Trafficking of HIV-1 RNA is Mediated by Heterogeneous Nuclear Ribonucleoprotein A2 Expression and...

# 2006 The AuthorsJournal compilation # 2006 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2006.00461.x

Traffic 2006; 7: 1177–1193Blackwell Munksgaard

Trafficking of HIV-1 RNA isMediated by HeterogeneousNuclear Ribonucleoprotein A2 Expression and Impactson Viral Assembly

Kathy Levesque1,2,†, Melanie Halvorsen1,2,†,

Levon Abrahamyan1,2, Laurent Chatel-

Chaix1,2,3, Viviane Poupon1,2,4, Heather

Gordon1,2,6, Luc DesGroseillers3, Anne

Gatignol2,5,6 and Andrew J. Mouland1,2,5,6,*

1HIV-1 RNA Trafficking Laboratory, 3755 Cote-Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E22Lady Davis Institute for Medical Research-Sir MortimerB. Davis Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E23Department of Biochemistry, Universite de Montreal,Montreal, Quebec, Canada H3C 1G44Current address: Montreal Neurological Institute, McGillUniversity, 3801 University Street, Montreal, Quebec,Canada H3A 2B45Department of Medicine, McGill University, Montreal,Quebec, Canada H3A 2B46Department of Microbiology & Immunology, McGillUniversity, Montreal, Quebec, Canada H3A 2B4*Corresponding author: Andrew J. Mouland,[email protected]†These authors contributed equally to this work.

Few details are known about how the human immuno-

deficiency virus type 1 (HIV-1) genomic RNA is trafficked

in the cytoplasm. Part of this process is controlled by the

activity of heterogeneous nuclear ribonucleoprotein A2

(hnRNP A2). The role of hnRNP A2 during the expression

of a bona fide provirus in HeLa cells is investigated in this

study. Using immunofluorescence and fluorescence

in situ hybridization techniques, we show that knock-

down of hnRNP A2 expression in HIV-1-expressing cells

results in the rapid accumulation of HIV-1 genomic RNA

in a distinct, cytoplasmic space that corresponds to the

microtubule-organizing center (MTOC). The RNA exits in

the nucleus and accumulates at the MTOC region as

a result of hnRNP A2 knockdown even during the expres-

sion of a provirus harboring mutations in the hnRNP A2-

response element (A2RE), the expression of which results

in nuclear retention of genomic RNA. We also demon-

strate that hnRNP A2 expression is required for down-

stream trafficking of genomic RNA from the MTOC in the

cytoplasm. Genomic RNA localization at the MTOC that

was both the result of hnRNP A2 knockdown and the

overexpression of Rab7-interacting lysosomal protein

had little effect on pr55Gag synthesis but negatively

influenced virus production and infectivity. These data

indicate that altered HIV-1 genomic RNA localization

modulates viral assembly and that the MTOC serves as

a central site to which HIV-1 genomic RNA converges

following its exit from the nucleus, with the host protein,

hnRNP A2, playing a central role in taking it to and from

this site in the cell.

Key words: AIDS, assembly, HIV-1, hnRNP A2, RNA

trafficking, virus–host interaction

Received 23 December 2005, revised and accepted for

publication 13 June 2006, published on-line 27 July 2006

HIV-1 infection is characterized by a lengthy latent period

before the onset of acquired immunodeficiency syndrome

(AIDS). During this period, abundant viral production is kept

in check by the immune system and cells that are killed by

infection are replaced. Despite mounting a strong early

immune response, HIV-1 expression progressively de-

pletes CD4þ T cells, a situation that leads to a progressive

weakening of the immune response to infection and the

onset of AIDS (1,2). HIV-1 gene transcription generates

a primary 9-kilobase pair (kbp) RNA that has three fates

dictated by a tight regulatory circuit and temporal activities

of viral proteins. The 9-kbp RNA is multiply spliced follow-

ing transcription to generate several 2-kbp RNAs that give

rise to regulatory proteins Tat, Rev and Nef. Tat accumu-

lates and is primarily responsible for high-level transacti-

vation of the integrated HIV-1 provirus. Once a threshold

level of Rev is reached, a molecular switch occurs to

promote the inhibition of splicing of the primary transcript.

The decreased splicing activity also produces singly-

spliced RNA species (4-kbp) (3). Rev binds the Rev-

responsive cis-acting element RNA (4) to promote the nuclear

export of the 9-kbp and singly-spliced 4-kbp HIV-1 RNAs.

The 9-kbp RNA is not only a substrate for the translation

machinery to generate structural (Gag) and viral enzymes,

but in addition, it is selected for encapsidation into new virus

particles. The 4-kbp RNAs are translated to produce auxiliary

proteins and the viral envelope protein, Env. Without Rev,

both of the latter RNA species are retained in the nucleus

and are presumably degraded by cellular mechanisms, and

as a consequence, viral production is severely impaired.

Nuclear export by Rev is also aided by the activities of

multiple host cell proteins. Several proteins, including

RNA-binding proteins, SR-splicing proteins, RNA helicases

and other proteins such as actin, influence HIV-1 Rev

activity to export RNA from the nucleus (5–7). Very little

is known about HIV-1 RNA trafficking following disengage-

ment of Rev in the cytosol. Recent data support the notion

that additional viral and host proteins engage to promote

1177

trafficking to sites of translation or assembly, identifying

roles for the cellular proteins, Sam68 and the human Rev-

interacting protein (hRIP) in these processes (5,8,9). Viral

Gag and motor proteins like KIF-4 also appear to play a role,

but the mechanisms of action remain uncharacterized

(10,11). We implicated heterogeneous nuclear ribonucleo-

protein A2 (hnRNP A2) in this step in a previous study in

which we identified HIV-1 RNA trafficking sequences in

several HIV-1 RNAs (known as the hnRNP A2-responsive

elements or A2RE) (12). We also showed that the interac-

tion of hnRNP A2 with the HIV-1 RNA via these sequences

was important for HIV-1 RNA trafficking from the nucleus,

whereby a single point mutation in the A2RE resulted in

nuclear sequestration of genomic RNA. Blocking this inter-

action also impacted on HIV-1 RNA, Gag and Vpr expression

patterns and the packaging of viral genomic RNA into virions

(13). Because RNA compartmentalization in the nucleus

only occurred late in the replication cycle, comparatively

normal levels of intracellular Gag levels accumulated. This

work suggested that hnRNP A2 acts at a specific time that

coincides with a late stage of the replication cycle.

In this study, we identify the roles of hnRNP A2 in the fate

of HIV-1 genomic RNA from the nucleus and into the

cytosol. We used small interfering (si) RNA to knockdown

hnRNP A2 gene expression in HIV-1-expressing HeLa

cells. Near-complete knockdown of hnRNP A2 had a sig-

nificant impact on HIV-1 RNA localization. The phenotype

was characterized by an accumulation of HIV-1 genomic

RNA at the microtubule-organizing center (MTOC), as

identified by fluorescence in situ hybridization (FISH) and

immunofluorescence (IF) analyses. This was also ob-

served in the context of the expression of an A2RE mutant

provirus that normally results in nuclear sequestration of

genomic RNA due to loss of association of hnRNP A2 to

the RNA. These new data reveal a nuclear retention

function for hnRNP A2. Kinetic studies and studies that

follow the fate of cytoplasm-localized HIV-1 RNA reveal that

the accumulation of RNA occurs very rapidly at the MTOC

and that HIV-1 genomic RNA egress within the cytoplasm

is dependent on hnRNP A2 expression. Gag expression

was not affected by hnRNP A2 knockdown and its traffick-

ing can be physically separated from that of the genomic

RNA by promoting the recruitment of dynein/dynactin

complex to the MTOC. This work represents the first study

to demonstrate that the fate of the HIV-1 genomic RNA

during proviral gene expression is mediated directly by

expression levels of the host cell protein, hnRNP A2.

Results

Efficient knockdown of hnRNP A2 expression is

achieved and has little effect on Gag synthesis or

HIV-1 RNA splicing

HeLa cells have been used in studies on viral RNA egress

(14,15), and successful depletion of heterogeneous

nuclear ribonucleoproteins (hnRNPs) by siRNA has

recently been documented in this cell line (16). To study

the involvement of hnRNP A2 in HIV-1 RNA trafficking, we

used 21-bp siRNA duplexes to specifically target its

expression. Cells were first transfected with siRNA du-

plexes and then cotransfected with proviral DNA, HxBRU

and the same siRNA duplexes 24 h later. The siRNA

duplexes were designed to target hnRNP A2 (siA2) and

hnRNP A1 (siA1). A nonsilencing siRNA (siNS) was also

included. At 30 h post-HxBRU transfection, protein expres-

sion was assessed byWestern analyses. To determine the

efficacy of the siRNA-mediated knockdown, hnRNPs were

identified using a pan-specific hnRNP polyclonal antibody

that recognizes predominantly hnRNP A1 and hnRNP A2

(16). Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) was used as a loading control (Figure 1A).

Whereas in mock, HxBRU or HxBRU þ siNS-transfected

cells, hnRNP A2 and A1 expression levels were constant

(Figure 1A, lanes 1–3); cells transfected with siA1 resulted

in an 85% reduction in hnRNP A1 expression (Figure 1A,

lane 4). When hnRNP A2 was targeted, it was also

specifically knocked down by 90% (lane 5). The siA2

treatment did not have an impact on the 55-kDa precursor

Gag (pr55Gag) synthesis in cells, whereas siA1 had a signifi-

cant negative impact on pr55Gag expression and viral

production when this siRNA was used alone or in combi-

nation with siA2 (Figure 1A, lane 4; data not shown). Viral

production levels were reduced by 29% (�11%, SEM) in

siA2-treated cells, and furthermore, a striking decrease in

infectivity was found in siA2-treated cells (65 � 6%, SEM;

Figure 1B). The results shown in Figure 1B represent

averages from 10 experiments each (pr55Gag synthesis

and viral production) and four experiments (viral infectivity).

There were no general effects on cell cycle distribution

when hnRNP A1 and hnRNP A2 were individually knocked

down [not shown; (16)].

Because hnRNPs have been shown to be involved in RNA

splicing in vitro, we therefore performed RNA splicing

analyses to determine if the knockdown of hnRNPs had

effects at this level. Cells were mock transfected or

transfected with HxBRU alone, HxBRU and siNS, and

siA2 or siA1. The knockdown was verified by Western

blot analyses (Figure 1C) and compared with the loading

control, GAPDH. Representative HIV-1 proteins encoded

by 4- and 2-kbp RNAs were also quantitated by Western

analyses, and this analysis showed that encoded proteins

gp160 and Rev levels were unaffected, indicating that the

expression of their RNAs was not affected in knockdown

conditions (Figure 1C). The RNA was isolated from cells

and first assessed by Northern analyses. There were no

differences between the relative levels of 9-, 4- and 2-kbp

RNAs in siNS and siRNA targeting hnRNP expression (data

not shown). In other experiments, RNA was subjected to

reverse transcription–polymerase chain reaction (RT-PCR)

to amplify 9-, 4- and 2-kbp HIV-1 RNA species using

specific sets of oligonucleotides as described in Materials

and Methods. The relative levels of genomic 9-kbp RNA

were virtually constant and the quantity and profiles of the

1178 Traffic 2006; 7: 1177–1193

Levesque et al.

