JVI Accepts, published online ahead of print on 28 April 2010

J. Virol. doi:10.1128/JVI.02264-09
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Nuclear RNA Export and Packaging Functions of Rev

2 Nuclear RNA Export and Packaging Functions of

3 HIV-1 Rev Revisited

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6 Maik Blißenbach, Bastian Grewe, Bianca Hoffmann, Sabine Brandt, Klaus Überla*

8 Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany

10 Running title: Nuclear RNA Export and Packaging Functions of Rev

11

12 Abstract: 153 words

13 Whole text: 7043 words

14

15

16 *Correspondent footnote:

17 Department of Molecular and Medical Virology

18 Ruhr-University Bochum

19 D-44780 Bochum, Germany

20 Tel.: 0049-234-3223189

21 Fax: 0049-234-3214352

22 E-mail: klaus.ueberla@rub.de

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 Nuclear RNA Export and Packaging Functions of Rev

1 Abstract

2 Although the viral Rev protein is necessary for HIV replication, its main function in the viral

3 replication cycle has been controversial. Re-investigating the effect of Rev on the HIV-1

4 RNA distribution in various cell lines and primary cells revealed that Rev enhanced

5 cytoplasmic levels of the unspliced HIV-1 RNA mostly 3- to 12-fold, while encapsidation of

6 the RNA and viral infectivity could be stimulated more than 1000-fold. Although this clearly

7 questions the general notion that the nuclear export of viral RNAs is the major function of

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8 Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of

9 the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference

10 with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to

11 enhanced accessibility of the cytoplasmic HIV-1 RNA to translation and encapsidation. This

12 might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.

13

14 Introduction

15 In contrast to simple retroviruses, HIV-1 as a lentivirus utilizes a number of trans-acting

16 regulatory proteins which fulfill important functions throughout the different phases of the

17 lentiviral replication cycle (recently reviewed in (16, 30, 34)). One of those regulators is the

18 16 kD HIV-1 Rev protein which is required for the expression of the structural proteins

19 during the late phase of the HIV-1 replication cycle. This effect of Rev has been attributed to

20 its nuclear export activity. After initial transcription of the integrated proviral genome “early”

21 transcripts are subjected to the cellular splicing machinery. Alternative splicing events caused

22 by the complex genome organisation of HIV-1 lead to expression of Rev from a multiply-

23 spliced mRNA. Once Rev is translated in the cytoplasm it is imported into the nucleus where

24 it binds to a viral RNA element, the Rev Response Element (RRE), which is present on

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 Nuclear RNA Export and Packaging Functions of Rev

1 singly-spliced and unspliced transcripts. Rev seems to circumvent further splicing and leads

2 to an increase of the cytoplasmic levels of unspliced and singly-spliced viral transcripts by

3 tethering these transcripts to the Crm1 export pathway ((13, 28), reviewed in (36)). The

4 unspliced and singly-spliced transcripts serve as templates for translation and the unspliced

5 RNA is also encapsidated into assembling virus particles.

6 The magnitude by which Rev enhances lentiviral RNA levels in the cytoplasm has been

7 controversial. Under some experimental conditions, unspliced viral RNA levels in the

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8 cytoplasm were only detectable in the presence of Rev while others observed just a 4-fold

9 enhancement of these cytoplasmic RNA levels by Rev (2, 8, 9, 12, 26, 36, 43, 45). Since Rev

10 was found to stimulate protein levels encoded by the Rev-dependent RNAs to a much larger

11 extent than the cytoplasmic levels of these RNAs, Rev also seems to stimulate translation ((6,

12 25, 35), recently reviewed in (17)). Consistently, Rev was furthermore shown to enhance the

13 association of the Rev-dependent viral RNA with polysomes (9).

14 Trying to develop RRE-deficient lentiviral vectors, we previously observed that the deletion

15 of the RRE led to a striking loss of infectivity despite nearly unchanged levels of unspliced

16 vector RNA in the cytoplasm of the producer cells (27). Further analyses revealed that Rev

17 only moderately enhanced unspliced vector RNA levels in the cytoplasm, but increased the

18 encapsidated RNA levels by two to three orders of magnitude (3). This effect was not due to

19 Rev-mediated stimulation of particle production, since a Rev-independent, codon-optimized

20 Gag-Pol expression plasmid lacking the RRE provided a constant excess of Gag (44).

21 The lentiviral vector RNA encapsidated in the experiments described above contained large

22 deletions of gag-pol and env also comprising cis-acting regulatory sequences (CRS) and

23 inhibitory sequences (INS) (8, 37–39). Since these sequences have been shown to modulate

24 Rev dependence, the minor effects of Rev on cytoplasmic lentiviral vector RNA levels could

25 simply be due to the absence of such regulatory sequences. Therefore, we have since

26 investigated the influence of Rev on almost full-length HIV-1 unspliced transcript levels in

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 Nuclear RNA Export and Packaging Functions of Rev

1 the cytoplasm and on the encapsidation process. Additionally, we have extended our analyses

2 of the function of Rev to infected T cell lines and primary PBMCs.

