bulk cultures revealed that the RSS
activity was statistically significant (p value ? 0.0001) (Fig. 1B).
Northern blot analysis using a sense strand GFP-specific probe
revealed low but detectable levels of the 700 nt GFP effector
RNA in the cells electroporated with the empty vector (Ve),
HC-Pro, P19, or Tat. By comparison, Turnip crinkle virus coat
protein (CP) caused the accumulation of effector dsRNA,
consistent with its role in preventing processing of dsRNAs (24,
L.Y., B.D., and P.d.H. contributed new reagents/analytic tools; S.Q., X.Z., L.Y., B.D., P.d.H.,
and K.B.-L. analyzed data; and S.Q., P.d.H., and K.B.-L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed at: 1925 Coffey Road, Columbus, OH
43210. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0806822106 PNAS ?
January 13, 2009 ?
vol. 106 ?
no. 2 ?
25). These results confirm the published observation that HC-
Pro and P19 do not affect the processing of long dsRNAs into
mature siRNAs (19, 21, 22, 26, 27). The results indicated that Tat
RSS activity is conserved across kingdoms and functions down-
stream of dsRNA duplexes.
TBSV P19 Suppresses RNA Silencing in Animal Cells Downstream of
miRNA Maturation. We next compared the RSS activity of Tat and
plant virus RSS in animal cells. The miR30-based luciferase
reporter system (28) contains 8 copies of the mir30 target
sequence (pCMV-Luc-8(x)-miR30) (Fig. 2A). Co-transfection of
HeLaT4 cells with the primary precursor miR30 (pri-premiR30)
expression plasmid pCMV-miR30 and the pCMV-Luc-8(x)-
miR30 reporter robustly silenced Luc activity (compare tan bars,
reduction by factor of 5, in Fig. 2B). Cotransfection with P19
suppressed miR30 activity by a factor of 7 (compare blue bar in
Fig. 2B). By comparison, downregulation of Dicer by Dicer-
specific siRNAs likewise eliminated miR30 activity (dicer
siRNA, blue bars, Fig. 2C) as compared to treatment with
scrambled siRNAs (sc, tan bars, Fig. 2C). Real-time RT PCR to
quantify steady state mRNA levels showed that the copy number
of luc mRNA did not change in response to P19 expression (data
We used the mirR30 assay to compare suppressor activity of
P19 with HIV-1 Tat, vaccinia virus E3L, influenza A virus NS1,
and adenovirus VA1. miR30 reduced Luc activity from pCMV-
luc8(x)-miR30 by a factor of 5 (blue bars, treatment group 1 and
2, Fig. 2D). Statistically significant partial restoration of Luc
activity was observed for E3L, NS1, P19, and Tat and VA1
completely restored Luc activity (blue bars, Fig. 2D, P ? 0.05).
Real-time RT PCR showed that the steady state level of luc
mRNA was similar among all samples (gray bars, Fig. 2D) and
indicated that the increased Luc activity was attributable to
reversal of translation repression.
Northern blotting with the antisense miR30 RNA probe on
71 nt premiR30 and 22 nt mature miR30 (Fig. 2E). Low but
detectable endogenous miR30 was detectable (treatment group
1 and 2) and abundant premiR30 and mature miR30 was
observed upon transfection of pCMV-miR30 (treatment group
3). Similar levels of premiR30 were detected in E3L, NS1, Tat,
and P19 (treatment groups 4–7) and a reduction was observed
in response to VA1 (treatment group 8). The ratio of premiR30
to mature miR30 was similar between empty vector control
(-suppressor) and E3L (1.2 and 1.4, respectively). The ratio was
reduced in response to NS1, Tat, and P19 (range 0.75 to 0.9),
indicating no significant block in the processing of premiR30 to
mature miR30. VA1 treatment resulted in baseline levels of
premiR30 and mature miR30 and the appearance of a smaller
species of premiR30 (treatment group 8) and supports the
observation that VA1 serves as a decoy substrate for exportin 5,
Dicer, and RISC (28, 29). The accumulation of premiR30 and
miR30 in the Tat treatment group indicated that, similar to the
plant viral RSS P19, Tat does not disrupt the maturation of
dsRNA, but reduces the efficiency of a downstream step in the
RNA silencing pathway.
determine whether the RNA silencing pathway inhibits gag
mRNA translation, Dicer was downregulated in HeLa T4 cells
by transfection with Dicer-specific siRNAs (30). Northern and
Western blotting detected significant Dicer mRNA downregu-
lation by 24 h and 48 h posttransfection (Fig. 3A), and Dicer
protein was downregulated in comparison to treatment with
non-silencing scrambled control (sc) (Fig. 3B, 48 h sample).