RT-PCR products obtained for the 4- and 2-kbp RNA

profiles in all conditions were similar (Figure 1D, panels

i–iii). These analyses show that there were no marked

effects of siRNA treatment on the relative levels of HIV-1

RNA species when the hnRNPs are individually knocked

down. Inclusion of radiolabeled deoxy-cytidine-5’-triphos-

phate (dCTP) in the last two cycles of the PCR reaction

allowed us to label the PCR products that represent singly-

and multiply-spliced HIV-1 RNAs and visualize them by

autoradiography in denaturing polyacrylamide gels as we

have performed previously (13,17). Analyses of these

radiolabeled RT-PCR products in denaturing gels further

demonstrated that there were no qualitative or quantitative

changes in the pattern of spliced RNA species following

hnRNP knockdown (not shown). The combination of

siA1 þ siA2 also had no effect on the pattern or levels of

HIV-1 RNA splicing (data not shown).

Depletion of hnRNP A2 results in paranuclear

localization of HIV-1 RNA

Because hnRNP A2 expression is critical for HIV-1 RNA

trafficking in murine cells, we next determined how hnRNP

A2 knockdown affected genomic RNA localization in cells

that express HIV-1. HeLa cells were mock transfected or

transfected with HxBRU and siNS, siA1 or siA2. hnRNP A1

and hnRNP A2 were identified by IF and were found to

have a predominant nuclear staining pattern in cells (red

fluorescence in Figure 2A–D). The HIV-1 genomic RNA

was identified by FISH. This analysis revealed a dispersed

and punctate distribution of genomic RNA in HxBRU þsiNS- and siA1-treated cells (Figure 2B,C), even when

hnRNP A1 was efficiently knocked down (Figure 2C,G:

two cells are shown: the cell on the left abundantly

expresses hnRNP A1 and the cell on the right in the same

panel shows efficient knockdown of hnRNP A1). In siA2-

treated cells, a significant reduction in the expression of

hnRNP A2 was observed, which was reflected in a marked

loss of red fluorescence staining intensity of hnRNP A2 in

cells (Figure 2D). In these cells, the distribution of RNA

was dramatically changed such that efficient hnRNP A2

knockdown resulted in an accumulation of HIV-1 genomic

RNA in the cytosol that was immediately adjacent to the

nucleus (paranuclear staining; Figure 2D,H). This RNA

staining pattern was radial and localized in a discrete,

cytoplasmic foci that did not coincide with the nuclear

membrane. This staining pattern of genomic RNA was

found in 94% of cells in which hnRNP A2was confirmed to

be knocked down by IF analysis. The histogram in Figure 2I

presents averages from seven independent knockdown

experiments in which several hundred cells were imaged

for siNS, siA1 and siA2. These data demonstrate a specific

effect of the depletion of hnRNP A2 on the accumulation of

Figure 1: Efficient knockdown of hnRNPs by siRNA. A) HeLa cells were mock transfected (lane 1) or transfected with HxBRU proviral

DNA alone (lane 2) or HxBRU and siRNA duplexes (siNS, nonsilencing control, lane 3; siA1, siRNA to knockdown hnRNP A1 expression,

lane 4; siA2, siRNA to knockdown hnRNP A2 expression, lane 5). GAPDH served as a loading control. The expression levels of hnRNPs and

pr55Gag were determined by Western blotting as described in Materials and Methods. B) The effects of siRNA treatment on pr55Gag

synthesis (black bars), viral production (gray bars) and viral infectivity (open bars) are represented in a histogram. The SEMs are presented

as error bars. Relative levels are shown, with the value for HxBRU set to 1.0. C) For RNA analyses in D, Western blot analyses for hnRNPs,

gp160 and Rev proteins. D) HIV-1 RNAs were reverse transcribed and PCR amplified to identify (i) 9-kbp RNA (genomic) (ii) 2-kbp RNA and

(iii) 4-kbp RNA species exactly as described (17). The expected size ranges of PCR products were obtained. The GAPDH RNAwas used as

a loading control for cellular RNA levels and the exclusion of RT or RNA in the RT reaction served as specificity controls. ND, viral infectivity

not determined for siA1-treated cells.

Traffic 2006; 7: 1177–1193 1179

hnRNP A2-Mediated HIV-1 RNA Trafficking

HIV-1 genomic RNA in this region of the cell. This RNA

localization phenotype was found in only 4% of cells

treated with siA1, with a confirmed knockdown by IF

analysis (Figure 2C). We therefore used the RNA staining

pattern as a reference to identify cells in which hnRNP A2

was knocked down in subsequent imaging and FISH

analyses. Knockdown of hnRNP A2 was confirmed by

Western and IF analyses for all experiments.

Knockdown of hnRNP A2 results in an accumulation of

HIV-1 genomic RNA at the MTOC

Because of the distinct distribution of genomic RNA

observed in siA2-treated cells (Figures 2, S1 and S2), we

next determined how the genomic RNA was localized in

relation to the staining pattern of a-tubulin, a component of

microtubules. We also wished to determine if the radially

distributed genomic RNA coincided with theMTOC. The IF

Figure 2: siA2 treatment results in a significant reduction in hnRNP A2 expression and a paranuclear distribution of genomic

RNA. HeLa cells were transfected as described in the legend of Figure 1. Cells were fixed at 30–36 h posttransfection and processed for

IF/FISH coanalyses for hnRNP expression and genomic RNA, respectively. The hnRNP A2 and hnRNP A1 were identified by IF using

purified rabbit antibodies to anti-hnRNP A2 (panels A, B, D–F and H) and anti-hnRNPA1 (panels C and G). The hnRNPA2 is detected as a red

fluorescence signal (panels A, B, E and F). The hnRNP A1 and hnRNP A2 expression levels are knocked down in siA1- and siA2-treated cells

(panels C and D, respectively) as shown by faint red fluorescence signals. The distribution of genomic RNA in siA1-treated cells is shown in

panels C and G, in siA2-treated cells in panels D and H. Panels E–H show merged images of hnRNP A2 in red, genomic RNA in green and

the nuclei in blue. I) The localization of genomic RNA found in siNS-, siA1- and siA2-treated cells is presented as averages from seven

independently performed experiments. White bar ¼ 10 mm in panels E–H.

1180 Traffic 2006; 7: 1177–1193

Levesque et al.

and FISH coanalyses were performed to identify the

tubulin-based cytoskeleton and genomic RNA in control

siNS-, siA1- and siA2-treated cells. In siNS-treated cells

and likewise in siA1-treated cells, genomic RNAwas found

to be distributed throughout the cells in a punctate staining

pattern as shown above (Figure 2). In siA2-treated cells,

the genomic RNA staining coincided with intense tubulin

staining in the pericentriolar region (Figure 3A–D). In siA2-

treated cells, genomic RNAwas also observed to reside on

microtubule tracks either emanating from the MTOC or at

distal sites near the periphery of the cell (data not shown).

Dual color analyses and black and white representations of

the IF and FISH images demonstrate that the area at which

the genomic RNA accumulates corresponds to the region

that overlaps the MTOC, a structure from which micro-

tubules polymerize (Figure 3D). A reduction in hnRNP A2

expression, but not that of the related hnRNP A1, results in

an accumulation of genomic RNA at this cellular site,

indicating that the HIV-1 genomic RNA may traffic to and

from the MTOC region during the gene expression stages

of virus replication.

We also explored whether the microtubule-disrupting

agent, nocodazole, altered the localization of genomic

RNA in siA2-treated cells. Cells were briefly treated with

nocodazole for 30 min before fixation to disrupt micro-

tubules but withminimal effects on the localization of other

organelles such as the Golgi (18). In these cells, nocoda-

zole disrupted the microtubule cytoskeleton as shown by

the resultant disaggregation of microtubule streaming in

cells (Figure 3E,H). This treatment resulted in a diffusion of

the genomic RNA from the MTOC region consistent with

a dependence on an intact microtubule cytoskeleton for

the anchoring of the genomic RNA at this region (Figure 3

E–H). To demonstrate this more clearly, hnRNP A2 expres-

sion in cells was identified by IF in mock- and siA2-treated

HxBRU-expressing cells (Figure 3I). hnRNP A2 was abun-

dantly expressed in mock-treated cells (Figure 3I, top

panel). The siA2 treatment resulted in a marked loss of

hnRNP A2 staining and a redistribution of genomic RNA to

the MTOC region (Figure 3I, middle panel). Again, brief

nocodazole treatment resulted in a loss in the MTOC-

localized genomic RNA and this was clearly observed in

hnRNP A2-depleted cells (Figure 3I, bottom panel), show-

ing that anchoring to the MTOC region depends on

polymerized microtubules. However, nocodazole treat-

ment also promotes a diffusion of late endosomes/lyso-

somes from the MTOC region (19), so these results may

also point to these vesicular organelles as sites of accu-

mulation of HIV-1 genomic RNA.

We next confirmed the localization of the genomic RNA by

costaining with an MTOC-resident protein and nucleator of

microtubules, g-tubulin. For this analysis, laser scanning

confocal microscopy was necessary to accurately localize g-tubulin and genomic RNA staining in cells. Similar to the

staining pattern shown in Figure 2, genomic RNA staining

was punctate throughout the cell (one representative cell is

shown in Figure 4, panels A–D). The g-tubulin staining was

discreet and characterized by a distinctive and intense

staining adjacent to the nucleus in most cells (indicated by

arrows in Figure 4). A black and white rendition is shown in

Figure 4C to provide better visualization of the g-tubulin/MTOC staining. A merged image of g-tubulin and HIV-1

genomic RNA in siNS-treated cells is shown in panel D. In

siA2-treated cells, the genomic RNA was predominantly

found in the distinct cytoplasmic region adjacent to the

nucleus (one representative cell is shown in Figure 4E). The

Figure 3: HIV-1 genomic RNA localizes to the MTOC in siA2-treated cells. Cells were transfected with HxBRU and siNS or siA2, and

at 30–36 h posttransfection, the cells were fixed and analyzed for microtubule staining using an anti-a-tubulin anti-serum and the

localization of genomic RNA (by FISH as green fluorescence). A–D) a-tubulin (red) and genomic RNA (green) staining in siA2-treated cells.

The MTOC is highlighted by the inset in panel D. Cells were treated with nocodazole for 30 min prior to fixation (panels E–H) and then

stained for a-tubulin as red fluorescence in all cells in panels E and G or more vividly in black and white in panel H. I) In complementary

experiments, hnRNP A2 expression (red) was analyzed by IF and the HIV-1 RNA by FISH (in green) in mock-transfected cells (top panel) or

cells transfectedwith HxBRUþ siA2 (middle panel), followed by nocodazole treatment (bottom panel) asmentioned above.White bar¼ 10

mm in panels D and H and panels presented in I.

