The mechanisms governing the encapsidation of retroviral genomic
RNAs have been the subject of intense research for many years.
The principal determinants for genomic RNA (vRNA) encapsidation
are an intact nucleocapsid (NC) domain in the precursor protein,
pr55Gag(termed Gag herein) and an intact packaging signal, psi,
within the vRNA (De Guzman et al., 1998). Recent work
demonstrates that long-range interactions, conformational changes
and the dimeric state of vRNA dictate the readiness for encapsidation
(D’Souza and Summers, 2005; Ooms et al., 2004; Paillart et al.,
2004), and the recent structure determination might help shed light
on this (Watts et al., 2009). For murine leukemia virus (MLV), a
network of interactions promotes sequence- and structure-specific
binding by NC to the psiRNA. The interaction between host proteins
and vRNA might also provide a molecular switch by suppressing
vRNA translation to favour encapsidation (Cimarelli and Luban,
1999). There is still some controversy about whether this type of
mechanism exists, because HIV-1 vRNA that is translated might
also be encapsidated (Butsch and Boris-Lawrie, 2000; Kaye and
Lever, 1999; Poon et al., 2002). Many questions remain concerning
how HIV-1 co-opts host protein function and machineries to traffic
and encapsidate its RNA genome.
Staufen1 is a double-stranded (ds)RNA-binding protein with roles
in RNA localization (St Johnston et al., 1991), translation (Dugre-
Brisson et al., 2005) and mRNA decay (Kim et al., 2005). It is also
a principal component of RNA transport ribonucleoproteins (RNPs)
(Villace et al., 2004), stress granules (SG) (Thomas et al., 2005),
APOBEC3G complexes (Chiu et al., 2006) and other types of RNPs
(Jonson et al., 2007; Snee and Macdonald, 2009). Staufen1 is mobile
in these contexts and its presence in several types of RNPs is
considered an important characteristic for performing its multiple
functions (Dahm et al., 2008). Recent in vivo work also supports
a role for Staufen in synapse development (Vessey et al., 2008).
In humans, there are two genes that encode Staufen1 and
Staufen2 (STAU1 and STAU2). Staufen2 is primarily found in
neuronal cells and has roles in RNA biogenesis and trafficking
(Duchaine et al., 2002; Monshausen et al., 2004). Staufen1 shares
these functional characteristics but STAU1 is more ubiquitously
expressed in human tissues. Moreover, each of these genes generates
a primary RNA transcript that can be differentially spliced to
produce identifiable isoforms. While some work has made in-roads
into deciphering the functional differences of the Staufen2 isoforms,
little information is known about the functional impact of the
expression of Staufen1 isoforms. Nevertheless, their existence
Novel Staufen1 ribonucleoproteins prevent formation
of stress granules but favour encapsidation of HIV-1
Levon G. Abrahamyan1,2, Laurent Chatel-Chaix1,2,3, Lara Ajamian1,2,4, Miroslav P. Milev1,2,4, Anne Monette1,2,4,
Jean-François Clément1,2, Rujun Song2,4, Martin Lehmann1,2, Luc DesGroseillers3, Michael Laughrea2,4,
Graciela Boccaccio5and Andrew J. Mouland1,2,4,6,*
1HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital,
Montréal, QC, H3T 1E2, Canada
2Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital, Montréal, QC, H3T 1E2, Canada
3Department of Biochemistry, Université de Montréal, Montréal, QC, H3C 3J7, Canada
4Department of Medicine, Division of Experimental Medicine and 6Department of Microbiology and Immunology, McGill University,
Montréal, QC, H3A 2B4, Canada
5Instituto Leloir, IIBBA CONICET and Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Buenos Aires C1405BWE, Argentina
*Author for correspondence (firstname.lastname@example.org)
Accepted 8 November 2009
Journal of Cell Science 123, 369-383 Published by The Company of Biologists 2010
Human immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus
particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA,
which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA
and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1
RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing
bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover,
SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We
demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which
Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly
of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This
work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved
in assembly, localization and encapsidation of vRNA.
Key words: Staufen1, HIV-1, Staufen1 HIV-1-dependent RNP, SHRNP, siRNA, Ribonucleoprotein, RNA encapsidation, Virus-host interaction, AIDS,
Journal of Cell Science
suggests multiple and possibly overlapping roles for these proteins
in RNA biogenesis and utilization.
Evidence for a role for Staufen1 in HIV-1 replication derives
from our observation that Staufen1 was incorporated in HIV-1 to
levels that corresponded to the quantity of vRNA (Mouland et al.,
2000). Staufen1 overexpression led to a several-fold increase in
vRNA encapsidation and a corresponding decrease in HIV-1
infectivity of progeny virions (Mouland et al., 2000). In later work,
we showed that Staufen1 was found in the HIV-1 RNP that contains
vRNA and precursor Gag (Chatel-Chaix et al., 2004). The depletion
of Staufen1 by siRNA led to defects in both virus production and
infectivity (Chatel-Chaix et al., 2004). We also found that Staufen1
modulates Gag assembly (Chatel-Chaix et al., 2007), probably due
to a direct interaction between the NC domain of Gag and a short
region in the N-terminus of Staufen1 (Chatel-Chaix et al., 2008).
The selectivity of Staufen1 to bind precursor Gag and vRNA
suggested that Staufen1 performs its role in the context of a
Staufen1-Gag-vRNA ternary complex and could be involved in the
initial recognition and selection of vRNA during the assembly of
In this report, a novel Staufen1 RNP is described whose formation
depends on HIV-1. Staufen1, vRNA and Gag colocalize in these
Staufen1 HIV-1-dependent RNPs (SHRNPs), the size of which
depends on existing Staufen1 levels in cells. A stable association
between Staufen1 and vRNA was found in colocalization studies
but Gag could nevertheless be displaced from SHRNPs. Staufen1
was found in various HIV-1- and stress-induced RNPs. A
relationship between the abundance of virion-associated vRNA and
Staufen1 and the apparent size of SHRNPs indicates that SHRNP
assembly is an important event in the selection, trafficking and
encapsidation of vRNA.
Staufen1 depletion enhances the formation of cytoplasmic
granules containing HIV-1 vRNA
Our early work in which we performed co-immunoprecipitation
analyses suggested that Staufen1 interacts with HIV-1 Gag in an
RNA-independent manner and forms a ternary complex with the
vRNA, excluding other HIV-1 mRNA species (Chatel-Chaix et al.,
2004). We developed a sequential, two-step, affinity-based
immunoprecipitation assay to provide further evidence that this
complex exists. We mock-transfected cells or transfected cells with
HIV-1 proviral DNA with or without a Staufen1-FLAG expression
construct and harvested cells for western blotting and binding
analyses. We readily detected Staufen1-FLAG and Gag by western
blotting and both the vRNA and gapdh mRNA by RT-PCR in HIV-
1-expressing cells (in INPUT). Cell extracts were first submitted
to an immunoprecipitation directed to the FLAG epitope. Staufen1-
FLAG was first eluted from FLAG-antibody coated beads in the
presence of an excess of FLAG peptide. Staufen1-FLAG was found
in the first eluate (Chatel-Chaix et al., 2004), and Gag co-purified
only when Staufen1-FLAG was coexpressed with HIV-1 (Fig. 1A)
attesting to the specificity of Gag immunoprecipitation with the anti-
FLAG resin. To examine whether the vRNA was carried through
and remained associated to Staufen1-Gag, an immunoprecipitation
was then performed using an anti-Gag antibody. In the final eluate,
a specific RT-PCR signal for vRNA was found only when both
Staufen1-FLAG and HIV-1 were coexpressed in cells, but not when
Staufen1-FLAG was excluded from the transfection. This
demonstrated the specificity with which vRNA co-precipitates with
the anti-FLAG and anti-Gag antibodies. The result provides
corollary biochemical evidence that a Staufen1-Gag-vRNA ternary
complex exists in HIV-1-producing cells. To confirm the existence
of this ternary complex and to better characterize it, we examined
the localization of Staufen1 in relationship to vRNA and Gag in
situ by using combined immunofluorescence and fluorescence in
situ hybridization analyses (IF/FISH) followed by laser scanning
confocal microscopy (LSCM). In control (siNS-treated) HIV-1-
expressing cells, Staufen1, Gag and vRNA exhibited colocalization
in the cytoplasm and prominent colocalization at the plasma
membrane. Imaging analyses demonstrated that these viral and host
components are found as a ternary complex in HIV-1-producing
cells (Fig. 1B). These observations extend our earlier work that
showed that Staufen1 associates selectively with these viral
components in cells (Chatel-Chaix et al., 2004; Mouland et al.,
2000). Whereas the diameter of Staufen1-positive punctae in mock-
treated (non-HIV-1-expressing) cells was determined to be less than
200 nm, in HIV-1-expressing cells Staufen1, Gag and vRNA were
generally found in small punctae of approximately 0.6-0.7 m (Fig.