These cells were transfected with HIV-1NL4–3and virion pro-
duction was measured at 24 h, 36 h, and 48 h post transfection.
Dicer downregulation increased virion production 3- to 4-fold
(Fig. 3C). Pulse labeling and Gag IP showed that the rate of Gag
protein synthesis was increased significantly by Dicer downregu-
lation (Fig. 3D, P ? 0.05). By comparison, gapdh translation was
not affected, as determined by GAPDH immunoprecipitation
(IP). Analysis of CEMx174 lymphocytes infected with HIV-
1NL4–3yielded a similar increase in Gag protein synthesis upon
Dicer knockdown (data not shown). Meanwhile, TCA-
precipitable counts measuring [35S]cysteine/methionine incor-
poration were similar between the samples treated with the
Dicer-specific siRNAs (3.3 ? 106? 8 ? 105cpm) and the sc
siRNAs (3 ? 106? 6 ? 105cpm). The results demonstrate that
inhibited processing of long dsRNA. (A) N. benthamiana culture cells proto-
plasts were transfected with GFP reporter plasmid, long GFP-specific dsRNA
and the empty vector (Ve) (1) or indicated RSS expression plasmid (2, 3, 4) and
monitored for GFP activity 3 days post electroporation. P19, Tat, and HC-Pro
restored GFP expression. (B) Quantitative analysis of bulk cultures is summa-
rized from 5 replicate experiments. Expression of indicated RSS increased the
with probe complementary to the antisense strand of gfp effector RNA. HC,
P19, and Tat do not induce accumulation of effector RNA. CP induced accu-
mulation of effector RNA. Lower panel is the gel stained with ethidium
Tat exhibits RSS activity in plant cells that is not attributable to
www.pnas.org?cgi?doi?10.1073?pnas.0806822106Qian et al.
HIV-1 replication in human cells is attenuated by RNA silencing
and that downregulation of the RNA silencing pathway signif-
icantly increases de novo Gag protein synthesis independently of
general effects on cellular protein synthesis.
Is Suppressed Equivalently by Tat and P19. We next evaluated
whether Tat and P19 RSS rescue gag mRNA translation. Flag-
tagged P19 was expressed in HelaT4 cells and P19 was consec-
utively verified by Western blotting at 12, 24, 36, and 48 h (Fig.
4A). At the 24 h time point, the cells were transfected with
HIV-1NL4–3and virion levels were measured by Gag p24 ELISA
on supernatant medium. P19 produced a significant increase in
virion level at each time point (Fig. 4B) but did not alter the copy
number of gag mRNA (Fig. 4C). Real-time RT PCR results
revealed no change in gapdh or c-myc mRNA in response to P19
or HIV-1 expression (Fig. 4C). At the 48 h time point, the rate
of Gag protein synthesis was assessed by pulse labeling and Gag
IP experiments. The increase in virion level by P19 expression
next compared virion production between HIV-1NL4–3and the
derivative provirus HIV-1/RSS that contains the K51A mutation
in the tat gene, which eliminates RSS activity but retains
transcriptional transactivation activity (16). Gag ELISA results
showed a significant reduction in virion production by a factor
of ten in response to K51A (tan bars, Fig. 4E). Expression of P19
was sufficient to complement the defect in virion production
from HIV-1/RSS and bolstered virion production from HIV-1
(blue bars, Fig. 4E). Real-time RT PCR data showed no
difference in the copy number of HIV-1/RSS gag mRNA nor
gapdh and c-myc RNA loading controls in response to P19 (Fig.