Traffic 2006; 7: 1177–1193 1181


MTOCwas identified by g-tubulin staining in red fluorescence(panel F) and a black and white rendition is shown in panel G.

In siA2-treated HIV-1-expressing cells, laser scanning confo-

cal imaging shows that the genomic RNAwas localized in the

direct vicinity of theMTOC as shown by a radial RNA staining

pattern emanating from the MTOC, identified by the distinct

and central g-tubulin staining in these cells [Figure 4, panel H

(and see inset showing magnified MTOC region)].

HIV-1 RNA accumulation occurs rapidly at the MTOC

A kinetic study was performed to determine how fast the

HIV-1 genomic RNA accumulated at the MTOC in siA2-

treated cells. Cells were transfected with either siNS or

siA2 and then with HxBRU. At 12, 20 and 30 h later, cells

were fixed and stained for hnRNP A2 and genomic RNA. At

the first time-point tested (12 h) in siNS-treated cells, the

genomic RNA was detectable at low levels and hnRNP A2

was abundantly expressed (Figure 5A). At the following

time-points in siNS-treated cells, genomic RNA became

more abundant and was distributed in a punctate staining

pattern (Figure 5B,C). When hnRNP A2 and genomic RNA

were identified by FISH/IF coanalyses in siA2-treated cells

(panels D–I), MTOC-localized genomic RNA was apparent

at 12 h in cells that were efficiently knocked down for

hnRNP A2 (identified by an arrow in Figure 5D). A set of

overexposed images in red, green and blue channels is

presented in order to first delineate the outline of the

nucleus of cells and second, to demonstrate the efficient

knockdown of hnRNP A2 as indicated by the faint red

fluorescence signal for hnRNP A2 in the HIV-1 RNA-

expressing cells shown at all time-points (Figure 5D–F).

The next set of panels represent normal exposed images,

and DAPI staining is included to characterize the distribu-

tion of HIV-1 RNA in relation to the nucleus (Figure 5G–I).

These results demonstrate again that in cells in which

hnRNP A2 expression is significantly knocked down, the

genomic RNA accumulates at the MTOC. The rapidity with

which this occurs suggests that hnRNP A2 serves key

roles in nuclear retention of RNA and genomic RNA

trafficking to the cytosol; moreover, these results indicate

that genomic RNA traffic might be blocked at this site in

these conditions.

The hnRNP A2 is required to traffic HIV-1 RNA from the

MTOC and maintain localization in the cytoplasm

Our previous work showed that hnRNP A2 expression is

necessary for trafficking of A2RE-containing HIV-1 RNAs

from the perikaryon into the dendrites of oligodendrocytes.

Furthermore, antisense-DNA-mediated suppression of

Figure 4: HIV-1 genomic RNA and g-tubulin staining patterns coincide in siA2-treated, HIV-1-expressing cells. HeLa cells were

mock transfected or transfected with HxBRU alone or with either siNS (panels A–D) or siA2 (panels E–H). At 30–36 h later, cells were

harvested forWestern blot analysis (top panel) and fixed and costained for HIV-1 RNA by FISH (panels A and E) and g-tubulin by IF (B and F).

Black and white renditions of the g-tubulin staining are shown in panels C and G and merged color images are shown in panels D and H.

Western blot analyses for hnRNPs and g-tubulin was performed to verify knockdown of hnRNP A2 (top panel). The g-tubulin was used as

a loading control. The inset in panel H magnifies the MTOC region in a cell (boxed). White bar ¼ 10 mm in panels C and G.

1182 Traffic 2006; 7: 1177–1193

Levesque et al.

hnRNP A2 expression in these cells blocked trafficking

to distal dendrite regions of these cells (A.J.M., E. Barbar-

ese, E. A. Cohen, J. Carson, unpublished). Therefore, in

order to determine if hnRNP A2 functions to promote HIV-

1 RNA transport from the MTOC in provirus-expressing

cells, an experiment was designed to examine the depen-

dence of hnRNP A2 expression on the fate of HIV-1 RNA in

the cytosol. To do this, cells were first transfected with

proviral DNA and expression of HIV-1 was allowed to

progress for 24 h. At this time-point, one set of cells was

fixed and stained for genomic RNA (Figure 6A). The

distribution of RNA was punctate in both nuclear and

cytoplasmic compartments. Cells were then treated with

siRNA (siNS or siA2), and 4 h later, the cells were treated

with Leptomycin B (LMB) to block nuclear RNA export.

The LMB will block genomic RNA export because this is

CRM1 dependent but will not affect hnRNP shuttling (20).

In this way, we examined the fate of the genomic RNA in

the cytoplasmic compartment following hnRNP A2 knock-

down. After 12 h of LMB treatment, cells were fixed and

stained for hnRNP A2 by IF to identify siA2 knockdown

cells and for genomic RNA by FISH. DAPI staining was

used to identify the nucleus. In all siNS-treated cells, HIV-1

RNA was nuclear following LMB treatment and RNA

staining was absent in the cytosol (Figure 6B,C). Presum-

ably at this time-point, most of the genomic RNA that had

accumulated in the cytosol during the first 24 h of HIV-1

expression had been trafficked out of the cell into new

virions, for instance, and/or had been turned over. In cells

exhibiting a significant knockdown of hnRNP A2 expres-

sion (Figure 6D,E), the genomic RNA focalized in a cyto-

plasmic region reminiscent of that observed in siA2-

treated cells. This genomic RNA distribution was observed

as early as at 8 h post-LMB treatment (data not shown). The

distribution of genomic RNA differed from the siA2 pheno-

type shown in Figures 2–6 such that genomic RNA also

accumulated in the nucleus demonstrating that there was

an efficient block to nuclear RNA export in LMB- and siA2-

treated cells. This analysis reveals that in LMB-treated

cells in which nuclear RNA export is blocked, the genomic

RNA found in theMTOC is derived from the cytosol. These

results also show that the MTOC region might and that

hnRNP A2 expression is critical for trafficking of the

genomic RNA away from the MTOC.

Figure 5: Time course of the accumulation of genomic RNA at the MTOC in siA2-treated cells. HeLa cells were transfected with

either siNS or siA2 and then with HxBRU. At 12, 20 and 30 h, the cells were fixed and processed for IF/FISH. The hnRNP A2 expression

levels are shown as red fluorescence signals in cells. Genomic RNA expression levels and distribution in siNS-treated cells are shown

(panels A–C). In siA2-treated cells, the localization of genomic RNA at theMTOCwas apparent at 12 h. Overexposed images are presented

in panels D–F to highlight the weak signals for hnRNP A2. Panels G–I show normal exposed merged images of genomic RNA and hnRNP

A2 and DAPI staining in blue. White bar ¼ 10 mm in panels C, F and I.

Traffic 2006; 7: 1177–1193 1183


Recruitment of HIV-1 genomic RNA to the MTOC by

Rab7-interacting lysosomal protein expression

The MTOC region has been shown to play host to late

endosomes, lysosomes and other cellular organelles.

These organelles are trafficked intracellularly by motor

proteins and adaptor proteins. Our previous work impli-

cated kinesin motor proteins in the translocation of HIV-1

RNAs. It remains unclear, however, whether these motor

proteins act to propel HIV-1 RNA in a vesicle-/membrane-

bound state or as naked ribonucleic protein (RNP) com-

plexes. Translocating RNP complexes (or RNA trafficking

granules) are known to contain RNAs, translation factors,

motor proteins and usually both plus-end- and minus-end-

directed motor proteins, kinesin and dynein (21). We were

interested to determine if the disruption of motor activities

was responsible for HIV-1 RNA trafficking in HeLa cells.

Because the expression of the Rab7-interacting lysosomal

protein (RILP) recruits dynein–dynactin motor complexes

to the MTOC region (19), we evaluated the localization of

genomic RNA following the expression of RILP in HIV-1-

expressing cells. Cells were transfected with HIV-1 proviral

DNA with or without a myc-tagged RILP-expression vector

(19) and processed for IF and FISH coanalyses. In this set

of experiments, cells were also treated with the complete

series of siRNAs that is listed in the headings of the ex-

pression blots shown in Figure 7A to determine if knock-

down conditions would affect the RILP-mediated effects.

The knockdown of hnRNP expression was very effective,

and both hnRNP A1 and hnRNP A2 were barely detectable

in the respective knockdown conditions (Figure 7A, lanes

3, 6 and 7). The myc-RILP was abundantly expressed in

cells (Figure 7A, lanes 4–7). The effects of siA1 on pr55Gag

synthesis are demonstrated here again showing that siA1

treatment resulted in a three-fold decrease in pr55Gag

synthesis levels (Figure 7A, lane 6 and Figure 1A). Equal

loading in the lanes is shown by the invariant levels of

GAPDH and g-tubulin expression in cell extracts (Figure 7

A). In siNS-transfected cells, the distribution of Gag and

Figure 6: hnRNP A2 is required for cytoplasmic trafficking of genomic RNA from the MTOC in HIV-1-expressing cells. HeLa cells

were transfected with HxBRU and HIV-1 expression proceeded from 0–24 h as shown in the scheme in the top panel. At this time, cells

were fixed and processed for FISH analyses (panel A) and other cells were transfected with either siNS (or siA1; not shown) as control or

siA2 to specifically target hnRNP A2 expression. Four hours later (time ¼ 28 h), cells were treated with LMB for 12 h and were then fixed

and processed for IF/FISH coanalyses (40 h). A) HIV-1 genomic RNA at 24 h. Following siRNA and LMB treatments, hnRNP A2 and

genomic RNA localization were assessed in cells. B,C) siNS-treated cells. D,E) siA2-treated cells. Knockdown was confirmed by Western

blot analysis (not shown). White bar ¼ 10 mm in panels A, C and E.

1184 Traffic 2006; 7: 1177–1193

Levesque et al.

genomic RNA was similar to what we have observed

previously and showed a predominantly punctate cellular

staining pattern (Figure 7B–D). The overexpression of

RILP severely affected the intracellular distribution of both

pr55Gag and genomic RNA. The RILP expression resulted

in the accumulation of pr55Gag at the cell periphery

(Figure 7E). Genomic RNA strikingly accumulated in a para-

nuclear region (Figure 6F), similar to the resultant distribu-

tion of genomic RNA at the MTOC in cells treated with

siA2 (Figure 2). Of note is that this localized distribution of

genomic RNA was seen in all cells that had been cotrans-

fected with HxBRU and RILP for any of the siRNAs tested

(Figures 7F and S3). Myc-RILP was distributed at the

MTOC consistent with previous findings that showed

localization with late endosomal/lysosomal membranes

[Figure 7H; (19,22)]. These analyses also revealed a high

Figure 7: RILP expression results in a redistribution of HIV-1 genomic RNA, but not Gag, to the MTOC region. HeLa cells were

transfected with HxBRU and the siRNAs as indicated in panel A with or without the myc-RILP-expression plasmid. Cell lysates were harvested

at 30–36 h post-transfection. A)Western blot analyses of hnRNPA2, hnRNPA1,myc-RILP, pr55Gag, g-tubulin andGAPDH.myc-RILP expression

was verified using an anti-myc antibody. GAPDHwas quantitated in cell lysates to normalize for gel loading. IF/FISH coanalyses revealed pr55Gag

(in red, panels B, D, E and G), genomic RNA (in green; panels C, D, F, G, I and J) and nuclei (by DAPI staining; panels C, D, F, G, I and J). Merged

images are shown in panels D, G and J. Genomic RNA andmyc-RILP were identified concomitantly in cells (panel J). Arrows in panel G indicate

punctate staining of genomic RNA at cell periphery. See Figure S3 for additional analyses. White bar ¼ 10 mm in panels B, D and H.