We next examined the effects on HIV-1 vRNA and Gag
localization of depleting cells of both Staufen1 isoforms by using
si55/63, or of the high molecular weight Staufen163kDaalone by
using si63. HeLa cells were mock-transfected or transfected with
proviral DNA and non-silencing control siRNA (siNS), si55/63 or
si63. Staufen1 depletions were always verified by RT-PCR and/or
western blotting (Fig. 1C). RT-PCR analyses showed that the
siRNAs specifically targeted Staufen1 mRNAs as expected, with
si55/63 targeting the expression of Staufen155kDaand Staufen163kDa
transcripts and si63 targeting uniquely the Staufen163kDa(T3)
transcript. The depletions of Staufen1 by si55/63 or si63 were
consistent, selective and highly efficient (depletions of 90-92%).
In our imaging analyses, we found an apparent doubling in the
size of granules for the RNPs that were enriched in vRNA, Gag
and the residual Staufen1 in si55/63-treated cells and these were
principally found in juxtanuclear and cytoplasmic domains (Fig.
1D,E). Strikingly, in 90±5% (mean ± s.e.m., n>250 cells) of the
cells, we only found Staufen1-containing RNPs in HIV-1-expressing
cells (white arrowheads, Fig. 1D) and not in cells in which HIV-1
expression was not detectable (yellow arrowheads, Fig. 1D, si55/63
panels). Residual levels of Staufen1 and Gag appeared in large bright
punctae due to accumulations of these proteins in these foci. We
also examined SHRNPs in Jurkat T cells, and found that these were
enlarged when Staufen1 was depleted by short hairpin (sh)RNA
(shSTF; data not shown); however, the paucity of cytoplasmic space
limited our conclusions from the results obtained using this cell
type. The localization of Staufen1, vRNA and Gag in Staufen163kDa-
depleted HeLa cells did not markedly change SHRNP appearance
in greater than 90% of cells examined (>200 cells, five experiments;
Fig. 1D, bottom panel). Identical findings for Staufen1 depletions
and SHRNP formation were obtained at 24 and 48 hours post-
transfection (Fig. 1E). These results demonstrate that larger
intracellular RNPs form that contain vRNA and the residual
Staufen1, primarily when both Staufen1 isoforms are silenced in
cells. We cannot completely rule out the possibility that the strong
signal intensities were due to coalescing RNPs or other organelles
at these foci. Because Staufen1, vRNA and Gag colocalize in
cytoplasmic punctae and at the periphery in HIV-1-producing cells,
we therefore named these RNPs Staufen1 HIV-1-dependent RNPs
Results from our Manders’ colocalization analyses were also
consistent, such that while Staufen1 and vRNA colocalized
Journal of Cell Science 123 (3)
Journal of Cell Science
Staufen1 HIV-1-dependent ribonucleoproteins
Fig. 1. Staufen1, vRNA and Gag are part of the same ternary complex and colocalize in cells. (A)HeLa cells were mock-transfected or transfected with pNL4-
3 proviral DNA without (–) or with (+) Staufen1-FLAG. A sequential Staufen1 and Gag binding assay was performed as described in Materials and Methods. Gag,
Staufen1-FLAG and GAPDH were quantified by western blotting in Input lanes and Staufen1-FLAG and Gag were identified following FLAG elution and Gag
immunoprecipitation (IP). vRNA and gapdh RNAs (loading control) were detected by RT-PCR in cellular extracts before analysis and in the final eluted IP
complexes. (B), HeLa cells were mock-transfected or co-transfected with the HxBRU proviral DNA and siNS, si55/63 or si63. Cells were harvested and fixed.
Staufen1 (red), vRNA (green) and Gag (pseudocoloured blue) were imaged by LSCM at 48 hours in mock- and siNS-treated cells. White arrowheads identify
regions of colocalization (magnifications are shown in insets). (C)Four Staufen1 transcripts are shown (T1-T4). The T3 variant encodes Staufen163kDadue to an
upstream ATG initiation codon (position 409). The other transcripts encode Staufen155kDa. siRNAs (si55/63, si63) or shRNAs (shNS, shSTF) are depicted above
the T3 transcript. The positions of forward and reverse primers used for semi-quantitative RT-PCR analyses to quantify Staufen1 mRNAs are shown below T3. On
the right is shown RT-PCR and western analyses of Staufen163kDaand Staufen155kDamRNAs and gene products in mock- and siRNA-treated cells. gapdh mRNA
and GAPDH are loading controls. PCR yields a 540 bp product for T3 or a 270 bp PCR product for the shorter Staufen1 mRNAs. (D)Staufen1 (red), vRNA (green)
and Gag (blue) were imaged at 48 hours in si55/63- and si63-treated cells. Large SHRNP foci are indicated by white arrowheads in si55/63-treated and/or HIV-1-
expressing cells. HIV-1-negative cells are indicated by yellow arrowheads. Insets represent high-resolution black and white depictions of cell regions (dashed
boxes). (E)Histograms show the average size of Staufen1 granules. The error bars represent s.d. Scale bars: 10m.
Journal of Cell Science
appreciably to 20±9.1% (mean ± s.e.m.) in siNS-treated cells, there
was a tripling to 58% of the proportion of Staufen1 colocalizing
with vRNA in conditions of si55/63 depletion (Table 1). This
increase also shows that there was little redistribution or loss of
Staufen1 from the vRNA-Gag complex in depletion conditions.
SHRNPs are in equilibrium with polysomes and are
distinct from stress granules
Viral infection can generate a cellular stress response (Gale et al.,
2000; McInerney et al., 2005) and Staufen1 is a component of SGs
(Thomas et al., 2005). Recent data show that Staufen1 might temper
stress responses in cells by controlling the equilibrium between the
formation of polysomes and SGs (Thomas et al., 2009). To
determine whether SHRNP assembly in Staufen1-depleted cells
depends on translation activity, a characteristic of SG assembly,
HeLa cells were transfected with HIV-1 proviral DNA and siRNAs
as above and left untreated, or briefly treated with puromycin (Pur)
or cycloheximide (CHX) to promote or inhibit SG assembly,
respectively. IF/FISH co-analyses were performed to identify
Staufen1, Gag or PABP1 (a marker for SG) and vRNA. On the one
hand, Pur treatment induced PABP1- and Staufen1-containing SG
and these were observed when HIV-1 was expressed and in
Staufen1-depleted cells (Fig. 2, panels 2-4). On the other hand, a
translation block at the level of elongation induced by CHX
treatment caused a uniform distribution of PABP1 as expected. The
distribution of Staufen1 was likewise dispersed in this condition
when HIV-1 was expressed (Fig. 2, panels 5-7). These data indicate
that SHRNP assembly is dependent on translation and appears to
be in equilibrium, like SG, with cellular mRNA translational
To determine whether vRNA was included in the Staufen1-
PABP1-containing foci, we next examined its localization under
these experimental conditions. Without drug treatment, HIV-1
expression did not elicit a stress response in cells because large
PABP1+ or Staufen1+ SG foci were not detectable (data not shown
and Fig. 3A, panel 2). In untreated cells, we observed identical
distributions for Staufen1, Gag and vRNA as shown in Fig. 1D for
siNS, si55/63 and si63 conditions, respectively (Fig. 3A, panels 2-
4). Pur treatment resulted in the formation of 2- to 5-m structures
containing Staufen1 in mock-transfected cells (Fig. 3A, panel 5)
and caused a near-complete recruitment of vRNA and Staufen1 to
Pur-induced SGs (Fig. 3A, panels 6-8) containing PABP1 (because
PABP1 is also colocalized with Staufen1; see Fig. 2, panels 2-4)
in HIV-1-expressing cells. Strikingly, Gag was almost completely
excluded from SGs in Pur-treated HIV-1-expressing cells,
demonstrating that the Staufen1-vRNA association is maintained
under Pur treatment. In Staufen1-depleted and Pur-treated cells, the
residual Staufen1 was also recruited to PABP1-positive SG foci
together with vRNA (Fig. 2, panel 4; Fig. 3A, panel 7). This
suggested that the prominent SHRNPs in Staufen1-depleted cells
(no drug added) can be remodelled and that Staufen1 and vRNA
can be recruited from Gag-containing SHRNPs to constitute SGs.