4F). These levels were also were unchanged in relation to HIV-1
(Fig. 4C). Pulse labeling and Gag IP demonstrated that HIV-
1/RSS leads to a significant reduction in the rate of Gag protein
activity is reduced by P19. (C) Luciferase activity from HelaT4 cells at 48 h posttransfection with indicated siRNA and plasmids determined that miR30 activity is
reduced by Dicer downregulation. Firefly Luc (F-Luc) activity from equivalent protein preparations. (D) Luciferase activity and luciferase RNA levels in 293 cells
transfected with indicated RSS and miR30 reporter plasmid and miR30 expression plasmid. Equivalent protein preparations were assays for Firefly Luc (F-Luc)
activity. (E) Northern blot analysis with antisense miR30-specific RNA probe that detected precursor miR30 (premiR30) and mature miR30. Small RNAs were
enriched and separated by 15% urea-PAGE. Lower is gel stained with ethidium bromide.
Tat suppresses miR30 function but does not block miR30 processing in human cells. (A) The luc RNA expressed from reporter plasmid pCMV-Luc-8(x)-
Qian et al. PNAS ?
January 13, 2009 ?
vol. 106 ?
no. 2 ?
synthesis (Fig. 4G Right) and that P19 expression significantly
increased the rate of Gag protein synthesis (Fig. 4G Left). As
summarized in Fig. 5, these IP data indicated that the K51A
mutation reduced Gag protein synthesis by a factor of 3 (tan
squares, HIV-1 ? pCAM; tan circles, HIV-1/RSS ? pCAM).
Similar to the Gag ELISA results, P19 expression bolstered Gag
to blue squares and circles). Measurement of TCA-precipitable
counts revealed similar levels of [35S]cysteine/methionine incor-
poration in the P19 treated cells (4.2 ? 106? 1 ? 106) and the
pCAM control (3.2 ? 106? 3.8 ? 105cpm). Statistical analysis
by Dunnett’s method determined no significant difference in de
novo cellular protein production (P ? 0.2273). Likewise, met-
abolic labeling with
difference in total cellular RNA synthesis during the 1 h labeling
period (P19 treatment: 5.2 ? 105? 1.8 ? 104and the pCAM
control: 6 ? 105? 1.3 ? 104cpm). The results demonstrated that
Tat and P19 increase the translatability of HIV-1 gag mRNA.
3H–uridine demonstrated no significant
Our data indicate that the production of HIV-1 Gag protein, and
thereby production of virus particles, is restricted by RNA
silencing, which confirms the results of Triboulet, et al. (17) and
de Vries, et al.(31). Viral strategies to counter RNA silencing
include RNA protection, silencing suppression, evasion, modu-
lation, and adaptation (4). Our results indicate that HIV-1 Tat
confers RNA silencing suppression, which counters host-
mediated inhibition of HIV-1 translation that is attributable to
cell-encoded miRNAs (17, 18). The observation that virion
production from an HIV-1 strain lacking RSS activity is severely
attenuated indicates that Tat RSS promotes acute HIV-1 infec-
tion. Heterologous plant virus P19 RSS is sufficient to overcome
translational RNA silencing and provides a mechanistic expla-
nation for the observation that Ebola V35 can replace Tat RSS
activity (32). HIV-1 Tat and TBSV P19 also cosegregate in their
loss of activity by point mutation of the dsRNA-binding domain.
Our results demonstrate that the RSS activity of Tat and P19
is modest in comparison to adenovirus VA1 RNA. While modest
in our system, P19 RSS activity is paramount to prevent host
attenuation of TBSV infection and pathogenesis (reviewed in
refs. 1, 2). Given our observation that P19 expression does not
reduce global cellular translation and that Tat RSS activity was
not recapitulated in a shRNA reporter assay (13), viral RSS
activity is not a global phenomenon and is targeted to select
The evidence demonstrating reversal of HIV-1 translation
repression by a plant RSS support the recent finding in Arabi-
dopsis that plant miRNAs operate by translational inhibition
(33). Our IP results, quantitative RNA analyses in human cells,
and functional analysis in N. benthamiana cells indicate that the
underlying mechanism of RNA silencing suppression by human
Tat and P19 do not reduce maturation of dsRNA duplexes
indicates a common mechanism to prevent guide strand pro-
gramming of RISC, and agrees with research by Lin and Cullen
(13) that Tat does not block premiRNA processing by Dicer. The
results indicate that influenza A virus NS1 RSS also relies on
si/miRNA-binding, whereas vaccinia virus E3L functions up-
stream and relies on binding to long dsRNAs, thereby preventing
their processing into mature si/miRNAs.