Traffic 2006; 7: 1177–1193 1185


degree of colocalization between RILP and the HIV-1

genomic RNA (from 78 to 93% as a range from four ex-

periments, calculated as described in Materials and Meth-

ods; Figure 7I,J). A small proportion of the HIV-1 genomic

RNA was also consistently observed in punctate staining

at the cell periphery with pr55Gag (Figure 7G, arrows; see

Discussion). The overexpression of RILP also resulted in a

60–75% reduction in virus production that was not

enhanced by any of the siRNA treatments (data not shown).

Discussion

hnRNP A2: Multiple roles in HIV-1 post-transcriptional

regulation

In this study, we demonstrate that hnRNP A2 plays a role

in HIV-1 genomic RNA trafficking using detailed imaging

and biochemical techniques. Several new conceptual

advances on HIV-1 egress and viral assembly are sup-

ported by this work. hnRNP A2, a bona fide RNA trafficking

protein in both lower and higher eukaryotes (23–25),

appears to be a dominant player at multiple steps of the

RNA trafficking pathway. In this study, we demonstrate

that the expression levels of hnRNP A2 play a central role

in mediating HIV-1 RNA trafficking out of the nucleus, to

and then from the MTOC (Figures 2–6). First, hnRNP A2

must bind the A2REs in the nucleus to contribute to HIV-1

RNA nucleocytoplasmic trafficking (13). Second, the

expression level of hnRNP A2 appears to mediate this

event and represents either a retention signal or a dominant

signal for nucleocytoplasmic RNA export (Figure S4). Third,

hnRNP A2 must be expressed to sufficient levels to allow

the trafficking of the genomic RNA from the MTOC region

in the cytoplasm (Figure 6). This last role does not appear

to be dependent on any particular late step of the HIV-1

replication cycle as genomic RNA accumulation at the

MTOC occurs as early as 12 h and as late as 36 h post-

transfection (Figure 5). These observations suggest that

hnRNP A2 is an engagement factor for genomic RNA

trafficking, with hnRNP A2 and its cognate cis-sequence

being central components in the regulation of this path-

way. The data presented here are consistent with the

concept that the fate of a messenger RNA (mRNA) is

determined by the proteins with which it interacts through-

out its travels in the nucleus and cytoplasm (26–28).

Predominantly, nuclear proteins such as hnRNP A2 are

also localized in the cytoplasm (29) and this explains their

described roles in cytosolic RNA trafficking (30,31).

The hnRNP A2 RNA trafficking pathway involves the

passage of RNA via the MTOC and this likely represents

one of the first points of convergence of genomic RNA

following its exit from the nucleus. RNA traffic via this

structure must be transient and rapid as there is little

evidence for accumulation of RNA at the MTOC in cells

that express hnRNP A2. Interestingly, genomic RNA

staining can be found to localize closely with g-tubulin in

cells (laser scanning confocal images presented in Figure 4

A–D). For type D retroviruses such as Mason�Pfizer

monkey virus, capsid assembly occurs at this site imme-

diately following Gag synthesis before the capsid is

trafficked through the cytoplasm and then out of the cell

at the plasma membrane (32,33). Interestingly, HIV-1 as

well as other retroviruses use this site in an early step

immediately following infection at which viral capsids or

RNP complexes (the preintegration complex in the case of

HIV-1) converge before nuclear entry (34,35). These re-

sults support the concept that theMTOC is a site via which

HIV-1 gets in and out of the nucleus (35). Nevertheless,

future work will be necessary to unequivocally identify

genomic RNA trafficking to and from the MTOC using

refined molecular and live cell imaging techniques.

hnRNP A2, but not hnRNP A1, functions in HIV-1 RNA

trafficking

The specificity of the RNA trafficking phenotype between

that of hnRNP A2 and hnRNP A1 is another interesting

aspect of this work. Whereas hnRNP A1 knockdown

significantly affects pr55Gag synthesis without any effect

on RNA localization (Figures 1 and 2), knockdown of

hnRNP A2 has virtually no effect on pr55Gag synthesis

but has a major impact on RNA localization and trafficking.

Our splicing data also demonstrate that there are no

marked effects on HIV-1 RNA levels or on RNA splicing

patterns in both conditions as demonstrated in two types

of assays (Figure 1 and data not shown) despite in vitro

and in vivo evidence that support a role for these hnRNPs

in splicing regulation (36–38). This is the first study to

demonstrate that compromised levels of hnRNP A1 neg-

atively affect pr55Gag synthesis that is not the result of

effects at the levels of splicing or RNA expression or

localization (Figures 1, 2 and 7). This reproducible effect on

pr55Gag synthesis might therefore be the result of an

influence on the HIV-1 internal ribosome entry site (IRES)

(39,40). Consistently, hnRNP A1 expression positively

influences translation initiation via an IRES in the fibroblast

growth factor mRNA (41). However, the degree to which

pr55Gag synthesis is blunted cannot be completely attribut-

able to an effect of hnRNP A1 on HIV-1 RNA IRES function.

The involvement of hnRNP A1 at multiple levels of retro-

viral posttranscriptional regulation is supported by several

studies (42,43), and further work will be required to define

how the depletion of hnRNP A1 leads to compromised

pr55Gag expression. Thus, the functions of hnRNP A1 and

hnRNP A2 during HIV-1 replication are now being resolved

to better define how their functions overlap and differ.

hnRNP A2: A role in genomic RNA retention in the

nucleus?

Our previous work revealed that the hnRNP A2–A2RE

interaction in the nucleus influences nuclear export of

genomic RNA (Figure 8, step 1a). Two sets of data shown

here suggest that hnRNP A2 has a role in nuclear retention

of genomic RNA. First, upon hnRNP A2 knockdown, we

generally observe lower levels of nuclear genomic RNA

(Figure 8, step 1b). This observation suggests that the

1186 Traffic 2006; 7: 1177–1193

Levesque et al.

reduction of hnRNP A2 expression results in rapid nuclear

export of the genomic RNA to theMTOC (Figures 2 and 5).

These results might point to a role of hnRNP A2 expression

in the balance between nuclear retention and export of

RNA. In cases when hnRNP A2 is knocked down during

expression of proviral gene (HxBRU or A2RE-2/A8G),

nuclear retention of the genomic RNA is overcome to

become cytosolically localized at the MTOC region

[Figures 2–6, S4 and 8 (steps 1b and 1c)]. Thus, in the

presence of an intact or mutated A2RE, the depletion of

hnRNP A2 serves as a dominant signal for nuclear export.

This even appears to be the case in a Rev-background

where the RNA is released into the cytoplasm when

hnRNP A2 is depleted by siRNA (A. J. M., manuscript in

preparation). The depletion of hnRNP A2 somehow alters

the nuclear HIV-1 RNP to overcome RNA retention in

the nucleus that is achieved by a variety of signals. It is

not clear at present if hnRNP A2 depletion has any

impact on CRM1-mediated RNA export or confers

CRM1-independent export of genomic RNA, but the data

presented in Figure 6 suggest that it does not as RNA

remains in the nucleus in the presence of LMB.

When the hnRNP A2–HIV-1 RNA interaction is prevented

by A2RE mutation, it is retained in the nucleus despite the

expression of Rev (Figure 8, step 1d), the key viral

mediator of nucleocytoplasmic RNA trafficking. This effect

is also coincident with the late expression phase at which

a threshold of Rev is reached and a switch between

splicing and nuclear export of unspliced RNA occurs

(13,44). The association of hnRNP A2 with the A2RE or

its expression levels appear to influence the nuclear export

of the Rev-dependent genomic RNA, a conclusion that

was hinted to in our previous study (13). One possibility is

that this could be via either a direct action by binding to Rev

or interaction with CRM1, as was shown for hRIP and the

RNA helicase, DDX3 (5,9,45). Alternatively, hnRNPs have

been shown to synergize with Rev (43), interact with the

Rev-responsive RNA element of HIV-1 (46) and interfere

with Rex RNA binding in HTLV-1 (47). The hnRNPs can also

interact with known regulators of retroviral RNA nucleo-

cytoplasmic export such as Sam68 (48). In this study, the

depletion of hnRNP A2may somehowmodulate the nuclear

HIV-1 RNP, making the genomic RNA more competent for

export. Nevertheless, it is clear from this work that hnRNP

A2 expression levels are important determinants in mediat-

ing genomic RNA export from the nucleus.

Nuclear export to the MTOC: Via dynein-dependent

translocation on microtubules?

Following nuclear export, we propose that HIV-1 genomic

RNA makes its way to the MTOC for subsequent rapid

trafficking toward the periphery. Mechanistically, this

could be by simple diffusion or the genomic RNA could

be propelled along microtubules toward the minus end of

themicrotubules in a dynein-dependent manner, leading to

efficient MTOC localization (? in Figure 8). The latter idea

appears to be the more probable, and in normal cells, this

Figure 8: Model for HIV-1 RNA traffickingmediated by hnRNPA2. Step 1: The association of hnRNP A2 to the HIV-1 RNA via the intact

A2RE acts to facilitate nuclear export of the RNA to the cytosol (step 1a). Knockdown of hnRNP A2 leads to enhanced nuclear export (step

1b, this article), even in the presence of a mutated A2RE (A2RE/A8G, step 1c). Mutation of the A2RE leads to a block in nuclear export (step

1d) perhaps by influencing Rev function or interference of the RRE. Following nucleocytoplasmic export (step 2), the RNA is possibly

trafficked toward the MTOC by a microtubule minus-end-directed motor, dynein (?). The RNA can be translated to make Gag (step 3) and

a small proportion can be concomitantly selected for encapsidation at or near the MTOC (step 4), followed by a rapid shuttling via Rab9-

containing vesicles (step 5) to the plasmamembrane by way of the TGN (step 6) at which Env can interact and rapidly shuttle the Gag–RNA

complex to sites of virus assembly (step 7) via vesicles that possess specific Rab proteins. In cases when hnRNP A2 is knocked down by

siRNA (steps 1b and 1c), the genomic RNA is released from the nucleus and accumulates at the MTOC region in the cytosol. hnRNP A2

expression is necessary for subsequent trafficking via an RNP complex that likely contains hnRNP A2 and adaptor proteins such as TOG

(step 5a). This complex likely translocates on microtubules because the recruitment of the dynein–dynactin complex by RILP expression

leads to sequestration of the majority of the genomic RNA to theMTOC region, as well as Rab7-containing LE. The RNA transported in this

complex is likely needed as the principal cofactor for virus assembly (step 7). LE, late endosomes.