4 Material and Methods

5 Plasmids

6 For the cloning of the proviral construct HIVRev-/4xMS2 the env ORF of pNL4-3Rev-/4xMS2

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7 (47) was inactivated by the deletion of bases 7251-7254 (according to GenBank Accession

8 Number AF324493). The rev ORF of the HIVRev-/4xMS2 construct was reactivated by site-

9 directed mutagenesis leading to HIVRev+/4xMS2. The expression plasmids encoding VSV-G

10 (pHIT/G), HIV-1 Tat (pcTat), HIV-1 Rev (pcRev), a fusion of the coat protein of phage MS2

11 and human nuclear shuttling factor TAP (pMS2-hTAP) and codon-optimized gag-pol of HIV-

12 1 (Hgpsyn) were kindly provided by M. Malim, J. Hauber, B. Cullen and R. Wagner,

13 respectively, and have been described previously (7, 14, 29, 44).

14

15 Cell Culture

16 Cell cultures of HEK 293T and TZM-bl were maintained in DMEM (Gibco), 10 % FCS,

17 Ciprofloxacin 20 µg/ml. Human T cell lines CEM-SS, HUT78 and Jurkat were cultured in

18 RPMI1640 (Gibco), 10 % FCS, 20 µg/ml Ciprofloxacin. PBMCs were isolated from the

19 blood buffy coat of three healthy donors using Ficoll400 centrifugation (400 x g, 30 min,

20 20 °C). PBMCs were cultured at 1x106 cells/ml in RPMI1640, 10 % FCS, 20 µg/ml

21 Ciprofloxacin and activated by addition of 5 µg/ml PHA and 100 IU/ml IL-2 for 72 h.

22

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 Nuclear RNA Export and Packaging Functions of Rev

1 Transfections

2 Transfections for the production of viral particles were done using the calcium phosphate

3 coprecipitation method. Briefly, 1.5x106 293T cells were grown in 25 cm2 flasks for 24 h and

4 were transfected with 2 µg pcTat, 2 µg Hgpsyn, and with or without 2 µg pcRev or pMS2-

5 hTAP, respectively. Unless stated otherwise, 500 ng of proviral construct per transfection

6 were used.

7 0.1 µg pCMV-GLuc-1 (Targeting Systems, El Cajon, CA) expressing Gaussia Luciferase was

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8 added to each transfection to control for transfection efficiency. The medium was changed

9 after 8 h to remove excessive plasmid DNA.

10 Transfections for Western blots were done using either calcium phosphate or PEI as described

11 in (1). A PEI/DNA (v/w) ratio of 1.5 was used. The total amount of DNA transfected was

12 adjusted to 10 µg per transfection by addition of calf thymus carrier DNA. Transfections were

13 normalized using Gaussia Luciferase as an indicator for transfection efficiency and cell

14 viability. The luciferase activities were used to adjust the RNA copy numbers of the RT-

15 qPCRs for transfection efficiency.

16

17 RNA Isolation

18 For RNA isolation, cells were detached and washed in 1 ml PBS. The pellet was resuspended

19 in 175 µl buffer RLN (50 mM TrisHCl, pH 8.0; 140 mM NaCl; 1.5 mM MgCl2; 0.5 %

20 Nonidet P-40 Substitute; 1000 U/ml RNase Inhibitor (Qiagen, Hilden, Germany); 1 mM

21 DTT) and incubated for 5 min on ice. Debris and nuclei were pelleted (300 x g, 2 min, 4 °C)

22 and the cytoplasmic fraction was transferred to 600 µl RLT buffer (Qiagen). For RNA

23 isolation from cell nuclei, the nuclear pellet was washed in 500 µl PBS, again pelleted for 3

24 minutes at 300 x g, and then resuspended in 600 µl RLT buffer. The pellet was homogenized

5
 Nuclear RNA Export and Packaging Functions of Rev

1 using Qiashredder columns (Qiagen) at 13000 x g for 30 sec. The flow-through was

2 transferred twice onto the same column.