This is consistent with the ability of Staufen to shuttle from RNA
trafficking granules to SGs in response to stress (Thomas et al.,
2005). Thus, the Pur-induced SGs containing Staufen1, vRNA and
PABP1 are probably bona fide SGs, and the association between
Staufen1 and vRNA remains stable in the context of both SHRNPs
and Pur-induced SGs. In the latter, however, Gag is not present,
and this was reflected by
immunoprecipitation assay (data not shown and Fig. 1A).
Consistently, we did not find a significant change in the
colocalization between Staufen1 and vRNA in untreated and Pur-
treated cells (Table 1). These results indicate that the Staufen1-
containing RNPs undergo remodelling and that the composition of
a SHRNP is distinct from that of a Pur-induced SG.
When cells were treated with CHX to block translation
elongation, highly dispersed distributions for PABP1 (Fig. 2), vRNA
and Gag were found. Few SG formed in all conditions and the
SHRNPs in Staufen1-depleted cells were less numerous (Fig. 3,
panels 9-12). The brief treatments with Pur or CHX had little impact
on steady-state levels of Gag protein (Fig. 3B). These results further
demonstrate a dependence on translation for SHRNP formation.
our sequential affinity-
HIV-1 prevents formation of SGs and processing bodies
The exposure of cells to arsenic (Ars) is known to induce the
formation of SGs that contain Staufen1, PABP1 and other proteins
such as HuR, G3BP and eIF3 (Anderson and Kedersha, 2008;
Kedersha et al., 2002; Thomas et al., 2005). Because SHRNPs and
SGs are related by virtue of the presence of Staufen1 in these
cytoplasmic foci, we determined what the outcome was on Staufen1,
Gag and vRNA localization by treating HIV-1-producing cells with
Ars. In mock-transfected cells we first confirmed that Ars treatment
induced visible SGs enriched in both Staufen1 and PABP1 (Fig.
4A). Strikingly, however, in cells expressing HIV-1 (i.e. staining
for vRNA and Gag; Gag staining is not shown), we were not able
to find typical Ars-induced SGs in either siNS or si55/63 conditions.
Here, vRNA and Staufen1 were dispersed and accumulated at a
juxtanuclear region in cells in about 95% of cells (Fig. 4B,C; white
arrowheads; n150-200). Thus, HIV-1 expression prevents the
formation of SG foci that are induced by Ars. The extent to which
Staufen1 and vRNA colocalized was not different to that found in
untreated cells (Table 1), indicating that a redistribution of these
SHRNPs had occurred. In 85% of transfected cells (n200), large
Staufen1 (and PABP1-positive, not shown) SGs were detectable in
HIV-1-negative cells (e.g. yellow arrowheads in Fig. 4B-D), similar
to those found in mock-treated cells (Fig. 4A, right panel). In
addition, a deficit in Staufen1 did not impede SG formation per se,
consistent with recent findings (Thomas et al., 2009), nor did it
change the abundance of SGs. However, HIV-1 changed the
characteristics and location of Staufen1-containing RNPs. These
data also demonstrate that HIV-1-expressing cells are resistant to
Ars-induced SG formation. Finally, PABP1 staining in these vRNA
and Staufen1 RNPs was virtually absent compared with that in the
surrounding cytoplasm, indicating that these RNPs did not bear this
hallmark SG marker (Fig. 4E). This was in contrast to the inclusion
Journal of Cell Science 123 (3)
Table 1. Staufen1 and vRNA colocalization* analyses
(% ± s.d.)
(% ± s.d.)Treatment
siNS + Pur
si55/63 + Pur
HIV-1 + Ars
Provirus pNL4-XX (Gag–, Gag/Pol–)
*Manders’ colocalization coefficients were calculated as described in
Materials and Methods.
Ars, arsenic; Pur, puromycin; nd, not determined.
Journal of Cell Science
Staufen1 HIV-1-dependent ribonucleoproteins
of PABP1 (and the exclusion of Gag) in Pur-induced SGs (Fig. 3).
The block to the formation of Ars-induced SGs by HIV-1 was
confirmed using other SG proteins such as eIF3 and G3BP
(Anderson and Kedersha, 2008) (supplementary material Fig. S1).
Moreover, the HIV-1-induced block to SG formation supports the
notion that SHRNPs are distinct in HIV-1 producing cells because
they also exclude four SG marker proteins tested in this work
(PABP1, G3BP, eIF3 and HuR) (Fig. 4 and supplementary material
It is known that SG formation and translational inhibition after
Ars treatment is dependent on eIF2phosphorylation, the reduction
of the translation ternary complex, and recruitment of several RNA-
binding proteins (Kedersha et al., 1999). Some viruses have
developed mechanisms to replicate during cellular stress even when
cellular translation is shut off by eIF2phosphorylation (McInerney
et al., 2005). We analyzed the levels of phosphorylated eIF2
(eIF2-P) in cells. This analysis revealed that Ars treatment
induced eIF2-P almost threefold (293%). Although HIV-1 alone
did not induce eIF2-P levels to any great extent (121%), HIV-1
did not markedly alter eIF2-P levels in Ars-treated cells either
(230% vs 293%), demonstrating that HIV-1 expression does not
protect cells from SG formation by inhibiting eIF2phosphorylation
The processing body (PB) represents another type of RNP
complex that plays a role in RNA storage and metabolism. PBs
have been posited to be important for replication of Ty
retrotransposons and for other RNA viruses (Beckham and Parker,
2008; Beliakova-Bethell et al., 2006). Staufen1 has also been found
in Drosophila PBs but not in those in mammalian cells (Barbee et
al., 2006; Thomas et al., 2009). We therefore attempted to clarify
whether the Staufen1-containing SHRNPs were related to PBs by
staining cells with a classical PB marker, DCP-1. HeLa cells were
transfected with HIV-1 DNA and siRNAs as described above, and
PBs were detected using an anti-DCP-1 antibody by indirect IF
and LSCM. As shown in Fig. 5A, there was no significant
colocalization between vRNA, Gag or Staufen1 and DCP-1 in
either control or Staufen1-depleted cells (Fig. 5A). We then
counted the number of PBs in cells in which we detected DCP-1
foci. In mock-transfected cells, the average number of PB foci in
a single LSCM focal plane through the middle of the cell (as judged
by confocal sectioning along the Z-axis) totalled 28±1 (mean ±
s.e.m.) foci per cell. By contrast, the number of PBs dramatically
reduced to 7±1 (mean ± s.e.m.), when we counted all cells
expressing DCP-1 at low and high levels in HIV-1-expressing cells.
In other imaging experiments, we observed few – if any – PBs in
HIV-1-expressing cells labelled with vRNA (Fig. 5B). Likewise,
the effects of HIV-1 on PB abundance are probably due to effects
on PB assembly because the abundance of DCP-1 in cells was
maintained (in both transfected HeLa and infected Jurkat T cells;
Fig. 5B and data not shown).