Traffic 2006; 7: 1177–1193 1187


would allow directed outbound trafficking of the genomic

RNA, implicating a switch in the motor activity from dynein

to kinesin-directed motoring toward the plus ends of

microtubules that might rely, for instance, on Gag synthe-

sis (see below). The observed accumulation of genomic

RNA at the MTOC in hnRNP A2-depleted cells indicates

that this first trafficking event is not disrupted but down-

stream trafficking is (see below). Further work will be

required to decipher the details surrounding these initial

trafficking events following nuclear export.

pr55Gag synthesis and genomic RNA selection occur

near the MTOC

The data presented in this study lead us to the conclusion

that the genomic RNA is translated in the vicinity of the

MTOC in HeLa cells (Figure 8, step 3). This conclusion is

supported by the data that show that pr55Gag synthesis

levels are similar in mock-, siA2- and RILP-treated cells,

despite almost complete MTOC localization of the geno-

mic RNA in the latter conditions (Figures 7 and S3). Further

support for localized synthesis of pr55Gag at the MTOC is

the published report that shows the return of pr55Gag to

late endosomal compartments when the expression of the

small GTPase, Rab9, is knocked down, a treatment that

prevents late endosome to trans-Golgi network (TGN)

pr55Gag trafficking (49). In addition, recent data point toward

the involvement of the TGN in sorting pr55Gag to the plasma

membrane by a TGN-associated E3 ubiquitin ligase named

human POSH (50). The results of this last study implied

that newly synthesized pr55Gag is quickly routed to the

TGN in wait of Env recycling from the plasma membrane.

This would be consistent with observations that a number

of transmembrane proteins such as M6PR, furin and

TGN38 traffic between late endosomes and the TGN and

are rapidly recycled to the plasma membrane (51).

Recent work has also identified the MTOC region as the

site at which, following synthesis, pr55Gag interacts and

selects its cognate genomic RNA (52) (Figure 8, step 4).

This report indicated that this is an infrequent event, and

overexpression was required to detect the Gag–RNA

interaction in the MTOC region. Consistently, we did not

detect any appreciable colocalization between RNA and

pr55Gag in this region in siA2 and RILP conditions. Because

these conditions enhance genomic RNA encapsidation

(Figure S5 and data not shown), our work also implicates

the MTOC region in genomic RNA encapsidation. This

might very well be due to the resultant abundance or

concentration of genomic RNA in this region, which would

favor genomic RNA capture by newly synthesized pr55Gag

(Figures S3 and S5). Genomic RNA trafficking and pr55Gag

synthesis must be linked at the molecular level and

spatially in the cell at some point (at the MTOC) because

the genomic RNA is translated to produce pr55Gag and

then must bind its own RNA for encapsidation (52–55).

The trafficking of genomic RNA and pr55Gag is character-

ized by a rapid transit to assembly domains, although each

of these viral components appears to be differentially

treated in cells. Following pr55Gag synthesis on cytosolic

polyribosomes in the MTOC region for instance, pr55Gag

could transit to the TGN in a Rab9-dependent manner,

followed by a rapid transit to the plasmamembrane (49,50)

(Figure 8, steps 5–7). This is consistent with the findings

that pr55Gag is found at the plasmamembrane immediately

following synthesis in HeLa cells (56). While this route may

be exploited by HIV-1, one significant difference exists

between our observations for RNA and pr55Gag trafficking

in this respect. If pr55Gag and the RNA are trafficked

together, it would be expected that they would exhibit

the same behavior with a given treatment. When we

overexpress RILP, however, pr55Gag and RNA are found

in completely opposite positions, with pr55Gag at the

periphery and its RNA at the MTOC (Figures 7 and S3).

While this finding supports a late endosome to TGN sorting

of the genomic RNA because dynein activity is required for

this sorting event (57), pr55Gag trafficking does not appear

to depend on dynein and is found at the plasma membrane

in these conditions. Nevertheless, only a small proportion

of the genomic RNA is selected for by pr55Gag in the

MTOC region as suggested (52), indicating that only

a minor proportion of the RNA co-traffics with pr55Gag to

reach assembly sites at the plasma membrane. Consis-

tently, calculations of the colocalization coefficient (total

amount of pr55Gag versus the amount found with genomic

RNA) indicate that only 8–25% of the pr55Gag signal is

found with genomic RNA in cells (e.g. Figure 7; calculated

range from 10 experiments). This is also reflected in our

imaging results that show that very little of the genomic

RNA is found to colocalize with pr55Gag at the plasma

membrane in normal (Figure 7C) and siA2 conditions

(Figure 7F) [see also Poole et al. (52)]. HIV-1 may co-opt

more than one sorting pathway to ensure pr55Gag and RNA

assembly, perhaps guaranteeing this by relying on inherent

membrane-binding signal of pr55Gag or on its ability to

directly associate to kinesin proteins during egress, for

example. Because viral assembly occurs at the plasma

membrane in HeLa cells (15,56), the data support impor-

tant roles for both rapid pr55Gag and RNA trafficking events

to the plasma membrane for assembly.

Now that pr55Gag is made and the genomic RNA is

selected for in the MTOC region, what would be the need

for subsequent hnRNP A2-dependent RNA trafficking from

the MTOC in the cytosol? Part of the answer lies in the

requirement for RNA in assembly. Numerous studies now

demonstrate the requirement for RNA for pr55Gag dimer-

ization and subsequent steps of assembly (58–60). Thus,

hnRNP A2-mediated RNA trafficking to distal sites is likely

needed later for RNA-dependent assembly, leading to

virion morphogenesis and capsid formation (61,62) and

this relies on the expression levels of hnRNP A2, dynein

and other auxiliary microtubule-binding proteins (23) (Fig-

ure 8, step 5a). Although HIV-1 and other retroviruses may

be able to use cellular or ribosomal RNAs as scaffolds for

assembly (59,63), the genomic RNA may be the ideal one

1188 Traffic 2006; 7: 1177–1193

Levesque et al.

for HIV-1. Consistently, while cellular pr55Gag synthesis is

not compromised in siA2- or RILP-overexpressing condi-

tions, viral production and infectivity are markedly reduced

in these two conditions (Figure 1B and data not shown).

The aberrant distribution of pr55Gag that results when the

hnRNP A2–A2RE interaction is blocked might also be

explained by the lack of genomic RNA availability in the

cytosol late in the replication cycle (13).

Conserved RNA trafficking mechanisms used by

eukaryotic and viral RNAs

A number of reports now indicate that hnRNP A2-

mediated RNA trafficking is selective and conserved from

Drosophila to man. Hrp48, the closest orthologue of

hnRNP A2 in vertebrates, mediates oskar mRNA traffick-

ing toward the plus end of microtubules during oogenesis

(24,25). Modulation of expression of hrp48 or the intro-

duction of germ line missense mutations in the gene do

not result in altered splicing activity (24,25), consistent

with our observations (Figure 1D). RNA localization also

requires the interdependent activities of kinesin and dynein

motor proteins. Not only do these motors share the same

binding site onmicrotubules (64) but also the kinesin-based

translocation depends on dynein activity in mRNA traffick-

ing in Drosophila (65). This is also true for hnRNP A2-

mediated RNA trafficking as these RNA granules contain

both kinesin and dynein motors (66–69). Consistently, the

recruitment of the dynein/dynactin complex by RILP abro-

gates HIV-1 genomic RNA trafficking to the periphery in all

conditions tested (Figures 7 and S3). While RILP expres-

sion causes the recruitment of late endosomes/lysosomes

to the MTOC region (19), this might suggest that HIV-1

RNA is trafficked to distal regions on these vesicles

(Figure 7). However, even though we do observe some

overlap of genomic RNA staining with Lamp-1 late endo-

somal membranes in siA2 knockdown conditions, in siNS-

treated cells, the genomic RNA shows little costaining with

this compartment (Figures S1 and S2). Our data do not rule

out the possibility that RNA trafficking is achieved in RNP

granules for which dynein activity is essential (65). Work in

murine leukemia virus also suggests that RNA is recruited

onto endosomes from a cytoplasmic compartment (70),

implicating both phenomena in retroviral RNA trafficking.

The combined activities of distinct sets of trans-acting

RNA-binding proteins, transport vesicles and motor pro-

teins will relay a given RNA to its final destination (28, 71–

73). HIV-1 appears to have co-opted conserved cellular

mechanisms in the trafficking of its genomic RNA that rely

in large part on hnRNP A2 expression.

Materials and Methods

Antibodies, genetic clones and reagentsMouse monoclonal and rabbit polyclonal anti-hnRNP A2 and anti-hnRNP A1

antisera were generous gifts from William Rigby (Dartmouth Medical

School, NH, USA), and a pan-specific hnRNP antiserum was generously

provided by Benoit Chabot (Universite de Sherbrooke, Sherbrooke, Que-

bec, Canada) (13); rabbit anti-M6PR was a generous gift from Paul Luzio

(Cambridge, UK); rabbit anti-Lamp-1 and anti-Lamp-2 were generous gifts

from Minora Fukuda (University of Medicine and Dentistry, NJ, USA) (74);

rabbit anti-Calnexin was purchased from Stressgen (Ann Arbor, MI, USA);

rat anti-a-tubulin was obtained from Abcam (Cambridge, MA, USA); rabbit

anti-myc was obtained from TechniScience (Montreal, Quebec, Canada)

and rabbit anti-g-tubulin, LMB and nocodazole were purchased from Sigma-

Aldrich. The proviral DNAs, HxBRU and A2RE A8G (harboring a single point

mutation in the second A2RE) have been described elsewhere (75,76).A

myc-epitope-tagged RILP expressor (pRILP-myc) was generously provided

by Dr Markus Thali (University of Vermont) (14) and was identified by IF and

Western analyses using the anti-myc tag monoclonal antibody 12CA5

(Roche, Montreal, Quebec, Canada) or rabbit anti-myc tag polyclonal

antiserum from TechniScience.