3 RNA isolation was done using the RNeasy Mini Kit (Qiagen) according to manufacturer´s

4 instructions followed by DNase digestion using the TurboDNA-free Kit (Ambion, Austin,

5 TX). The amount of total RNA extracted from the cytoplasmic and nuclear fraction was

6 determined by the Quant-iT RiboGreen RNA quantitation kit (Invitrogen, Karlsruhe,

7 Germany).

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8 For RNA isolation from virus particles, supernatants of transfected cells were loaded on top

9 of a 30 % sucrose cushion and ultracentrifuged at 150000 x g for 2 h at 4 °C. Virus particles,

10 pelleted from 5 ml of cell culture supernatant were resuspended in 150 µl PBS. RNA was

11 isolated from the resuspended viral particles using QIAamp Viral RNA Mini Kit (Qiagen) and

12 eluted in 45 µl followed by DNase digestion using the TurboDNA-free Kit.

13

14 Quantitative RT-PCRs

15 HIV-1 unspliced RNA levels were determined using the QuantiTect Probe RT-PCR Kit

16 (Qiagen). Primer sequences homologous to a region of gag were taken from the Amplicor

17 HIV-1 Monitor test (31) and are specific for the unspliced HIV-1 RNA. Primers are pSK145

18 (AGT GGG GGG ACA TCA AGC AGC CAT GCA AAT) and pSKCC1B (TAC TAG TAG

19 TTC CTG CTA TGT CAC TTC C). Serial dilutions of an in vitro transcript were prepared as

20 a RNA standard with known copy numbers. The sensitivity of the assay was below 100

21 RNA copies/PCR, the inter-assay variability was 3 %. Cross reaction of the PCR with the

22 codon-optimized Hgpsyn was not detected even after adding 108 DNA copies of Hgpsyn. This is

23 consistent with the 7 to 8 mismatches between the primer sequences and their target sequence

24 of the codon-optimized gene. HIV-1 unspliced RNA copy numbers in 500 ng cytoplasmic or

25 nuclear RNA or 1 µl of the RNA extracted from the viral particles (corresponding to 6 – 26 ng

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 Nuclear RNA Export and Packaging Functions of Rev

1 p24) were determined in direct comparison to serial dilutions of the RNA standard.

2 Elimination of transfected plasmid DNA was routinely confirmed by simultaneously

3 analysing aliquots of one or two representative samples without adding the reverse

4 transcriptase. Since non-encapsidated extracellular RNA is rather unstable we did not perform

5 any additional RNase treatment.

6 Subcellular fractionation was also controlled with the QuantiTect Probe RT-PCR Kit

7 (Qiagen) using primers preGAP-DHE6s (CCA CCA ACT GCT TAG CAC C) and preGAP-

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8 DHE6a (CTC CCC ACC TTG AAA GGA AAT) (4) homologous to the exon 6/intron 6

9 junction and the intron 6 of the unprocessed pre-mRNA of the glyceraldehyd 3-phosphate

10 dehydrogenase gene, respectively. 500 ng of extracted nuclear and cytoplasmic RNA and

11 serial dilutions of the nuclear RNA were subjected to the real-time RT-PCR. This allowed the

12 calculation of the percentage of preGAPDH RNA in the cytoplasm relative to the nuclear

13 preGAPDH RNA levels.

14

15 Protein analyses

16 For the analysis of the purity of cytoplasmic and nuclear fractions two flasks of 293T cells

17 were transfected as described above. One half of the pooled cells was used to prepare total

18 cell lysates by the addition of 500 µl of the stringent BLP lysis buffer (50 mM Tris-HCl (pH

19 7.4); 150 mM NaCl; 40 mM NaF; 5 mM EDTA; 5 mM EGTA; 1 % (v/v) Nonidet P-40;

20 0.1 % (w/v) Natriumdesoxycholat; 0.1 % (w/v) SDS). The other half was fractionated as

21 described for the RNA analyses. Fractions were brought to a final volume of 500 µl using the

22 BLP buffer.

23 Equal amounts of total protein were subjected to SDS-PAGE prior to the staining of the

24 Western blots with antibodies to Histone H2B (Epitomics, Burlingames, CA), α-Tubulin

25 (Rockland, Gilbertsville, PA), and GFP (Santa Cruz Biotechnology, Heidelberg, Germany).