Materials and Methods
Plasmids. HIV/RSS was constructed by PCR-based site-directed mutagenesis of
pNL4–3 to introduce K51A (16). P19 eukaryotic expression plasmid
(34) with primers containing terminal ClaI and BamHI restriction sites [sup-
porting information (SI) Table S1]. Plant expression plasmid pRTL2Tat was
constructed by SacI and BamHI restriction of pRTL2 (35) and pCMV-Tat-1 (36)
(a gift of Andrew Rice) isolation from agarose and ligation. All constructions
were verified by sequencing. Previously described plasmids are pRTL2:smGFP,
HC-Pro, and CP (24), VA1 (28); NS1 and E3L (23, 32). The pCMV-Luc-8(x)-
miR30(p) and pCMV-miR30 was a gift of Bryan Cullen (28).
kidney cells and HeLaT4 (CD4?HeLa) cells were cultured in DMEM with 10%
medium 1640/10% FBS. Transient transfections of 1 ? 105HelaT4 cells in
triplicate in 6-well plates featured 1 ?g of p19FL or empty pCAM vector and
0.2 ?g of pGL3 firefly luciferase transfection control in Fugene6. After 2 days,
transfectants were subcultured and transfected with 1 ?g of HIV-1NL4–3or
HIV/RSS proviral plasmid. Culture media were harvested for HIV-1 Gag P24
P-40 lysis buffer (20 mM Tris?HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, and 1%
Nonidet P-40). Ten ?l of lysate was assayed in Luciferase reagent (Promega)
and relative light units were used to standardize minor differences in trans-
Virus stocks for infections were generated by transfection of 1 ? 106
HEK293 cells with HIV-1NL4–3or HIV-1/RSS and infection of stock culture of
CEMx174 T cells. After 48 h, cells were harvested on Ficoll Hypaque and
cultured with naïve CEMx174 cells at a 1:10 ratio. Fluorescence-activated cell
sorting (FACS) analysis of intra-cellular Gag expression was performed with
anti-p24 KC57-FITC antibody (Beckman-Coulter) and Fix and Perm (CALTAG).
PI units x106
30 9012030 60 90 120
53.5 122.5 10511922.7 363157.6
121116.515 1312 1015
HIV-1 Gag (pg/ml x103)
Hours post-siRNA treatment
structural protein in human cells. (A) Northern blot of total cellular RNA from
HelaT4 cells transfected with dicer siRNA (dicer) or scrambled siRNA (sc)
determined downregulation of dicer mRNA at both 24 and 48 h posttreat-
ment. (B) Immunoblot with Dicer and Grp78 antiserum determined down-
regulation of Dicer protein at 48 h. (C) Down-regulation of Dicer enhanced
Gag production in HeLaT4 cells transfected with HIV-1NL4–3. Gag ELISA was
performed on cell-free medium from 3 independent transfections. Gag levels
were normalized to cotransfected Luciferase. (D) IP assay determined that
Dicer downregulation increases rate of synthesis of Gag P55 but not GAPDH.
Down-regulation of Dicer enhances the production of the HIV-1
www.pnas.org?cgi?doi?10.1073?pnas.0806822106 Qian et al.
Protein Analysis. Cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 0.1% SDS,
1% Triton-X, 150 mM NaCl, 1% deoxycholic acid, 2 mM PMSF) and 50 ?g
protein was subjected to SDS/PAGE and transferred to nitrocellulose mem-
brane. Immunoblotting antibodies detected Flag and ?-actin (Abcam). Visu-
alization was performed with Luminol reagent (Santa Cruz Biotechnology).
The IP and TCA precipitation protocols are described previously (37).
beta-actin antiserum determined expression of P19-Flag fusion protein at indicated times posttransfection. (B) Gag production from HeLaT4 cells transfected
or empty vector (pCAM). Gag levels were normalized to cotransfected Luciferase. (C) P19 expression did not change steady state levels of HIV-1 gag, c-myc, or
gapdh transcripts. Evaluation of total cellular RNA preparations from 3 replicate transfections by reverse transcription and real-time PCR with HIV-1 gag, c-myc,
and gapdh specific primers. (D) IP assay demonstrated that P19 expression increases rate of synthesis of HIV-1 Gag P55 but not GAPDH. (E) Gag production from
did not change steady state levels of HIV/RSS gag, c-myc, or gapdh transcripts. Evaluation of total cellular RNA preparations from 3 replicate transfections by
of HIV/RSS Gag P55 but not GAPDH.