Cell culture and transfectionsHeLa cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen,

Burlington, ON, USA) supplemented with 10% fetal bovine serum and 1%

of Pen/Strep antibiotics at 378C in a humidified atmosphere containing 5%

CO2. The siA2 50-AAGCTTTGAAACCACAGAAGA and siA1 50-AATGGG-

GAACGCTCACGGACT siRNA duplexes were used at 25 nM to knockdown

hnRNP gene expression as described (16). These and the siNS siRNA

(50-AATTCTCCGAACGTGTCACGA) duplexes were synthesized by Qiagen-

Xeragon (Flanders, NJ, USA). The siRNA transfections were performed

exactly as described in Chatel-Chaix et al. (17), except HeLa cells were

used. Exponentially growing HeLa cells were trypsinised, and 3 � 105 to

3.5 � 105 cells were seeded into six-well plates. Cells were also seeded

onto glass cover slips in six-well plates for imaging studies. Twenty-four

hours later or when the cells reached 70% confluency, siRNA duplexes

(25 nM) were transfected using Lipofectamine 2000 (Invitrogen) according

to the manufacturer’s instructions (17). At 24 h post-transfection, a second

transfection was performed using 0.5 mg HxBRU. Thirty hours later, the

cells were washed with ice-cold PBS and lysed in NTEN buffer (100 mM

NaCl, 10 mM Tris, pH 7.5, 1 mM ethylene-diamine-tetraacetic acid, 0.5%

Nonidet P-40) for protein analyses or fixed as described below for imaging

analyses. In experiments using myc-RILP, 0.5 mg of pRILP-myc (14) was

added to transfections with HxBRU (or at the time of the second trans-

fection) to cause recruitment of the dynein/dynactin complex to the MTOC

region and to disrupt kinesin-driven RNA trafficking and processed for IF/

FISH coanalyses 30 h later. In some experiments, nocodazole was used to

disrupt microtubules. This was added to 5 mM 30 min before cell fixation and

IF and FISH analyses. This treatment time was chosen to disrupt micro-

tubules and not Golgi integrity (18). In studies using LMB (Sigma-Aldrich),

cells were first transfected with HxBRU for 24 h to allow expression and

accumulation of genomic RNA in cytosol and then transfected with either

siNS or siA2 duplexes as described (17). Four hours later, LMB was added

to 2 nM for an additional 8 or 12 h after which cells were processed for

Western and IF/FISH analyses. This treatment completely blocked HIV-1

genomic RNA nuclear export as determined by visualization in empirical

FISH experiments while maintaining cell integrity and viability (not shown).

Western blot analysesAt 30 h post-transfection, cells were washed with ice-cold PBS and lysed in

NTEN buffer. Cytosolic extracts were quantified for protein content by the

micro Bradford assay (BioRad, Mississauga, ON, USA). Equal quantities of

protein were loaded onto gels and hnRNPs, pr55Gag, GAPDH and g-tubulinlevels were assessed by Western blot analyses.

Quantitation of virus production and infectivity

studiesVirus production was quantitated on neat harvested supernatant or purified

and concentrated virus by p24 enzyme-linked immunosorbent assay and

infectivity determinations were performed on equal quantities of virus using

CEM–LTR–GFP indicator cells as described (13,17).

Traffic 2006; 7: 1177–1193 1189


IF and FISH analysesThe IF and FISH analyses have been recently described in detail elsewhere

(13). Briefly, HeLa cells were fixed at the specified time-points posttrans-

fection depending on the experiment (in 4% paraformaldehyde in PBS for

20 min, followed by permeabilization with 0.2% Trition-X-100 for 10 min).

Cells were washed with PBS and blocked with 10% dry milk in PBS and

incubated with each of the antibodies described above. Secondary fluo-

rophore-conjugated antisera (Alexa Fluor 488 and 564) were obtained from

Molecular Probes (Eugene, OR, USA). For FISH/IF coanalyses experiments,

the FISH analysis was performed first. Following fixation and permeabiliza-

tion, cells were treated with deoxyribonuclease I (DNase I; Invitrogen) and

washed in PBS. The digoxigenin-labeled RNA probe to identify genomic

RNAwas prepared as described previously (13). In several experiments, the

nucleic acid stain 4’,6-diamidino-2-phenylindole (DAPI) (Molecular Probes,

OR, USA) was used at 1:500 following FISH analysis.

Imaging analysisMost of the imaging of cells was performed on an Olympus BX-51

fluorescence microscope equipped with an UPlanFI 100X oil objectif

(Center Valley, PA, USA). Alexa Fluor 488 nm, 594 nm and DAPI images

were obtained using 460–490 nm, 510–550 nm and 330–385 nm bandpass

emission filters, respectively. Red, green and DAPI images were sequen-

tially captured in black and white with the Spot camera (Diagnostics

Instruments, Sterling Heights, MI, USA) using Spot Advanced Software

and Image-Pro-Plus version 4.0.1. Images were pseudo-colored using

Adobe Photoshop CS (Adobe Systems, San Jose, CA, USA) in RGB mode

and then merged. Phase contrast images were captured to examine cell

morphology and cell intactness and were obtained with the same micro-

scope by transmitted visible light. Images were captured at a resolution of

512 pixels. All merged digitized images were imported into Adobe Illustrator

CS for figure montage shown in this article. In several experiments, the

subcellular distribution of RNA and proteins was confirmed by laser

scanning confocal microscopy using a Carl-Zeiss (Mississauga, ON, USA)

LSM5 Pascal microscope exactly as described before (13). For all micro-

scopy analyses, at least 50 cells per experimental condition in each

experiment were examined and representative cells are shown here.

Settings for image capture were kept constant in each channel when

comparing staining intensities between siNS- and siA2- or siA1-treated

cells. In knockdown cells in particular, the gain was adjusted in order to

balance the signal intensities from knockdown and nonknockdown cells in

the same microscope field of vision and then the slides containing cells

from other transfection conditions were examined using the identical

microscope settings. In many of the images presented in this article,

silenced and nonsilenced cells (for hnRNPs) are presented in the same field

of vision. Most experiments involving extensive image analysis were

performed at least seven times. To provide an index of colocalization

between viral, cellular antigens and genomic RNA in cells, we used

Colocalization Pro Software (Boise, IO, USA) to calculate the overlap

coefficient according to Manders. This was performed in several experi-

ments as indicated in the text in at least 100 cells per treatment to obtain an

average estimation. Colocalization coefficients were also calculated to

determine the relative amount of colocalized RNA and pr55Gag in relation

to the total pr55Gag signal in cells. Size bars were precisely calculated using

Carl-Zeiss LSM Image Browser software (Mississauga, Ontario, Canada).

RNA extraction and RT-PCR analysisHeLa cells were transfected asmentioned above, and total RNAwas isolated

for splicing, RT-PCR and denaturing gel analyses. Cells were washed with

PBS and lysed on ice in NTEN buffer containing Complete protease inhibitor

cocktail (Roche) for 30 min. Lysates were subjected to a 30 min centrifuga-

tion at 14 000x g, 48C. An aliquot of the supernatant was kept for Western

blot analyses for hnRNP A1, hnRNP A2, HIV-1 Gag and GAPDH. Before RNA

extraction, cell lysates were treated with 40 U of RNaseout and 114 U of

DNase I for 5 min at room temperature. Total RNA was extracted from cell

lysates using Trizol LS (Invitrogen) according to the procedure suggested by

the manufacturer. Five micrograms of glycogen (Roche, Montreal, Quebec,

Canada) was used as carrier for RNA precipitation.

Resulting RNAs were subjected to RT-PCR amplification for the 4-kbp,

singly spliced and 2-kbp, multiply spliced HIV-1 RNA species using specific

primer combinations exactly as described previously (13,17) except that the

RNA PCR Core Kit (Applied Biosystems, Streetsville, ON, USA) was used

for the two-step reverse transcription (RT) and PCR reactions. A negative

control included the exclusion of RT in the RT step. After 20 and 30 cycles,

15% of the total RT-PCR products were visualized on 1% agarose gel using

Gel Doc System and quantitated by the Quantity One Software version

4.4.1 (BioRad). Inverted images of the ethidium-bromide-stained gels are

shown in the article. For a more sensitive detection of each of the 4- and

2-kbp HIV-1 RNA species, 20% of RT-PCR products at 20 cycles were

subjected to two additional PCR cycles in the presence of [32P]dCTP and

labeled PCR products were resolved on polyacrylamide/urea gels as

described (13,17).

Cell cycle and apoptosis determinationsCell cycle analysis was performed using fluorescence activated cell sorting

as described (76,77).

Acknowledgments

We thank Jean-Francois Clement for contributions in preliminary experi-

ments; Markus Thali for sharing data prior to publication; William Rigby,

Benoit Chabot, Minora Fukuda and Paul Luzio for antibodies; Fred Maxfield

and Markus Thali for helpful discussions and expression vectors; Kimberly

Hu for technical assistance; and Alan Cochrane for reagents and for critical

reading of the manuscript. A. J. M. is supported by Canadian Institutes of

Health Research (CIHR) New Investigator Award and this work was

supported by grants from the CIHR, the Canadian Foundation for AIDS

Research and the Canadian Foundation for Innovation to A. J. M.

Supplementary Materials

Figure S1: The localization of genomic RNA in siA2-treated cells in

relation to Golgi and late endosomal membranes. HeLa cells were

transfected with HxBRU and siA2. IF/FISH coanalyses at 30–36 h post-

transfection. In all images, the green fluorescence signal and blue are the

genomic RNA and DAPI staining of the nucleus, respectively. The Golgi

apparatus, TGN and late endosomal membranes were identified as red

fluorescence signals by fluorescence microscopy using Bodipy-ceramide

(panels A–C), an anti-M6PR antibody (panels D–F) and an anti-Lamp-1

antibody (panels G–I). Panels A, D and G show the distribution of RNA

(green fluorescence) in relation to the nuclei; panels B, E and H show the

distribution of the genomic RNA in relation to the staining pattern of the

organellar markers and panels C, F and I show the merges of all three

components. Arrows in panels G–I indicate genomic RNA in a radial

distribution pattern in the perinuclear space and the asterisk is placed for

orientation. White bar ¼ 10 mm in panels A, D and G. (See supplementary

materials for results.)

Figure S2: Perinuclear distribution of Lamp-1 late endosomal com-

partments. HeLa cells were mock transfected or transfected with HxBRU

proviral DNA and siNS (panel A), siA1 (not shown) or siA2 (panels C and D).

Cells were processed for IF/FISH coanalyses. A rabbit anti-Lamp-1 was

used to identify late endosomal compartments in all series of cells (red

fluorescence in panels A–D). Green fluorescence represents the genomic

RNA and DAPI staining identifies the nuclei (in blue). Merged images are

shown above. Lamp-1 is expressed in all cells and is found in a discrete

patch or slightly diffused region immediately adjacent to the nucleus in

most cells. Genomic RNA was found widely distributed in a cell in

a punctate staining pattern (panel B) and siA2 treatment to knockdown

1190 Traffic 2006; 7: 1177–1193

Levesque et al.

hnRNP A2 expression resulted in the perinuclear distribution of genomic

RNA that was found in close proximity to the late endosomal compartments

(panels C and D). White bar ¼ 10 mm.