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 Nuclear RNA Export and Packaging Functions of Rev

1 For the examination of Gag expression levels, transfected cells were lysed in 500 µl BLP.

2 After adjusting to the same total protein content, lysates were separated by SDS-PAGE and

3 p24CA was detected using antibody 183-H12-5C (NIH AIDS Research and Reference

4 Reagent Program).

6 Infections

7 VSV-G pseudotyped viruses for the infectivity assay were prepared as follows: 293T cells

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8 were grown to 75 % confluence in a 175 cm2-flask and transfected with 2 µg pcRev, 5 µg

9 Hgpsyn, 2 µg pcTat, 4 µg pHIT/G, and 10 µg of HIVRev-/4xMS2 or HIVRev+/4xMS2,

10 respectively. 1.5 µg pEGFP-C1 and calf thymus carrier DNA to a total of 40 µg DNA were

11 added to each transfection. Transfection medium was changed after 8 h. Supernatants were

12 collected after 48 h and 72 h, filtered through a 0.45 µm filter, and stored at -80 °C in aliquots

13 until use. Infectivity was measured on TZM-bl cells using ß-galactosidase staining as follows:

14 5x104 TZM-bl were seeded on a 24-well plate and grown for 24 h. Cells were treated with

15 200 µl serial dilutions of the virus containing supernatant for 4 h. The medium was changed

16 and cells were grown for 2 days until fixation using 0.5 % glutaraldehyde in PBS. After

17 repeated washing in PBS, cells were stained with 5-bromo-4-chloro-3-indolyl ß-D-

18 galactopyranoside. For infection, T cells or PBMCs were pelleted and resuspended in the

19 virus containing supernatant, thereby adjusting the MOI to 0.1. The medium was changed

20 after 4 h. Cells were cultured for a further 36 h until RNA isolation.

21

22 Statistical analyses

23 To determine whether HIV-1 RNA levels in the cytoplasm and the particle differ significantly

24 in the presence or absence of Rev or MS2-TAP, a two-sided T-test was used on the

25 logarithmically transformed values for the RNA copy numbers.

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 Nuclear RNA Export and Packaging Functions of Rev

1 Results

2 To study the effect of Rev on unspliced HIV-1 RNA levels in the cytoplasm and genomic

3 RNA encapsidation in the context of authentic viral sequences, HIVRev-/4xMS2, a proviral

4 construct with inactivating point mutations in rev, was used (Fig. 1A). The construct also

5 contained a 4 bp deletion in env preventing virus replication. In addition, a part of the nef

6 open reading frame was replaced by four copies of a RNA stem loop of the bacteriophage

7 MS2 to allow complementation of Rev by heterologous export factors (47).

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8 The HIVRev-/4xMS2 construct was transfected into 293T cells in the presence or absence of a

9 Rev expression plasmid. A Tat expression plasmid was always included to enhance

10 expression levels. Since Rev has a strong effect on Gag-Pol expression from proviral

11 constructs, we provided excess levels of Gag-Pol in trans by cotransfection of Hgpsyn. The

12 gag-pol open reading frame of this plasmid has been optimized to mammalian codon usage

13 without changing the amino acid sequence (44). Mutating approximately every fourth

14 nucleotide of gag-pol rendered the Gag-Pol expression completely independent of Rev. As

15 expected, transfection of HIVRev-/4xMS2 led to detectable levels of Gag expression only if co-

16 transfected with the Rev expression plasmid (Fig. 1B). The addition of Hgpsyn led to a further

17 increase of Gag expression levels which were clearly independent of the presence or absence

18 of Rev (Fig. 1B). Thus, any effect of Rev on RNA encapsidation should not be due to the

19 enhancement of Gag-Pol expression.

20 To determine an influence of Rev on the packaging efficiency of proviral RNA we performed

21 a quantitative RT-PCR-based packaging assay. Briefly, 293T cells were co-transfected with

22 HIVRev-/4xMS2, a Tat expression plasmid and Hgpsyn in the presence or absence of a Rev

23 expression plasmid. After 48 h, virus particles were pelleted through a 30 % sucrose cushion

24 and the particle-associated RNA was extracted. In parallel, cells were lysed under standard

25 conditions to isolate cytoplasmic RNA. Quantitative RT-PCR was performed with primers

26 targeting gag in order to measure the amount of full-length HIV-1 RNA in the cytoplasm and

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 Nuclear RNA Export and Packaging Functions of Rev

1 in the viral particles. Hgpsyn encoded RNA is not detected in this PCR due to its extensive

2 codon optimization. As shown in Figure 2, Rev increased the cytoplasmic levels of the HIV-1

3 unspliced RNA after transfection of 1 µg HIVRev-/4xMS2 from 8.9x107 to 4.9x108 copies/µg