Ars treatment of cells dramatically induces the number of visible
PB foci in cells. In addition, it promotes the formation of Staufen1-
and PABP1-positive SGs. The relationship between SHRNPs and
PBs was therefore analyzed after stress induction by Ars. We could
not find PBs at the juxtanuclear region in Ars-treated cells, which
was where vRNA, Gag and Staufen1 were found (Fig. 5C, panels
1,2). In mock-transfected cells, PABP1 staining was dispersed and
PBs were detectable (Fig. 5B, panels 3,4), whereas in mock-
transfected cell treated with Ars, we observed PBs in close
proximity to numerous PABP1-positive SGs (Fig. 5C, panels 5,6,
indicated by yellow arrowheads), confirming that SGs and PBs
are dynamically linked (Kedersha et al., 2005). Our results
Fig. 2. SHRNPs are not SG. HeLa cells were mock-transfected (panels 1,2) or transfected with HxBRU DNA and siNS (panel 3) or si55/63 (panel 4). Cells
presented in panels 2-4 were incubated with Pur before fixation. Insets show black and white renditions for PABP1 and Staufen1 staining in boxed areas. Cells
shown in panels 5-7 were treated with CHX before fixation. Cells were fixed and stained for Staufen1 (red), PABP1 (green) and Gag (not shown). Large yellow
granules in panels 2-4 represent colocalized Staufen1-PABP1 foci induced by Pur treatment. Arrows identify SG induced by Pur. Scale bars: 10m.
Journal of Cell Science
demonstrate that in HeLa cells, vRNA and Gag do not localize to
PBs, and SHRNPs do not contain DCP-1 and therefore are
probably not PBs. Cumulatively, our results suggest that HIV-1
impairs the formation of constitutive or stress-induced silencing
foci. By regulating the equilibrium between SGs, PBs and other
RNPs, HIV-1 could efficiently exploit the cellular machinery to
ensure efficient virus assembly when cells are exposed to different
types of stresses.
Journal of Cell Science 123 (3)
Fig. 3. Pur induces the formation of large
granules containing Staufen1 and vRNA
but not Gag. HeLa cells were transfected as
in Fig. 1. (A)Combined IF/FISH co-analyses
for Staufen1 (red), Gag (blue) and vRNA
(green) in untreated cells (panels 1-4) or cells
treated with Pur (panels 5-8) or CHX (panels
9-12). Staufen1, Gag and vRNA accumulated
in large granules in si55/63 conditions (white
arrowheads in panel 3 and corresponding
insets). In Pur-treated cells, Staufen1 was
found in SG (red granules in panel 5
indicated by yellow arrowheads). The yellow
granules in panels 6-8 correspond to
Staufen1 and vRNA colocalization in SG
induced by Pur treatment, but Gag was
excluded from these SG (white arrowheads
in panels 6-8 and corresponding insets).
Insets are 1.5? magnifications.
(B)Corresponding protein expression levels
for Staufen1, Gag and GAPDH (loading
control). Scale bars: 10m.
Journal of Cell Science
Staufen1 HIV-1-dependent ribonucleoproteins
Gag expression is decreased after Staufen1 depletion
The depletion of both Staufen1 isoforms by si55/63 resulted in
markedly diminished Gag expression levels (53±5%, mean ±
s.e.m., five experiments; Fig. 6). Staufen163kDadepletion alone had
little effect on Gag expression (84±2.5%, mean ± s.e.m., five
experiments) and both treatments had little effect on gp120 (Env)
expression levels in the context of pNL4-3 (Fig. 7 and data not
shown). The effects on Gag expression cannot be explained by the
activation of the Jak-Stat pathway and the expression of IFN-
inducible genes like PKR, by changes in the cell cycle or by changes
in Gag protein stability (data not shown). Because we have shown
that Staufen1 can enhance translation of certain mRNAs (Dugre-
Brisson et al., 2005) and can stabilize polysomes (Thomas et al.,
2009), these changes are probably due to effects of Staufen1
depletion on HIV-1 vRNA translation.
Staufen1 and vRNA assemble without Gag
Gag has a central role in assembly and virus structure and this is
substantiated by its ability to bind and co-opt many cellular
proteins, including Staufen1 (Chatel-Chaix et al., 2004). Our
observation that Gag could be rapidly excluded from Staufen1-
vRNA RNPs by a brief Pur treatment revealed that these RNPs are
dynamic and plastic structures. We therefore determined whether
Staufen1-vRNA RNPs could form in the absence of Gag expression
and also questioned whether PABP1 could replace Gag when it is
not expressed. In order to do this, we expressed a pNL4-3-based
proviral DNA (pNL4-XX) in which Gag expression is prevented
due to the introduction of two stop codons in the gag open reading
frame. This vector expresses all HIV-1 RNAs, and aborted
translation of the vRNA prevents the synthesis of Gag and Gag-
Pol proteins but the translation of 4 and 2kb RNAs is not impaired
Fig. 4. HIV-1 prevents SG formation. (A)HeLa
cells were transfected as in Fig. 1. Cells were
incubated with Ars and then allowed to recover
before fixation. (A)Staining for Staufen1 in mock-
treated cells (left panel) or for Staufen1 and PABP1
in cells treated with Ars (right panel). Arrows
identify typical SG enriched in Staufen1 and
PABP1. Insets show black and white renditions of
Staufen1 and PABP1 foci. (B,C)Staufen1 and
vRNA localization in Ars-treated, HIV-1-expresing
cells transfected with siNS or si55/63. Insets show
staining of Staufen1 and vRNA. White arrowheads
indicate accumulations Staufen1 and vRNA RNPs
in the juxtanuclear region in HIV-1-expressing
cells. (D)Normal shading was applied to original
confocal images of cells generated using Imaris
software for Ars-treated, HIV-1 expressing cells
showing PABP1 staining (red) and vRNA staining
(green). HIV-1-expressing cells are indicated by
white arrowheads. Yellow arrowheads identify
Ars-induced SG in non-producing cells. Dashed
lines demarcate image limit. (E)Intensity plots for
PABP1 and vRNA staining from point A to B
(shown in D) in cell are shown. (F)Corresponding
western blot analyses for eIF2-P (P-eIF2),
eIF2, Gag and GAPDH are shown. Numbers
below the eIF2 bands indicate relative levels of
eIF2-P and are derived from the ratio of eIF2-P
to eIF2 signal intensities ?100%. eIF2-P levels
found in untreated, mock-transfected cells were set
to 100%. GAPDH is loading control. Scale bars:
Journal of Cell Science
(Ajamian et al., 2008). Following expression of pNL4-XX in siNS
and si55/63 conditions, cells were harvested and processed for
western blotting and IF/FISH co-analyses for Staufen1 and vRNA.
gp120 (Env) was stably expressed but Gag and Gag-Pol were not,
as expected (Fig. 7A). Some 26% of cellular Staufen1 colocalized
with vRNA (Table 1). In cells in which Staufen1 was depleted, we
noted again larger Staufen1 punctae in pNL4-XX-expressing cells
and a doubling of the extent to which Staufen1 colocalized with
vRNA (to 60±10%, mean ± s.d.), consistent with a lowered
abundance of cellular Staufen1 and little increase in the total amount
of Staufen1 colocalizing with vRNA (Table 1). This also indicated
that the association of Staufen1 to vRNA was probably not
significantly affected when it was in limiting supply. We did not
detect any significant plasma membrane localization when Staufen1
was depleted in cells (Fig. 7B), indicating that Gag expression might
be involved in the localization of SHRNPs. This would be consistent
with our recent findings showing a dependence of Gag on HIV-1
RNP localization (Lehmann et al., 2009).