Expression of P19 enhanced production from HIV-1 and HIV/RSS. (A) Immunoblot of HelaT4 cells transfected with indicated plasmids with Flag or
Qian et al. PNAS ?
January 13, 2009 ?
vol. 106 ?
no. 2 ?
Statistical Analysis. One-way analysis of variance model was applied to log Download full-text
base 2-transformed data. Dunnett’s method analyzed the mean difference
among multiple groups.
N. benthamiana Protoplast Assays. Isolation of N. benthamiana cultured cell
protoplasts and electroporation are described in detail by Qi, et al. (24).
presence or absence of 5 ?g of double stranded GFP effector RNA by electro-
poration. The assays were performed in triplicate wells of 6-well plates with 5
?g of suppressor plasmid. At 3 days postelectroporation, GFP fluorescence
intensity was measured by CytofluorTM 2350 Fluorescence Measurement
System with the plate reader software (Millipore).
Northern Blot Analyses. For detection of plant GFP effector RNA, 5 ?g of total
RNA was separated on 5% PAGE with 8M urea and 0.5X TBE. For detection of
separated by 15% PAGE/8M urea/0.5X TBE. The small RNA preparations were
isolated from total cellular RNA with differential ethanol precipitation using
miRvana protocol (Ambion). The RNAs were transferred to Hybond-XL nylon
membrane (Amersham Biosciences) and subjected to UV crosslinking. The
(Ambion) overnight, washed twice in 2? SSC/0.1% SDS for 15 min and twice
in 0.2? SSC/0.1% SDS for 15 min. Hybridization and washing were performed
at 65 °C and at 37 °C for detection of large RNA species and small RNA species,
respectively, and visualized by PhosphorImaging. To generate32P-UTP-
labeled antisense miR30 probe, 1 ?g of PCR product containing T7 promoter
was in vitro transcribed by T7 RNA polymerase.
Real-Time RT PCR. The RT-PCR protocol (37) used random hexamers and
Sensiscript reverse transcriptase (Qiagen) and 100 ng RNA. Ten percent of the
actin (Table S1) in Lightcycler (Roche).
ACKNOWLEDGMENTS. We thank members of the K.B.-L. laboratory for crit-
This work was supported by National Institutes of Health National Cancer
Institute R01CA108882; P01CA16058; P30CA100730.
1. Li WX, Ding SW (2001) Viral suppressors of RNA silencing. Curr Opin Biotechnol
2. Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–
3. Cullen BR (2006) Is RNA interference involved in intrinsic antiviral immunity in mam-
mals? Nat Immunol 7:563–567.
4. Yeung ML, Benkirane M, Jeang KT (2007) Small non-coding RNAs, mammalian cells,
and viruses: Regulatory interactions? Retrovirology 4:74.
5. Sullivan CS, Ganem D (2005) MicroRNAs and viral infection. Mol Cell 20:3–7.
6. Voinnet O (2005) Induction and suppression of RNA silencing: Insights from viral
infections. Nat Rev Genet 6:206–220.
7. Cullen, BR (2006) Viruses and microRNAs Nat Genet 38 Suppl:S25–S30.
8. Yeung ML, Bennasser Y, Jeang KT (2007) miRNAs in the biology of cancers and viral
infections. Curr Med Chem 14:191–197.
9. Zamore PD, Haley B (2005) Ribo-gnome: The big world of small RNAs. Science
10. Bartel DP (2004) MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell
11. Sen GL, Blau HM (2005) Argonaute 2/RISC resides in sites of mammalian mRNA decay
known as cytoplasmic bodies. Nat Cell Biol 7:633–636.
12. Jagannath A, Wood MJ (2008) Localization of Double-stranded siRNA to Cytoplasmic
13. Lin J, Cullen BR (2007) Analysis of the interaction of primate retroviruses with the
human RNA interference machinery. J Virol 81:12218–12226.