4 extracted RNA. In contrast to this modest increase, Rev enhanced packaged HIV-1 RNA copy

5 numbers approximately 4500-fold. To exclude a potential non-linear relationship between the

6 HIV-1 genomic RNA concentration in the cytoplasm and the packaging of this RNA into

7 virus particles, the amount of HIVRev-/4xMS2 DNA transfected was decreased in the presence

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8 of Rev and increased in its absence. This led to experimental conditions under which the

9 cytoplasmic HIV-1 unspliced RNA concentrations were higher in the absence of Rev than in

10 its presence (Fig. 2). Even under these conditions, encapsidation was at least 350-fold higher

11 in the presence of Rev. The packaging defect in the absence of Rev was not due to inefficient

12 particle production, since the amount of p24 detected in the pelleted particles in the absence

13 of Rev was only 1.2 to 4.3-fold lower than with Rev (Fig. 2). This is consistent with previous

14 observations that the budding of the virus particles is independent of the amount of viral RNA

15 (15, 24). Thus, the major function of Rev seems to be the enhancement of packaging of the

16 genomic HIV-1 RNA, while there is only a modest effect on cytoplasmic levels of this HIV-1

17 RNA.

18 Only modest effects of Rev on cytoplasmic RNA levels of RRE-containing transcripts have

19 been observed before, particularly in the context of subgenomic constructs (9, 11, 26) but

20 have also been questioned due to concerns of contamination of cytoplasmic fractions with

21 nuclear RNAs (28). We therefore performed various experiments to determine the purity of

22 the cytoplasmic fractions. 293T cells were transfected as before including an EGFP

23 expression plasmid and the cytoplasmic fraction was recovered as described above. In

24 addition, the nuclear fraction was collected after a washing step of the nuclear pellet. Total

25 cell lysates as well as the nuclear and cytoplasmic fractions were then analyzed by Western

26 blot for EGFP, histone protein H2B and α-tubulin content (Fig. 3A). As expected, histone

10
 Nuclear RNA Export and Packaging Functions of Rev

1 H2B was detected in the nuclear fraction but not in the cytoplasmic fraction, while α-tubulin

2 was present in the cytoplasmic fraction but not in the nuclear fraction. EGFP could be

3 detected in both fractions confirming proper loading. Histone H2B could be more tightly

4 associated with the nucleus than nuclear RNA and is therefore perhaps not a good marker for

5 the contamination of the cytoplasmic fraction with nuclear RNA. We therefore determined the

6 ratio of pre-mRNA levels of the endogenous Glyceraldehyde 3-phosphate dehydrogenase

7 (preGAPDH) gene in the cytoplasm and the nucleus. RNA was extracted from the nuclear and

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8 cytoplasmic fractions of transfected 293T cells. The same amounts of extracted nuclear and

9 cytoplasmic RNA and serial dilutions of the nuclear RNA were subjected to real-time RT-

10 PCR with primers spanning intronic sequences of preGAPDH. Similar crossing points of the

11 real-time RT-PCR results for the cytoplasmic RNA samples and a 1:30 dilution of the nuclear

12 RNA preparation indicated that the concentration of the preGAPDH RNA in the cytoplasmic

13 RNA was approximately 3 % of the concentration of the nuclear RNA (Fig. 3B). Assuming

14 an exclusive nuclear localization of the preGAPDH RNA, these results indicate that 3 % of

15 the extracted cytoplasmic RNA is actually derived from the nucleus. This nuclear

16 contamination could only bias the results regarding the effect of Rev on the cytoplasmic RNA

17 levels of the unspliced HIV-1 RNA, if the concentration of this RNA in the nucleus was

18 substantially higher than in the cytoplasm. Therefore, we also quantified the HIV-1 full-length

19 RNA copy numbers in RNA extracted from the nucleus and cytoplasm of HIVRev-/4xMS2

20 DNA transfected cells. In the presence and absence of Rev the concentration of the unspliced

21 transcript in the cytoplasmic RNA preparation was higher than in the nuclear preparation (Fig.

22 3C). This excludes the possibility that the full magnitude of the enhancing effect of Rev on

23 the cytoplasmic levels of the full-length HIV-1 RNA is masked by a contamination with

24 nuclear RNA.