We then determined whether Gag could be included in the
Staufen1-vRNA RNP if Gag expression was rescued in trans. In
this case, Rev is synthesized from pNL4-XX and a gag RNA is
derived from a Rev-dependent Gag expressor supplied in trans
(Lingappa et al., 2006). Gag was only expressed when pNL4-XX
was coexpressed and, in si55/63 conditions, the decrease in Gag
synthesis was again observed (Fig. 7A). Confocal imaging analyses
for Staufen1, vRNA and Gag revealed that whereas the
Journal of Cell Science 123 (3)
Fig. 5. SHRNPs are distinct from PBs, and Gag and vRNA are not localized to PBs. (A)Staining for PB in HIV-1-expressing cells transfected with siNS,
si55/63 or si63. Panels 4-6 show 6? magnifications of boxed areas. White arrowheads indicate large granules containing vRNA and Gag in Staufen1-depleted
cells. (B)Left: DCP-1 (red) was localized in a HIV-1-expressing cell (vRNA in green). Right: DCP-1 expression levels were evaluated by western blotting with
GAPDH as a loading control. Percentages indicate relative levels of DCP-1. (C)Panels 1 and 2: Staining for DCP-1 (red), vRNA (green) and Gag (blue) in HIV-1-
expressing (siNS) and Ars-treated cells. vRNA, Gag and DCP-1 staining in the juxtanuclear region is shown in Ars-treated cells with DCP-1 staining (panel 1; 6?
magnification of boxed region in panel 2). Panels 3-6 show DCP-1 and PABP1 staining in mock-transfected cells without or with Ars-treatment. SG are indicated
by yellow arrowheads (compare panels 3,4 with panels 5,6). Scale bars: 10 m (A1-3, C1,3,5); 5 m (A4-6, C2,4,5,6).
Journal of Cell Science
Staufen1 HIV-1-dependent ribonucleoproteins
colocalization between Staufen1 and vRNA was maintained, Gag
was not recruited to Staufen1-vRNA RNPs when it was expressed
in trans (Fig. 7C). Furthermore, the larger RNPs that were found
to be devoid of Gag had low immune reactivity for PABP1 (Fig.
7C), and this was found in the majority of cells (70%) in three
experiments. Therefore, in order for a SHRNP to form that contains
Gag, there might be a requirement for cis translation of the gag
mRNA (i.e. vRNA) and the co-translational recruitment of Gag to
Staufen1-vRNA complexes. This could also prevent the formation
of typical SG in HIV-1-expressing cells by excluding PABP1. This
result also supports the notion that SHRNP formation requires
Staufen1 depletion enhances vRNA encapsidation
Finally, we studied the consequences of Staufen1 depletion and the
remodelling of SHRNPs on the HIV-1 replication cycle. Because
Staufen1 is a virion-associated protein (Mouland et al., 2000; Kozak
et al., 2006), we determined whether there were any compositional
differences between purified HIV-1 derived from control (siNS) and
Staufen1-depleted cells. Cells were harvested for western blot and
RT-PCR analyses. Virus was purified from cell supernatants and
quantified by a sensitive ELISA that detects the mature Gag product,
p24; this result was routinely confirmed by western blotting
analyses for virion-associated Gag (Fig. 9B and data not shown).
vRNA was isolated from cells, and equal quantities of virus were
quantified by semi-quantitative RT-PCR. Whereas cellular levels
of vRNA remained relatively unchanged, si55/63 enhanced the
abundance of vRNA in virus particles (Fig. 8A). This enrichment
of virion-associated vRNA was reproducible in different settings,
using different proviral strains and siRNA targets and being
measured by different methods (data not shown). Indeed, using
shRNA to deplete intracellular Staufen1 (Fig. 1C), we confirmed
that vRNA encapsidation was enhanced over twofold during the
expression of an isogenic protease-negative provirus (HxBRU Pr–;
Fig. 8B; corresponding expression blots are shown in Fig. 9B). The
increases in vRNA encapsidation in both si55/63 and shSTF
conditions ranged from 1.7- to 4.5-fold (3.6±0.5-fold, mean ± s.e.m.,
eight experiments). Thus, the depletion of Staufen1 from HIV-1-
expressing cells consistently results in enhanced vRNA
Because vRNA encapsidation might be coupled to dimerization
(Ooms et al., 2004), we then tested whether Staufen1 influences
vRNA dimerization. Despite an efficient knockdown of Staufen1
expression by 90% (the corresponding western blots for this
experiment are shown in Fig. 6), the melting curves for vRNA
dimers derived from siRNA-treated cells were found to be similar
(Fig. 8C), ruling out an influence of Staufen1 on vRNA dimerization
To examine the protein content of virus in these conditions, we
performed western blotting on extracts from cells and purified virus.
Equal protein quantities (for cell extracts) and equal p24-equivalents
of virus were loaded onto SDS-PAGE gels. Staufen155kDawas
efficiently depleted by 90% in si55/63-treated cells when compared
Fig. 6. Staufen1 depletion downregulates Gag expression. HeLa cells were
transfected as described in Fig. 1. Numbers below pr55Gag(Gag) western
blotting lanes correspond to relative levels of Gag compared to siNS condition
(calculated for this experiment).
Fig. 7. Staufen1-vRNA RNPs form in the absence of Gag expression. HeLa
cells were mock-transfected or transfected with proviral DNA (pNL4-XX,
harbouring two mutations to prevent Gag synthesis) and siNS or si55/63 and
were harvested and fixed 36-40 hours later for western blotting and IF/FISH
analyses. (A)Western blotting for Staufen1, GAPDH (loading control) and
gp120 (Env). (B)Cells were stained for Staufen1 (red) and vRNA (green) in
siNS and si55/63 conditions expressing pNL4-XX. (C)Gag expression was
rescued with GagRRE when expressed in trans with pNL4-XX and cells were
stained for Staufen1 (red), vRNA (green) and Gag (blue; left panel) or for
Staufen1 (red), PABP1 (green) and Gag (blue; right panel). HIV-1-expressing
cells are indicated by white arrowheads and HIV-1-negative cells are identified
by yellow arrowheads. UT, untransfected cell. Scale bars: 10 m.
Journal of Cell Science
to mock- or siNS-treated cells. Cellular Gag levels were diminished
to a similar extent in si55/63 conditions (Fig. 9A). si55/63 treatment
led to a several-fold increase in the abundance of virus-associated
Staufen163kDaas shown in western blots for Staufen1 (Fig. 9A).
Actin levels in the viruses were constant regardless of treatment.
The presence and absence of actin and heterogeneous nuclear
(hn)RNP A2/B1 in purified virus served as positive and negative
virion-associated protein controls, respectively (Liu et al., 1999;
Mouland et al., 2001). In an independent experiment, the abundance
of ribosomal L7 and the major nonsense-mediated decay protein,
Upf1, two proteins that have been identified in Staufen1 complexes
(Brendel et al., 2004; Kim et al., 2005) and the HIV-1 RNP (Ajamian
et al., 2008), were also increased in highly purified virus from
Staufen1-depleted cells (Fig. 9A, right panel). These results show
that Staufen1 depletion induces a selective salvaging of the
Staufen163kDaisoform in virus.
We also depleted Staufen1 by shSTF during the expression of a
protease-negative provirus. In this experiment, Staufen1 expression
was depleted by 85% (Fig. 9B). The depletion of both Staufen1
isoforms again promoted the preferential encapsidation of
Staufen163kDain virus (1.9-fold increase; Fig. 9B). Western blot
analyses for virion-associated Gag (pr55Gag) demonstrated equal
loading of virus. Levels of Staufen155kDadetected in viral lysates
did not markedly change and the relative abundance of the two
Staufen1 isoforms in cells and virus was reversed. This result
indicates that Staufen163kDais available for encapsidation and that
when Staufen1 expression is compromised, it is salvaged, like the
vRNA, into de novo synthesized virus particles, probably via the
formation of SHRNPs in these conditions.