Figure S3: The RILP induces a perinuclear distribution of genomic

RNA in all siRNA conditions. HeLa cells were mock transfected (not

shown; see Figure 7) or transfected with HxBRU proviral DNA and siNS

(panels A-C), siA1 (panel D) or siA2 (panel E). RILP was coexpressed to

recruit the dynein/dynactin motor complexes to the MTOC. Cells were

processed for IF/FISH coanalyses. A rabbit anti-hnRNP A2 was used to

identify hnRNP A2 expression in all series of cells (red fluorescence in

panels A–E). Green fluorescence represents the genomic RNA and DAPI

staining identifies the nuclei (in blue). Merged images are shown above. A

merged image including phase contrast (PC; panel A) of a cell that was

transfected with HxBRU, siNS and vector control only in the same

experiment is shown to demonstrate wild-type distribution of genomic

RNA before RILP expression (panel A). In siNS- and siA1-treated cells,

hnRNP A2 levels are not modulated and is expressed in all cells (panels B–

D), whereas siA2 treatment resulted in a near-complete reduction in the

hnRNP A2 expression in four of the five cells shown (panel E). The HIV-1

genomic RNA was recruited to the MTOC in all conditions when RILP was

expressed. White bar ¼ 10 mm.

Figure S4: The siA2 treatment of HIV-1-/A2RE-expressing cells pro-

motes nucleocytoplamic transport of genomic RNA. Cells were trans-

fected as described in legend Figure 1, except that the a provirus harboring

amutation in the A2RE-2/A8Gwas transfected alone or with the siRNAs as

indicated. A) Western blot analyses reveal efficient knockdown of

hnRNPs. B–E) HIV-1 genomic RNA is identified by FISH (as green

fluorescence) in cells expressing the HIV-1/A2RE provirus alone B) or with

siNS C), siA2 D) or siA1 E). White bar ¼ 10 mm. (See supplementary

material for results.)

Figure S5: The siA2 treatment modulates genomic RNA content in

virions. Because the siA2 RNA localization phenotype is quite dramatic, we

investigated how siA2 treatment impacted on the genomic RNA content in

virions. A) HeLa cells were transfected with HxBRU alone or HxBRU and

siNS, siA1 or siA2. Virus was isolated from cells at 36 h posttransfection,

concentrated by ultracentrifugation, and quantitated by p24 enzyme-linked

immunosorbent assay (ELISA) (17,75). A) p24 virus equivalents per milliliter

are presented in the histogram. Virus was treated with DNase I (13) and

then solubilized in 1% sodium dodecyl sulphate and deproteinized with

proteinase K. The purified RNA was separated in denaturing formaldehyde/

agarose gels and blotted onto a nylon membrane. Northern blot analysis

was performed using a probe that corresponds to a Apa1/Sph1 HIV-1 Gag

DNA fragment that will recognize genomic RNA (75). Genomic RNA signals

were quantitated as described previously (13). This analysis revealed that

siA2 treatment enhanced genomic RNA content by 30% and the genomic

RNA content corresponded directly to p24 levels for HxBRU, siNS and siA1

conditions, as shown by the p24/RNA ratios that are near 1. B) To

independently confirm this result, virus was prepared in an identical manner

in two additional experiments. Virus was normalized for p24 content by

ELISA, and RNA was isolated from pelleted virus [2750 ng equivalents (17)]

and was then solubilized in an equal volume of guanidium thiocyanate

buffer. Aliquots were denatured and slot blotted onto membranes. The

membrane was probed as described above. The O.D. of the resultant

signals were quantitated by densitometry, and the mean genomic RNA

content between the two experiments is shown below blots. These

analyses show that siA2 mediated a 30–60% increase in virion-associated

genomic RNA, suggesting that siA2 impacted on virion RNA content as

shown in A, and this affected negatively on infectivity.

Supplemental materials are available as part of the online article at http://

www.blackwell-synergy.com

References

1. Coffin JM. HIV population dynamics in vivo: implications for genetic

variation, pathogenesis, and therapy [Comments]. Science 1995;267:

483–489.

2. HoDD,Neumann AU, Perelson AS, ChenW, Leonard JM,MarkowitzM.

Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1

infection [Comments]. Nature 1995;373:123–126.

3. Pomerantz RJ, Seshamma T, Trono D. Efficient replication of human

immunodeficiency virus type 1 requires a threshold level of Rev:

potential implications for latency. J Virol 1992;66:1809–1813.

4. Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR. The HIV-1 rev

trans-activator acts through a structured target sequence to activate

nuclear export of unspliced viral mRNA. Nature 1989;338:254–257.

5. Yedavalli VS, Neuveut C, Chi YH, Kleiman L, Jeang KT. Requirement of

DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function. Cell

2004;119:381–392.

6. Kjems J, Askjaer P. Rev protein and its cellular partners. Adv Pharmacol

2000;48:251–298.

7. Hofmann W, Reichart B, Ewald A, Muller E, Schmitt I, Stauber RH,

Lottspeich F, Jockusch BM, Scheer U, Hauber J, Dabauvalle MC.

Cofactor requirements for nuclear export of Rev response element

(RRE)- and constitutive transport element (CTE)-containing retroviral

RNAs. An unexpected role for actin. J Cell Biol 2001;152:895–910.

8. Yu Z, Sanchez-Velar N, Catrina IE, Kittler EL, Udofia EB, Zapp ML. The

cellular HIV-1 Rev cofactor hRIP is required for viral replication. Proc

Natl Acad Sci U S A 2005;102:4027–4032.

9. Sanchez-Velar N, Udofia EB, Yu Z., Zapp ML. hRIP, a cellular cofactor

for Rev function, promotes release of HIV-1 RNAs from the perinuclear

region. Genes Dev 2004;18: 23–24.

10. Tang Y, Winkler U, Freed EO, Torrey TA, Kim W, Li H, Goff SP., Morse

HC III. Cellular motor protein KIF-4 associates with retroviral Gag.

J Virol 1999;73:10508–10513.

11. Kim W, Tang Y, Okada Y, Torrey TA, Chattopadhyay SK, Pfleiderer M,

Falkner FG, Dorner F, Choi W, Hirokawa N., Morse HC III. Binding of

murine leukemia virus Gag polyproteins to KIF4, a microtubule-based

motor protein. J Virol 1998;72:6898–6901.

12. Mouland AJ, Xu H, Cui H, Krueger W, Munro TP, Prasol M, Mercier J,

Rekosh D, Smith R, Barbarese E, Cohen EA, Carson JH. RNA

trafficking signals in human immunodeficiency virus type 1. Mol Cell

Biol 2001;21:2133–2143.

13. Beriault V, Clement JF, Levesque K, Lebel C, Yong X, Chabot B, Cohen

EA, Cochrane AW, Rigby WF, Mouland AJ. A late role for the

association of hnRNP A2 with the HIV-1 hnRNP A2 response elements

in genomic RNA, Gag, and Vpr localization. J Biol Chem 2004;

279:44141–44153.

14. Nydegger S, Foti M, Derdowski A, Spearman P, Thali M. HIV-1 egress

is gated through late endosomal membranes. Traffic 2003;4:902–910.

15. Ono A, Freed EO. Cell-type dependent targeting of HIV-1 assembly to

the plasma membrane and the multivesicular body. J Virol 2004;

78:1552–1565.

16. Patry C, Bouchard L, Labrecque P, Gendron D, Lemieux B, Toutant J,

Lapointe E, Wellinger R, Chabot B. Small interfering RNA-mediated

reduction in heterogeneous nuclear ribonucleoparticule A1/A2 proteins

induces apoptosis in human cancer cells but not in normal mortal cell

lines. Cancer Res 2003;63:7679–7688.

17. Chatel-Chaix L, Clement J-F, Martel C, Beriault V, Gatignol A, Des-

Groseillers L, Mouland AJ. Staufen is part of the HIV-1 Gag ribo-

nucleoprotein complex and is involved in the generation of infectious

viral particles. Mol Cell Biol 2004;24:2637–2648.

18. Honda A, Al-Awar OS, Hay JC, Donaldson JG. Targeting of Arf-1 to the

early Golgi by membrin, an ER-Golgi SNARE. J Cell Biol 2005;

168:1039–51.

Traffic 2006; 7: 1177–1193 1191


19. Jordens I, Fernandez-Borja M, Marsman M, Dusseljee S, Janssen L,

Calafat J, Janssen H, Wubbolts R, Neefjes J. The Rab7 effector protein

RILP controls lysosomal transport by inducing the recruitment of

dynein-dynactin motors. Curr Biol 2001;11:1680–1685.

20. Lichtenstein M, Guo W, Tartakoff AM. Control of nuclear export of

hnRNP A1. Traffic 2001;2:261–267.

21. Cochrane AW, McNally MT, Mouland AJ. The retrovirus RNA traffick-

ing granule: from birth to maturity. Retrovirology 2006;3:18.

22. Wu M, Wang T, Loh E, Hong W, Song H. Structural basis for

recruitment of RILP by small GTPase Rab7. EMBO J 2005;24:

1491–1501.

23. Kosturko LD, Maggipinto MJ, D’Sa C, Carson JH, Barbarese E. The

microtubule-associated protein, TOG, binds to the RNA trafficking

protein, hnRNP A2. Mol Biol Cell 2005.

24. Yano T, Lopez de Quinto S, Matsui Y, Shevchenko A, Shevchenko A,

Ephrussi A. Hrp48, a Drosophila hnRNPA/B homolog, binds and

regulates translation of oskar mRNA. Dev Cell 2004;6:637–648.

25. Huynh JR, Munro TP, Smith-Litiere K, Lepesant JA, St Johnston D. The

Drosophila hnRNPA/B homolog, Hrp48, is specifically required for

a distinct step in osk mRNA localization. Dev Cell 2004;6:625–635.

26. Kim VN, Dreyfus G. Nuclear mRNA binding proteins couple pre-mRNA

splicing and post-splicing events. Mol Cell 2001;12:1–10.

27. Kim VN, Yong J, Kataoka N, Abel L, Diem MD, Dreyfuss G. The Y14

protein communicates to the cytoplasm the position of exon-exon

junctions. EMBO J 2001;20:2062–2068.

28. Kress TL, Yoon YJ, Mowry KL. Nuclear RNP complex assembly

initiates cytoplasmic RNA localization. J Cell Biol 2004;165:203–211.

29. Kamma H, Portman DS, Dreyfuss G. Cell type-specific expression of

hnRNP proteins. Exp Cell Res 1995;221:187–196.

30. Farina KL, Singer RH. The nuclear connection in RNA transport and

localization. Trends Cell Biol 2002;12:466–472.

31. Gu W, Pan F, Zhang H, Bassell GJ, Singer RH. A predominantly nuclear

protein affecting cytoplasmic localization of beta-actin mRNA in fibro-

blasts and neurons. J Cell Biol 2002;156:41–51.

32. Sfakianos JN, LaCasse RA, Hunter E. The M-PMV cytoplasmic

targeting-retention signal directs nascent Gag polypeptides to a peri-

centriolar region of the cell. Traffic 2003;4:660–670.