25 Over-expression by transient transfection of epithelial 293T cells might lead to a spill-over of

26 nuclear HIV-1 RNA into the cytoplasm in a Rev-independent manner. Therefore, we also

11
 Nuclear RNA Export and Packaging Functions of Rev

1 studied the effect of Rev on cytoplasmic RNA levels under rather natural expression levels in

2 lymphoid cell lines which is considered to be a more relevant model for HIV infection. The

3 CEM-SS, HUT78 and Jurkat lymphoid T cell lines were infected with VSV-G pseudotyped

4 HIV particles transferring either Rev-deficient HIVRev-/4xMS2 genome or a Rev-proficient

5 HIVRev+/4xMS2 genome. The rev defect of the HIVRev-/4xMS2 construct was complemented

6 in trans during the production of the pseudotypes by co-transfection of a Rev expression

7 plasmid. Vector titers were determined on TZM-bl cells in order to normalize the infectious

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8 dose to a multiplicity of infection of 0.1. Forty hours after infection cytoplasmic RNA was

9 isolated as described above and the HIV-1 genomic RNA levels were compared between

10 HIVRev-/4xMS2 and HIVRev+/4xMS2 infected T cell lines. Although all three cell lines were

11 infected with the same stocks of pseudotypes, the effect of Rev on unspliced cytoplasmic

12 HIV-1 RNA levels varied substantially (Fig. 4). In CEM-SS cells Rev enhanced the levels of

13 the genomic transcript in the cytoplasm nearly 60-fold, while the 3- to 9-fold increase

14 observed in Jurkat and HUT78 cells is similar to our observations in transfected 293T cells.

15 Therefore, the HIVRev-/4xMS2 and HIVRev+/4xMS2 pseudotypes were also used to infect

16 activated human peripheral blood monocytes (PBMCs) at a multiplicity of infection of 0.1.

17 Determining the unspliced HIV-1 RNA levels in the cytoplasm of the infected PBMCs

18 revealed that Rev increased these HIV-1 RNA levels just 3-fold (Fig. 4).

19 The nuclear export activity of Rev can be functionally replaced by constitutive transport

20 elements (5, 18, 19, 33) or tethering of heterologous export factors to the HIV-1 RNA (46,

21 47). To determine whether this is also valid for the packaging effect of Rev the HIVRev-

22 /4xMS2 DNA was co-transfected with an expression plasmid for a fusion protein consisting

23 of the MS2 coat protein and the human Tip associated protein (hTAP), a cellular factor known

24 to shuttle mRNA from the nucleus to the cytoplasm. Since the HIVRev-/4xMS2 RNA contains

25 four copies of the MS2 stem loop in nef, the MS2-hTAP fusion protein can bind to the HIV-1

26 RNAs. Similar to a previous study (46), the co-transfection of MS2-hTAP with HIVRev-

12
 Nuclear RNA Export and Packaging Functions of Rev

1 /4xMS2 DNA increased p24 levels approximately 140-fold (see Fig. 5B). However, co-

2 transfection of MS2-hTAP enhanced unspliced HIV-1 RNA levels in the cytoplasm only

3 marginally, while encapsidation of the HIV-1 genomic RNA into particles encoded by co-

4 transfected Hgpsyn was enhanced 280-fold (Fig. 5A). The tethering of hTAP to the MS2 stem

5 loops was important, since encapsidation of HIVRev- genomic RNA lacking the MS2 stem

6 loops was strongly impaired even in the presence of MS2-hTAP. Although Rev enhanced

7 encapsidation to an even larger extent, the tethering of hTAP to the HIV-1 genomic RNA can

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8 clearly rescue the packaging defect of a Rev-deficient HIV-1. To measure the effect of Rev

9 and MS2-hTAP on HIV-1 infectivity, VSV-G pseudotyped HIVRev-/4xMS2 particles were

10 produced in the presence or absence of Rev or MS2-hTAP (Fig. 5B). Without Rev and MS2-

11 hTAP the infectious titer was below the detection limit. Adding Rev or MS2-hTAP increased

12 the infectious titer by at least 3000- and 500-fold, respectively. Since Rev and MS2-hTAP

13 also increased the secretion of Gag from HIVRev-/4xMS2 transfected cells 533- and 140-fold,

14 respectively, the increase in infectivity could simply be due to enhanced particle production

15 rather than the enhancement of RNA encapsidation. Therefore, we again included Hgpsyn in

16 the co-transfection experiments. Adding Hgpsyn in the presence of Rev or MS2-hTAP did not

17 further increase the infectious titer (Fig. 5B). In the absence of MS2-hTAP and Rev, Hgpsyn

18 allowed the transfer of the HIVRev-/4xMS2 construct, but the infectious titer was

19 approximately 100-fold lower than in the presence of Rev. Since transfection of Hgpsyn leads

20 to higher Gag expression levels than the co-transfection of HIVRev-/4xMS2 and the Rev

21 expression plasmid, the infectivity correlates better with encapsidation efficacy than with Gag

22 expression levels.