In mammalian cells, Staufen proteins are found in RNPs of
molecular weight 440-670 kDa that are thought to represent RNA
trafficking granules and to be involved in the repositioning and
translational activity of polysome-bound mRNAs (Brendel et al.,
2004; Kohrmann et al., 1999; Mallardo et al., 2003). Our earlier
observations that indicated a relationship between Staufen1 and the
selection of vRNA by Gag are followed up in this study in which
we now provide evidence for a role for Staufen1 in vRNA
encapsidation. First, we demonstrate that HIV-1 recruits Staufen1
to form distinct, intracellular HIV-1-dependent SHRNPs and that
these appear as larger foci in intracellular domains when Staufen1
is in limiting supply. Second, the striking correlation between the
formation of larger, visible SHRNP foci and an increased abundance
of vRNA in virions suggests that SHRNPs might represent vRNA-
containing scaffolds through which vRNA is trafficked and
Journal of Cell Science 123 (3)
Fig. 8. Virological determinations in cells treated with siNS, si55/63 or
si63. (A)Cellular and viral RNA were analysed by RT-PCR. HxBRU DNA
was used to generate a standard curve. Numbers above histograms represent
the fold induction over siNS treatment control from the experiment shown.
The inset shows PCR product for cell-associated vRNA levels following ten
additional PCR cycles. (B)RT-PCR analysis for virus derived from cells
expressing HxBRU Pr–. Negative controls included the exclusion of RNA
(–RNA) in the RT reaction and the exclusion of cDNA products (–cDNA) in
the PCR reaction. 1 ng proviral DNA (+DNA) served as a positive control for
the PCR. (C)Dimerization analysis of vRNA isolated from virus from siNS
(dashed line) and si55/63-treated (solid line) cells.
Fig. 9. Cellular and viral protein expression levels following either siNS or
si55/63 treatment. (A)Cells were mock-transfected (lane 1) or co-transfected
with the HxBRU proviral DNA and either siNS (lanes 2 and 4) or si55/63
(lanes 3 and 5). Cell extracts and purified virus were prepared. Cell extracts
(lanes 1-3) and corresponding virus (lanes 4,5) were subjected to western
analyses for Staufen1, MAp17, actin and hnRNPA2/B1. Equivalent loading of
viral extracts is shown by similar signal intensities of Gag proteins in the viral
samples (lanes 4,5). Right: an independent experiment was performed exactly
as described in A. Western blotting was performed for Staufen1 and Gag
proteins, MAp17, ribosomal protein L7, GAPDH and Upf1. (B)Western
blotting for Staufen1 and Calnexin (CNX, loading control) and Staufen1 and
pr55Gagin viral extracts from cells expressing HxBRU Pr–. (C) The stacked
histogram reveals the reversal in the relative abundance of the two Staufen1
isoforms in cell and viral extracts (averages from five experiments).
Journal of Cell Science
Staufen1 HIV-1-dependent ribonucleoproteins
encapsidated into assembling HIV-1. Staufen1-containing RNPs
form with or without the presence of Gag and are bona fide RNPs
rather than preformed viral particles.
The colocalization of Staufen1 with vRNA and Gag, and the
increase in the apparent size of SHRNPs that correlated with vRNA
encapsidation levels (Figs 1 and 8), both suggest that Staufen1 is
a relevant host factor during the selection of HIV-1 vRNA during
assembly. The observed changes in size and content of SHRNPs
are probably due to the accumulation of vRNA, whose fate and
trafficking depend on Staufen1. A notable analogy can be drawn:
in cases in which decapping enzymes have been depleted, small
and poorly visible PBs become supraphysiologic in size and
accumulate RNA substrates (Franks and Lykke-Andersen, 2007).
Whether the increased size of SHRNPs in Staufen1-depleted cells
represents an increase in size of the Staufen1 RNP or represents
the coalescing of multiple SHRNPs remains to be determined.
However, when we observe SHRNPs, HIV-1 expression alone can
induce compositional changes to Staufen1 RNPs in cells (Ajamian
et al., 2008), and modulation of Staufen1 levels enhances insoluble
Gag complexes (Chatel-Chaix et al., 2007). SHRNP localization
was also found to be partly coincident with the perinuclear region
where vRNA and Gag have been shown to physically interact (Poole
et al., 2005) and to which targeting of vRNA has been shown to
lead to enhanced vRNA encapsidation (Levesque et al., 2006). From
a mechanistic standpoint, the formation of larger RNPs could explain
the deregulation of assembly and enhanced encapsidation of vRNA,
Staufen163kDaand other Staufen1-binding proteins found in the HIV-
1 RNP such as Upf1 (Ajamian et al., 2008). In addition, it could
explain the deleterious effects we have demonstrated on infectivity
(Chatel-Chaix et al., 2004). This is not without precedence. For
example, Resh and colleagues showed that Gag transits through
intracellular bodies before being trafficked to the plasma membrane
(Martinez et al., 2008), and the selective knockdown of cellular
RNA-binding proteins blocks HIV-1 RNA transport at distinct
intracellular loci (Levesque et al., 2006). This type of selectivity
of a Staufen RNP was also found in Drosophila in the Sponge-
Body (Snee and Macdonald, 2009). Moreover, the ‘T-body’ RNP
harbours the Ty1 retrotransposon genomic RNA and a Gag-like
protein and was suggested to serve as a scaffold for viral RNA
trafficking and assembly (Malagon and Jensen, 2008). Furthermore,
large intracellular RNPs form during flavivirus replication and these
contribute to genomic RNA encapsidation (Emara and Brinton,
2007). Other RNPs that contain Staufen are relevant for RNA
trafficking and for virus restriction (Chiu et al., 2006; Gallois-
Montbrun et al., 2007; Kozak et al., 2006; Menager et al., 2009).
The SHRNP probably represents an RNP that is specifically
engineered by HIV-1 to constitute an intermediary compartment to
which vRNA is trafficked for selection by Gag. Further study of
the SHRNP will be important in order to understand in what context
it is found in cells and to develop a more complete understanding
of the fate of the genomic RNA during HIV-1 assembly.
Virus infection can induce a cellular stress response and the
formation of SGs (Gale et al., 2000; McInerney et al., 2005) but,
conversely, several viruses have developed mechanisms to
counteract cellular stress responses that can impose severe
limitations to viral replication. Although both SG and SHRNP
assembly depend on active translation, the SHRNPs that we have
identified after Staufen1 depletion as well as in cells with normal
levels of Staufen1 are distinct from the known Staufen1-containing
SGs identified in earlier work (Thomas et al., 2005) (supplementary
material Fig. S2). Pur, however, leads to the remodelling of
SHRNPs by displacing Gag from Staufen1-vRNA-containing RNPs,
thus demonstrating that SHRNPs are plastic in nature as are other
Staufen-containing RNPs (Snee and Macdonald, 2009). Knockdown
of Staufen1 enhances SG formation in murine cells but SGs in this
particular case are devoid of Staufen1, leading to the notion that
this host protein monitors stress responses in cells without being
crucial to SG assembly (Thomas et al., 2009). This result perhaps
Fig. 10. HIV-1 affects cellular silencing foci and
promotes the formation of specific RNA
granules. (A)PBs and SGs are present in non-
infected cells. Silencing foci size and number
depends on stress levels, and both PBs and SGs are
in dynamic equilibrium with polysomes. The
double-stranded RNA-binding protein Staufen1
downregulates SG formation. (B)After HIV-1
infection, PBs and stress-induced SGs do not form
and specific particles containing vRNA, Gag and
cellular Staufen1 (termed SHRNPs) form leading
to the encapsidation of vRNA. Gag is dispensable
for their formation. SHRNP assembly is in
equilibrium with translating polysomes and
resembles the behaviour of cellular silencing foci
in A. Staufen1 depletion enhances the apparent
size of SHRNP foci in cells representing either
coalesced SHRNPs or enlarged SHRNPs (shown)
to facilitate vRNA encapsidation.
Journal of Cell Science
parallels what is actually occurring in HIV-1-expressing cells in
which the assembly of SHRNPs is favoured over that of SG. The
Staufen1-, vRNA- and Gag-containing SHRNPs did not contain
PABP1, indicating the preference towards SHRNP formation even
when oxidative stress was induced by Ars in HIV-1-producing cells
(Figs 4, 5 and supplementary material Fig. S1). Consistently, HIV-
1 protease cleaves PABP1 and this could provide further substantive
evidence that HIV-1 needs to deactivate PABP1 function (Alvarez
et al., 2006) to favour the assembly of SHRNPs during the late
stages of replication.