14. Pfeffer S, et al. (2005) Identification of microRNAs of the herpesvirus family. Nat
15. Omoto S, et al. (2004) HIV-1 nef suppression by virally encoded microRNA. Retrovirol-
16. Bennasser Y, Le SY, Benkirane M, Jeang KT (2005) Evidence that HIV-1 encodes an
siRNA and a suppressor of RNA silencing. Immunity 22:607–619.
17. Triboulet R, et al. (2007) Suppression of microRNA-silencing pathway by HIV-1 during
virus replication. Science 315:1579–1582.
CD4(?) T lymphocytes. Nat Med 13:1241–1247.
19. Vargason JM, Szittya G, Burgyan J, Tanaka Hall TM (2003) Size selective recognition of
siRNA by an RNA silencing suppressor. Cell 115:799–811.
20. Omarov RT, Ciomperlik JJ, Scholthof HB (2007) RNAi-associated ssRNA-specific ribo-
nucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete
siRNA-containing effector complex. Proc Natl Acad Sci USA 104:1714–1719.
21. Silhavy D, et al. (2002) A viral protein suppresses RNA silencing and binds silencing-
generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21:3070–3080.
22. Ye K, Malinina L, Patel DJ (2003) Recognition of small interfering RNA by a viral
suppressor of RNA silencing. Nature 426:874–878.
23. Bucher E, Hemmes H, de Haan P, Goldbach R, Prins M (2004) The influenza A virus NS1
24. Qi Y, Zhong X, Itaya A, Ding B (2004) Dissecting RNA silencing in protoplasts uncovers
novel effects of viral suppressors on the silencing pathway at the cellular level. Nucleic
Acids Res 32:e179.
25. Qu F, Ren T, Morris TJ (2003) The coat protein of turnip crinkle virus suppresses
posttranscriptional gene silencing at an early initiation step. J Virol 77:511–522.
26. Kasschau KD, Carrington JC (1998) A counterdefensive strategy of plant viruses:
suppression of posttranscriptional gene silencing. Cell 95:461–470.
27. Llave C, Kasschau KD, Carrington JC (2000) Virus-encoded suppressor of posttranscrip-
tional gene silencing targets a maintenance step in the silencing pathway. Proc Natl
Acad Sci USA 97:13401–13406.
28. Lu S, Cullen BR (2004) Adenovirus VA1 noncoding RNA can inhibit small interfering
RNA and MicroRNA biogenesis. J Virol 78:12868–12876.
29. Andersson MG, et al. (2005) Suppression of RNA interference by adenovirus virus-
associated RNA. J Virol 79:9556–9565.
30. Hutvagner G, et al. (2001) A cellular function for the RNA-interference enzyme Dicer
in the maturation of the let-7 small temporal RNA. Science 293:834–838.
Int J Biochem Cell Biol
32. Haasnoot J, et al. (2007) The Ebola virus VP35 protein is a suppressor of RNA silencing.
PLoS Pathog 3:e86.
33. Brodersen P, et al. (2008) Widespread translational inhibition by plant miRNAs and
siRNAs. Science 320:1185–1190.
34. Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA
silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes
35. Restrepo MA, Freed DD, Carrington JC (1990) Nuclear transport of plant potyviral
proteins. Plant Cell 2:987–998.
36. Rice AP, Carlotti F (1990) Mutational analysis of the conserved cysteine-rich region of
the human immunodeficiency virus type 1 Tat protein. J Virol 64:1864–1868.
37. Hartman TR, et al. (2006) RNA helicase A is necessary for translation of selected
messenger RNAs Nat Struct Mol. Biol 13:509–516.
0 3060 90120150
35S-cysteine/methionine labeling time (min)
(PI units x103)
HIV/RSS + p19
HIV/RSS + pCAM
HIV-1 + p19
HIV-1 + pCAM
activity. Quantification of Gag IP assay results of Fig. 4C and F. Introduction of
K51A mutation in HIV/RSS reduces Gag production and P19 expression in-
creases rate of HIV-1 and HIV/RSS Gag protein synthesis to similar levels.
Tat is the viral RSS and the plant viral RSS P19 can replace Tat RSS
www.pnas.org?cgi?doi?10.1073?pnas.0806822106Qian et al.