23

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 Nuclear RNA Export and Packaging Functions of Rev

1 Discussion

2 The observation that the tethering of TAP to the HIV-1 RNA enhances the encapsidation of

3 the genomic RNA to a much larger extent than the cytoplasmic RNA levels indicates that the

4 packaging function of Rev can be replaced by a heterologous protein. This is consistent with

5 the recent observation that the replacement of the RRE by a constitutive transport element

6 (CTE) leads to efficient genomic RNA encapsidation (32). Since the tethering of TAP to Rev-

7 dependent RNA can also replace the nuclear export function of Rev (46), both activities of

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8 Rev seem to be mechanistically linked. However, this raises a number of questions regarding

9 the precise mechanism by which Rev and TAP enhance the encapsidation process. How can

10 both the Rev-mediated nuclear export via the CRM-1 pathway and the MS2-TAP triggered

11 nuclear export render the cytoplasmic HIV-1 genomic RNA accessible to packaging, while

12 the same HIV-1 genomic RNA reaching the cytoplasm in the absence of Rev or MS2-TAP is

13 poorly encapsidated? This is particularly puzzling, since without a tethered export factor, the

14 unspliced HIV-1 RNA is believed to reach the cytoplasm via the default TAP-mediated

15 mRNA export pathway. One potential explanation could be that the rapid tethering of the

16 unspliced HIV-1 RNA to either of the two nuclear export pathways prevents the formation of

17 a ribonucleoprotein (RNP) complex that is incompetent for translation and encapsidation

18 despite being exported to the cytoplasm. Evidence for the rapid interaction of Rev with the

19 newly synthesized target RNA has been previously obtained (20). In addition to Rev and

20 tethered TAP, CTEs were also shown to enhance the nuclear export and translation of Rev-

21 dependent RNAs (18, 19, 33) as well as the infectivity of lentiviral vector particles (48). Since

22 it is unlikely that different nuclear export factors and constitutive transport elements trigger

23 the formation of RNP complexes mediating the same pattern of pleiotropic effects

24 (enhancement of nuclear export, translation and packaging), the suppression of the formation

25 of an inhibitory RNP complex by different RNA export factors and constitutive transport

26 elements might be the more plausible mechanism. Whether any of the known cellular co-

14
 Nuclear RNA Export and Packaging Functions of Rev

1 factors of Rev (recently reviewed in (40)) is involved in the suppression of the formation of

2 such an inhibitory RNP remains to be determined. The formation of a poorly accessible HIV-

3 1 RNA protein complex in the absence of Rev might also explain an apparent discrepancy

4 between full-length transcript levels in the cytoplasm as observed by in situ hybridisation and

5 cellular fractionation experiments, respectively. In the absence of Rev there is hardly any

6 cytoplasmic unspliced HIV-1 RNA detectable by in situ hybridisation (10, 23). In contrast, we

7 and others have observed substantial levels of unspliced HIV-1 RNA in the cytoplasm by

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8 cellular fractionation experiments. Extensive control experiments for the fractionation argue

9 against the assumption that high levels of full-length transcript detected in the cytoplasmic

10 RNA preparation in the absence of Rev are due to the contamination with nuclear RNA. This

11 clearly raises the possibility that the unspliced RNA present in the cytoplasm without Rev

12 escapes efficient detection by in situ hybridisation. The mild denaturing conditions during in

13 situ hybridisation might not be sufficient to resolve a tightly packed RNP complex that is

14 formed in the absence but not the presence of Rev.

15 The magnitude of the effect of Rev on genomic HIV-1 RNA levels is not only influenced by

16 the method used to measure the amount of HIV-1 RNA but also by the type of cells analyzed.

17 In infected lymphoid cell lines, Rev enhanced cytoplasmic RNA levels from 3- to 60-fold.

18 The latter value was obtained in CEM-SS cells. Interestingly, this cell line was also used in a

19 previous study demonstrating that Rev is absolutely required for nuclear export (28).

20 However, since Rev only enhances cytoplasmic RNA levels in primary PBMCs by a factor of

21 3, this strong dependence on Rev seems to be a particular property of CEM-SS cells.