How and why HIV-1 dramatically impairs the assembly of Ars-
induced SGs (Fig. 4, 5 and supplementary material Fig. S1) but not
that of Pur-induced SGs remain important questions. Ars and Pur
elicit the formation of SG by distinct mechanisms. For example,
Pur induces SG by disassembling polysomes whereas Ars induces
SGs by eliciting eIF2 phosphorylation to result in a block to the
recycling of the translation ternary complex (Farny et al., 2009;
Kedersha et al., 2000). Because HIV-1 expression did not promote
the phosphorylation of eIF2 in basal conditions, nor did it impair
Ars-induced eIF2 phosphorylation (Fig. 4), the inhibition of SGs
is probably not due to a block to eIF2 phosphorylation as reported
for other viruses (Gale et al., 2000; Schneider and Mohr, 2003).
Future work will help unravel how HIV-1 blocks SG assembly and
whether other retroviruses have the same effect. The phenotype we
observed in this study is similar to a stress-resistant phenotype
characterized for Semliki Forest Virus (McInerney et al., 2005).
Likewise, HIV-1 has evolved strategies to prevent a stress response
that might lead to apoptosis and cell death (Dayton, 2008). We
propose that SG formation is blocked by HIV-1 to prevent
deleterious effects on viral replication and this might be achieved
by preventing the recruitment of key components required for SG
Given that polysomes, SGs and PBs are believed to be in a
dynamic equilibrium (Eulalio et al., 2007; Kedersha et al., 2000)
and share several components [Staufen1 for instance (Kedersha et
al., 2005)], we examined the relationship between SHRNPs and
PBs, the latter representing cytoplasmic RNPs involved in mRNA
metabolism and storage (Bruno and Wilkinson, 2006). Although it
was only our intent to rule out that SHRNPs were not PBs, in this
manuscript we are able to report two novel and salient findings.
First, vRNA, Gag and even Staufen1 were not detectable in PB foci
(Fig. 5; and data not shown) and, second, a dramatic decrease in
the abundance of PBs was observed in HIV-1-expressing cells and
often, PBs were not detectable (Fig. 5). The decrease in size and
abundance of PBs has also been noted when expression levels of
other cellular proteins are modified or when the translational
apparatus is stabilized (Eulalio et al., 2007). Likewise, Staufen1-
containing APOBEC3G complexes that could restrict HIV-1 do not
cofractionate with PB components (Chiu et al., 2006), and
intimations from recent work suggest that PBs might be deleterious
to HIV-1 replication (Nathans et al., 2009). We speculate that HIV-
1 expression suppresses PB formation or promotes their disassembly
by sequestration and/or relocalization of cellular proteins involved
in Ars-induced SG and PB assembly or integrity. Recent findings
support this idea in that a correlation between the expression of
heat-shock protein 72 and SG and PB assembly was drawn (Mazroui
et al., 2007). Interesting parallels can also be drawn between our
results and those of others in which SGs and PBs cannot form in
flavivirus-infected cells due in part to interaction between viral and
SG components (Emara and Brinton, 2007). HIV-1 has probably
evolved strategies to protect the vRNA from translational silencing
and/or degradation in PBs to allow for Gag expression and vRNA
selection for encapsidation. HIV-1 inhibits siRNA and miRNA
silencing pathways (Bennasser et al., 2005; Triboulet et al., 2007)
that actually prevent PB formation (Eulalio et al., 2007), supporting
our observation that HIV-1 co-opts RNA degradation machineries
to favour viral expression (Ajamian et al., 2008).
The RNA trafficking function of Staufen1 relies on its association
with a dimeric RNA in the context of an RNP in Drosophila
(Ferrandon et al., 1997), although we could not find a link between
vRNA dimerization and encapsidation levels (Fig. 8). Because
dimerization and encapsidation are coupled processes in HIV-1
(Paillart et al., 2004), it is possible that Staufen1 influences
encapsidation via its association to dimeric vRNA. Indeed, MLV
genomic RNA selection is influenced by the capacity of Gag to
bind vRNA (D’Souza and Summers, 2004). In line with this notion
is a recent report that shows that the anti-viral effect of APOBEC3G
is controlled by its capacity to bind viral RNA (Soros et al., 2007).
The depletion of Staufen1 led to a decrease in cellular Gag gene
expression but this was not due to changes in steady-state vRNA
levels (Fig. 8) or Gag protein stability (data not shown). Staufen1
might therefore act as a molecular switch to arrest translation and
to favour vRNA encapsidation, a proposed role for EF1 described
in an earlier study (Cimarelli and Luban, 1999). An effect on vRNA
translation might rely on the ability of Staufen1 to associate to
vRNA, because we show that in the presence and absence of Gag
and during stress conditions, Staufen1 remains associated with
vRNA (Figs 1-4, 7; and data not shown). Alternatively, the close
association of Staufen1 to Gag could play a role (Chatel-Chaix et
al., 2004). Support for this idea is found in work that shows that
Gag levels control the equilibrium between vRNA translation and
encapsidation via translational inhibition of vRNA for HIV-1
(Anderson and Lever, 2006) and for the Idefx retrotransposon
(Meignin et al., 2003). The dynamic link that has been established
between translationally silent PBs and Staufen1 RNPs (like that
between PBs and SG) (Zeitelhofer et al., 2008) supports the
implication of Staufen1 RNPs in translational derepression. The
formation of an HIV-1-specific RNP harbouring the components
important for vRNA translation and encapsidation is an attractive
idea. It will be interesting to determine the compositional differences
between these RNPs and the efficiency with which vRNA is
translated in SHRNPs.
To our knowledge, few studies have attempted to differentiate
between the functions of the Staufen1 isoforms, although evidence
indicates that they might be isoform-specific (Duchaine et al., 2000;
Duchaine et al., 2002; Macchi et al., 2004). The selective depletion
of the larger Staufen163kDahad little effect on Gag expression levels,
on the formation of large SHRNPs or on vRNA encapsidation levels
(Figs 1, 8; and data not shown). This isoform is generally less
abundant than Staufen155kDaand a more-subtle phenotype might
not be detected using our methods. Conversely, our siRNA and
shRNA experiments also show that Staufen163kDais preferentially
recruited into HIV-1 particles when Staufen1 is depleted in cells.
In light of the reproducibility of these results using several
knockdown strategies, future work will be needed to understand
how and why Staufen163kDais preferentially salvaged into virions
and the functional relationships that exist between the various
This work leads to a model in which distinct RNPs form that
contain vRNA, Gag, Staufen1 and, probably, other cellular proteins.
However, very much like cellular silencing foci (Fig. 10A), in HIV-
1 expressing cells these so-called SHRNPs are also in equilibrium
Journal of Cell Science 123 (3)
Journal of Cell Science
Staufen1 HIV-1-dependent ribonucleoproteins
with translating polysomes, because both CHX and Pur affect their
assembly. As is the case for SGs, apparent SHRNP size is enhanced
by limiting Staufen1 levels (Fig. 10B), probably by mechanisms
that involve the stabilization of polysomes. Although SHRNPs could
be considered as HIV-1-specific silencing foci, their formation
would serve as a scaffold for efficient packaging of the two vRNA
molecules selected per virus particle. The inhibition of cellular
silencing foci including SGs and PBs is consistent with the ability
of HIV-1 to titrate key cellular components in order to assemble
functional, virus-specific SHRNPs.