33. Sfakianos JN, Hunter E. M-PMV capsid transport is mediated by Env/

Gag interactions at the pericentriolar recycling endosome. Traffic

2003;4:671–680.

34. Petit C, Giron ML, Tobaly-Tapiero J, Bittoun P, Real E, Jacob Y, Tordo

N, De The H, Saib A. Targeting of incoming retroviral Gag to the

centrosome involves a direct interaction with the dynein light chain 8.

J Cell Sci 2003;116:3433–3442.

35. McDonald D, Vodicka M, Lucero G, Svitkina Y, Borisy G, Emerman M,

Hope TJ. Visualization of the intracellular behavior of HIV in living cells.

J Cell Biol 2002;159:441–452.

36. Kashima T, Manley JL. A negative element in SMN2 exon 7 inhibits

splicing in spinal muscular atrophy. Nat Genet 2003;34:460–463.

37. Bilodeau PS, Domsic JK, Mayeda A, Krainer AR, Stoltzfus CM. RNA

splicing at human immunodeficiency virus type 1 3’ splice site A2 is

regulated by binding of hnRNP A/B proteins to an exonic splicing

silencer element. J Virol 2001;75:8487–8497.

38. Caputi M, Mayeda A, Krainer AR, Zahler AM. hnRNP A/B proteins are

required for inhibition of HIV-1 pre-mRNA splicing. EMBO J 1999;

18:4060–4067.

39. Buck CB, Shen X, Egan MA, Pierson TC, Walker CM, Siliciano RF. The

human immunodeficiency virus type 1 gag gene encodes an internal

ribosome entry site. J Virol 2001;75:181–191.

40. Brasey A, Lopez-Lastra M, Ohlmann T, Beerens N, Berkhout B, Darlix

JL, Sonenberg N. The leader of human immunodeficiency virus type 1

genomic RNA harbors an internal ribosome entry segment that is active

during the G2/M phase of the cell cycle. J Virol 2003;77:3939–3949.

41. Bonnal S, Pileur F, Orsini C, Parker F, Pujol F, Prats AC, Vagner S.

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribo-

some entry site trans-acting factor that modulates alternative initiation

of translation of the fibroblast growth factor 2 mRNA. J Biol Chem

2005;280:4144–4153.

42. Kress E, Baydoun HH, Bex F, Gazzolo L, Duc Dodon M. Critical role of

hnRNP A1 in HTLV-1 replication in human transformed T lymphocytes.

Retrovirology 2005;2:8.

43. Najera I, Krieg M, Karn J. Synergistic stimulation of HIV-1 rev-depend-

ent export of unspliced mRNA to the cytoplasm by hnRNP A1. J Mol

Biol 1999;285:1951–1964.

44. Pomerantz RJ, Trono D, Feinberg MB, Baltimore D. Cells nonproduc-

tively infected with HIV-1 exhibit an aberrant pattern of viral RNA

expression: a molecular model for latency. Cell 1990;61:1271–1276.

45. Neville M, Stutz F, Lee L, Davis LI, Rosbash M. The importin-beta

family member Crm1p bridges the interaction between Rev and the

nuclear pore complex during nuclear export. Curr Biol 1997;7:

767–775.

46. Xu Y, Reddy TR, FischerWH,Wong-Staal F. A Novel hnRNP specifically

interacts with HIV-1 RRE RNA. J Biomed Sci 1996;3:82–91.

47. Dodon MD, Hamaia S, Martin J, Gazzolo L. Heterogeneous nuclear

ribonucleoprotein A1 interferes with the binding of the human T cell

leukemia virus type 1 rex regulatory protein to its response element.

J Biol Chem 2002;277:18744–18752.

48. Yang JP, Reddy TR, Truong KT, Suhasini M, Wong-Staal F. Functional

interaction of Sam68 and heterogeneous nuclear ribonucleoprotein K.

Oncogene 2002;21:7187–7194.

49. Murray JL, Mavrakis M, McDonald NJ, Yilla M, Sheng J, Bellini WJ,

Zhao L, Le Doux JM, Shaw MW, Luo CC, Lippincott-Schwartz J,

Sanchez A, Rubin DH, Hodge TW. Rab9 GTPase is required for

replication of human immunodeficiency virus type 1, filoviruses, and

measles virus. J Virol 2005;79:11742–11751.

50. Alroy I, Tuvia S, Greener T, Gordon D, Barr HM, Taglicht D, Mandil-

Levin R, Ben-Avraham D, Konforty D, Nir A, Levius O, Bicoviski V, Dori

M, Cohen S, Yaar L et al. The trans-Golgi network-associated human

ubiquitin-protein ligase POSH is essential for HIV type 1 production.

Proc Natl Acad Sci U S A 2005;102:1478–1483.

51. Maxfield FR, McGraw TE. Endocytic recycling. Nat Rev Mol Cell Biol

2004;5:121–132.

52. Poole E, Strappe P, Mok HP, Hicks R, Lever AM. HIV-1 Gag-RNA

interaction occurs at a perinuclear/centrosomal site; analysis by confo-

cal microscopy and FRET. Traffic 2005;6:741–755.

53. Butsch M, Boris-Lawrie K. Translation is not required to generate virion

precursor RNA in human immunodeficiency virus type 1-infected T

cells. J Virol 2000;74:11531–11537.

54. LeBlanc JJ, Beemon KL. Unspliced Rous sarcoma virus genomic RNAs

are translated and subjected to nonsense-mediated mRNA decay

before packaging. J Virol 2004;78:5139–5146.

55. Poon DT, Chertova EN, Ott DE. Human immunodeficiency virus type 1

preferentially encapsidates genomic RNAs that encode Pr55(Gag):

functional linkage between translation and RNA packaging. Virology

2002;293:368–378.

56. Rudner L, Nydegger S, Coren LV, Nagashima K, Thali M, Ott DE.

Dynamic fluorescent imaging of human immunodeficiency virus

type 1 gag in live cells by biarsenical labeling. J Virol 2005;79:

4055–4065.

57. Itin C, Ulitzur N, Muhlbauer B, Pfeffer SR. Mapmodulin, cytoplasmic

dynein, and microtubules enhance the transport of mannose 6-phos-

phate receptors from endosomes to the trans-golgi network. Mol Biol

Cell 1999;10:2191–2197.

58. Alfadhli A, Dhenub TC, Still A, Barklis E. Analysis of human immuno-

deficiency virus type 1 Gag dimerization-induced assembly. J Virol

2005;79:14498–14506.

1192 Traffic 2006; 7: 1177–1193

Levesque et al.

59. Ott DE, Coren LV, Gagliardi TD. Redundant roles for nucleocapsid and

matrix RNA-binding sequences in human immunodeficiency virus type

1 assembly. J Virol 2005;79:13839–13847.

60. Huseby D, Barklis RL, Alfadhli A, Barklis E. Assembly of human

immunodeficiency virus precursor gag proteins. J Biol Chem

2005;280:17664–17670.

61. Derdowski A, Ding L, Spearman P. A novel fluorescence resonance

energy transfer assay demonstrates that the human immunodeficiency

virus type 1 Pr55Gag I domain mediates Gag-Gag interactions. J Virol

2004;78:1230–1242.

62. Larson DR, Ma YM, Vogt VM, Webb WW. Direct measurement of

Gag-Gag interaction during retrovirus assembly with FRET and

fluorescence correlation spectroscopy. J Cell Biol 2003;162:

1233–1244.

63. Muriaux D, Mirro J, Harvin D, Rein A. RNA is a structural element in

retrovirus particles. Proc Natl Acad Sci U S A 2001;98:5246–5251.

64. Mizuno N, Toba S, Edamatsu M, Watai-Nishii J, Hirokawa N,

Toyoshima YY, Kikkawa M. Dynein and kinesin share an overlapping

microtubule-binding site. EMBO J 2004;23:2459–2467.

65. Duncan JE, Warrior R. The cytoplasmic dynein and kinesin motors have

interdependent roles in patterning the Drosophila oocyte. Curr Biol

2002;12:1982–1991.

66. Carson JH, Barbarese E. Systems analysis of RNA trafficking in neural

cells. Biol Cell 2005;97:51–62.

67. Kanai Y, Dohmae N, Hirokawa N. Kinesin transports RNA: isolation and

characterization of an RNA-transporting granule. Neuron 2004;43:

513–525.

68. Song J, Carson JH, Barbarese E, Li FY, Duncan ID. RNA transport in

oligodendrocytes from the taiep mutant rat. Mol Cell Neurosci

2003;24:926–938.

69. Ling SC, Fahrner PS, Greenough WT, Gelfand VI. Transport of

Drosophila fragile X mental retardation protein-containing ribonucleo-

protein granules by kinesin-1 and cytoplasmic dynein. Proc Natl Acad

Sci U S A 2004;101:17428–17433.

70. Basyuk E, Galli T, Mougel M, Blanchard JM, Sitbon M, Bertrand E.

Retroviral genomic RNAs are transported to the plasma membrane by

endosomal vesicles. Dev Cell 2003;5:161–174.

71. Yoon YJ, Mowry KL. Xenopus Staufen is a component of a ribonucleo-

protein complex containing Vg1 RNA and kinesin. Development 2004;

131:3035–3045.

72. Kiebler MA, DesGroseillers L. Molecular insights into mRNA transport

and local translation in the mammalian nervous system. Neuron

2000;25:19–28.

73. Cohen RS. The role of membranes and membrane trafficking in RNA

localization. Biol Cell 2005;97:1–13.

74. Carlsson SR, Roth J, Piller F, Fukuda M. Isolation and characterization

of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2.

Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem

1988;263:18911–18919.

75. Mouland AJ, Mercier J, Luo M, Bernier L, DesGroseillers L, Cohen EA.

The double-stranded RNA-binding protein Staufen is incorporated in

human immunodeficiency virus type 1: evidence for a role in genomic

RNA encapsidation. J Virol 2000;74:5441–5451.

76. Yao XJ, Mouland AJ, Subbramanian RA, Forget J, Rougeau N,

Bergeron D, Cohen EA. Vpr stimulates viral expression and induces

cell killing in human immunodeficiency virus type 1-infected dividing

Jiurkat T cells. J Virol 1998;72:4686–4693.

77. Mouland AJ, Coady M, Yao XJ, Cohen EA. Hypophosphorylation of

poly(A) polymerase and increased polyadenylation activity are associ-

ated with human immunodeficiency virus type 1 Vpr expression.

Virology 2002;292:221–230.

78. Pelchen-Matthews A, Kramer B, Marsh M. Infectious HIV-1 assembles

in late endosomes in primary macrophages. J Cell Biol 2003;162:

443–455.

Traffic 2006; 7: 1177–1193 1193


Trafficking of HIV-1 RNA is Mediated by Heterogeneous Nuclear Ribonucleoprotein A2 Expression and...

Documents

Transcript of Trafficking of HIV-1 RNA is Mediated by Heterogeneous Nuclear Ribonucleoprotein A2 Expression and...