Materials and Methods
Cell culture, proviral, siRNA and shRNA transfections
Transfection of HEK293T and HeLa cells and details on the proviral constructs
(HxBRU, pNL4-XX) and siNS and siRNA sequences were described previously
(Ajamian et al., 2008; Chatel-Chaix et al., 2004; Mouland et al., 2000). siRNA
duplexes and shRNAs were used to knockdown Staufen1 gene expression. The
Staufen1 RNA is alternatively spliced to generate four transcripts. Transcript variant
3 (T3) is the longest and encodes the Staufen163kDathat contains an additional 81
amino acids in the N-terminus. The transcripts T1, T2 and T4 encode Staufen155kDa
(Fig. 1C). si55/63 (Chatel-Chaix et al., 2004) and si63 (5?-TGAATGGGTCT -
ACCTGCATTT-3?) were synthesized (Qiagen-Xeragon) and used at 10 nM. We also
performed parallel experiments using additional siRNAs targeted against different
regions of Staufen1 mRNA, and an alternative strategy to knockdown Staufen1 by
shRNA. Silencing and non-silencing shRNA (shSTF and shNS) were expressed from
an RNA polymerase-II-driven DNA expression vector, as described (Chatel-Chaix
et al., 2007). The similarity of results with the multiple siRNAs utilized increases
the likelihood that the observed phenotypes were the result of Staufen1 knockdown
and not due to off-target effects (Scacheri et al., 2004). Gag expression was rescued
in some experiments using a Rev-dependent Gag expressor (from Jaisri Lingappa,
University of Washington, Seattle, WA) (Lingappa et al., 2006). Pur and CHX (Sigma-
Aldrich) were used at 0.25 mg/ml for 4 hours before fixation. HeLa cells were
incubated for 30 minutes with Ars (0.5 mM) at 37°C, washed and incubated for
additional 30 minutes at 37°C in fresh medium and then fixed.
At 12-48 hours post-transfection, cells were lysed and equal amounts of protein from
cleared post-nuclear supernatant (40 or 50 g protein) were subjected to SDS-PAGE
and western blotting as described (Chatel-Chaix et al., 2004).
IF, FISH and LSCM
IF and combined two-colour IF/FISH analyses were performed as described (Beriault
et al., 2004; Levesque et al., 2006). LSCM and image analysis were performed using
a Zeiss Pascal LSM5. LSM Image Browser software (Carl Zeiss, Germany) was used
to estimate the size of SHRNPs. At least 50 SHRNPs were measured for each condition
in at least three experiments. vRNA-containing RNPs that were bigger than 2 m
were excluded from the statistical analysis. PB numbers were calculated by counting
DCP-1-positive foci in at least 50 cells from the 10-15 optical fields for each
experimental condition. 6? magnifications (Fig. 7) were acquired by LSCM. For
other images, higher pixel resolution images to reproduce more detail and subtle
colour transitions were obtained by increasing the number of pixels for selected regions
in Adobe Photoshop (Adobe). For combined IF/FISH analyses our initial protocol
was modified as described (Lehmann et al., 2009): after FISH with a digoxigenin-
labelled probe, vRNA was visualized by staining with biotinylated anti-digoxigenin
(Sigma-Aldrich), following by anti-biotin monoclonal antibody conjugated with Alexa
Fluor 488 (Invitrogen). The vRNA was visualized in a colour of choice (green or
red) due to the Alexa Fluor moiety on this secondary antibody. LSCM was performed
at 1024?1024 pixel resolution using the same equipment and settings. Colocalization
analyses were performed using Manders’ coefficient as described (Ajamian et al.,
2008) from at least 15 cells per experimental condition in three experiments. Intensity
plots were generated by Imaris software (Bitplane) in the measurement pane through
the centre focal plane, as described elsewhere (Lehmann et al., 2009).
A rabbit anti-Staufen1 RLS1 antibody, that recognizes both Staufen1 isoforms was
used in IF and western analyses (Thomas et al., 2005). This does not react against
Staufen2 in immunofluorescence or western blot analyses. Antibodies to Gag
(CA/p24, pr55Gag), MA/p17 polyclonal sheep serum (NIH AIDS Reference and
Reagent Program from Michael Phelan), GAPDH, hnRNP A2/B1, anti-tubulin and
calnexin are described elsewhere (Beriault et al., 2004; Chatel-Chaix et al., 2004;
Levesque et al., 2006; Mouland et al., 2000). Anti-Upf1 and anti-DCP1 were provided
by Jens Lykke-Anderson (University of Colorado, Boulder, CO). Anti-PABP1 was
provided by Nahum Sonenberg (McGill University, Montreal, Canada), anti-HuR
and anti-Ras-GAP SH3 domain binding protein (G3BP) were provided by Imed
Gallouzi (McGill University, Montreal, Canada) and anti eIF3 was purchased from
Santa Cruz Biotechnology. Anti-actin, anti-eIF2-P, anti-pan-eIF2were purchased
from Abcam; anti-L7, anti-EF1, anti-PABP1 and anti-GAPDH were purchased from
Novus Biologicals, Upstate, Sigma-Aldrich and TechniScience, respectively. A goat
anti-gp120 (Env) was obtained from the NIH (#385, from Kathelyn Steimer). The
secondary antibodies were anti-rabbit-Alexa-Fluor-594, anti-mouse-Alexa-Fluor-
488, -594 or -647 and anti-sheep-Alexa-Fluor-647 (Invitrogen).
vRNA dimerization analyses
Dimerization analysis was performed using RNA extracted from 5 g p24-equivalents
virus as described (Laughrea and Jette, 1997).
RT-PCR of Staufen1 and HIV-1 RNAs
Total RNA or viral RNA was extracted by Trizol reagent and subjected to SuperScript
One-Step RT-PCR (Beriault et al., 2004; Chatel-Chaix et al., 2004). Virus was
quantified by p24-ELISA and equal amounts of virus were used in subsequent RNA
extraction. RNA was treated with DNAse I, repurified and used in RT-PCR. Staufen1
primers were designed to amplify portions of the T3 and T2 transcripts to generate
540 bp and 270 bp PCR products, respectively: sense primer (nt 140-169) and antisense
primer (nt 670-699) of the Staufen1 cDNA (NM017453). gapdh RNA was quantified
by RT-PCR as described (Levesque et al., 2006).
Sequential affinity-based immunoprecipitation and RT-PCR
HeLa cells were mock-transfected with FLAG epitope-tagged Staufen1 expressor
(Staufen1-FLAG; (Chatel-Chaix et al., 2008) or transfected with proviral DNA (pNL4-
3) and Staufen1-FLAG. At 30-36 hours post-transfection, total cell lysates were
collected in a Nonidet P40 lysis buffer (Chatel-Chaix et al., 2007). An aliquot
representing 3% of the cell lysate (input) was used for western blotting for Staufen1-
FLAG, Gag and GAPDH. 1 mg protein was immunoprecipitated with FLAG-agarose
beads (Sigma-Aldrich) and then eluted with excess FLAG peptide (Sigma-Aldrich)
according to the manufacturer’s instructions. 10% of this eluate was assessed by
western blotting to measure the recovery of Staufen1-FLAG and then Gag was
immunoprecipitated from the first eluate with affinity-purified mouse anti-p24
monoclonal antibody (Ajamian et al., 2008). The immune complex was then eluted
by 0.1 M glycine-HCl (pH 2.5). The RNA was purified using Trizol LS (Invitrogen).
10% of each immunoprecipitate was collected for western blotting analysis. 1 g of
input total cellular RNA and 50% of that isolated from the immunoprecipitate were
used for RT-PCR. RT-PCR was performed for vRNA and gapdh mRNA as described
We thank Imed Gallouzi for critical reading of the manuscript and
helpful discussions and Jens Lykke-Andersen, Nahum Sonenberg, Imed
Gallouzi, William Rigby, Koren Mann, David Ott, Jaisri Lingappa,
Kathelyn Steimer, Michael Phelan and the NIH AIDS Reference and
Reagent Program for reagents and antibodies. Anne Monette and Lara
Ajamian are recipients of Canadian Institutes of Health Research
(CIHR) Doctoral fellowships and A.J.M. was supported by a CIHR
New Investigator and FRSQ Chercheur-boursier Senior career awards.
This work was supported by grants from the Canadian Foundation for
Innovation and the CIHR (MOP-38111, MOP-56794, OPC-83178) to
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