22 Thus, the results of the present study challenge the general notion that the main function of

23 Rev is simply to mediate nuclear export of viral RNAs. Interestingly, the nuclear export

24 pathway of Gag encoding RNAs has also been observed to modulate the assembly of Gag

25 proteins into particles (21, 22, 41, 42). Therefore, it rather seems that Rev modulates the RNP

26 complex formed in the nucleus on RRE-containing viral RNAs, thereby either leading to a

15
 Nuclear RNA Export and Packaging Functions of Rev

1 particular subcytoplasmic compartmentalization or affecting the accessibility of the unspliced

2 HIV-1 RNA to translation and encapsidation.

3 The detection of substantial levels of unspliced HIV-1 RNA in the cytoplasm in the absence

4 of Rev and therefore probably also during the early phase of the viral replication cycle could

5 severely affect HIV-1 spread and persistence. If the structural proteins were indeed produced

6 during the early phase of replication, infected cells could be lysed by antiviral effector

7 mechanisms prior to the release and spread of progeny virus. Therefore, the Rev dependence

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8 of nuclear export and translation of the unspliced and singly-spliced HIV-1 RNAs might have

9 actually evolved in order to increase the efficiency, by which expression of structural proteins

10 during the early phase of the viral replication cycle is prevented. In the late phase of the

11 replication cycle, Rev could then promote coordinated particle production and RNA

12 encapsidation.

13

14 Acknowledgements

15 This work was supported through a grant from the German Research Foundation to K.Ü.

16 (Ue45/11-1). B.G. is supported by the graduate course GRK 1045 funded by the German

17 Research Foundation. The funders had no role in study design, data collection and analysis,

18 decision to publish, or preparation of the manuscript.

19 We are grateful to T. Grunwald, N. Ternette, V.V. Temchura and B. Tippler for experimental

20 help and would like to thank M. Malim, J. Hauber, B. Cullen and R. Wagner for providing

21 plasmids and D. Cosgrove for critical reading of the manuscript. TZM-bl cells were obtained

22 from the EVA Centre for AIDS Reagents, NIBSC, UK and were donated by JC Kappes, X

23 Wu, and Tranzyme Inc. The following reagent was obtained through the NIH AIDS Research

24 and Reference Program, Division of AIDS, National Institute of Allergy and Infectious

16
 Nuclear RNA Export and Packaging Functions of Rev

1 Diseases: HIV-1 p24 Hybridoma (183-H12-5C), catalog number 1513 from Bruce Chesebro

2 and Hardy Chen.

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22

22
 Nuclear RNA Export and Packaging Functions of Rev

1 Figure Legends

2 Figure 1: Rev-(in)dependent Gag-Pol expression.

3 A) Map of plasmids HIVRev-/4xMS2 and Hgpsyn. The proviral HIVRev-/4xMS2 construct

4 contains the HIV-1 genome with inactivating point mutations in rev and env. In addition, parts

5 of nef gene are replaced by 4 repeats of an RNA stem loop targeted by the MS2 coat protein

6 (marked by the white box). Inactive ORFs are shown as black bars, active ORFs appear grey.

7 The arrow indicates the region of the HIV-1 genome detected by the RT-qPCR.

Downloaded from jvi.asm.org at NAT INST OF HEALTH LIB on May 21, 2010
8 Hgpsyn encompasses a codon-optimized gag-pol ORF flanked by a strong heterologous

9 promoter (CMV) and a polyadenylation (pA) signal. The codon-optimized ORF is hatched.

10 B) Western blot analysis with an HIV-1 p24 capsid antibody of total cell lysates transfected

11 with proviral DNA (HIVRev-/4xMS2) with or without the codon-optimized Gag-Pol

12 expression construct (Hgpsyn) in the presence or absence of a Rev expression plasmid (upper

13 panel). A GFP expression plasmid was also co-transfected to control for transfection

14 efficiency by Western blot analysis with an anti-GFP antibody (lower panel).

15

16 Figure 2: Rev enhances packaging of HIV-1 genomic RNA.

17 Unspliced HIV-1 RNA copy numbers in the cytoplasmic RNA fraction and in the RNA

18 extracted from viral particles are shown. The indicated amounts of HIVRev-/4xMS2 DNA were

19 co-transfected with a constant amount of Hgpsyn in the presence or absence of Rev as

20 indicated. Values shown are the means and the SEMs of 3 (*=2) experiments. The fold-

21 enhancement of full-length HIV-1 RNA levels in the cytoplasm and the particles by Rev is

22 given for the 1 µg dose of HIVRev-/4xMS2. Statistical analysis of these results revealed that

23 the difference between the HIV-1 RNA copy numbers is statistically significant (p < 0.01) in

23
 Nuclear RNA Export and Packaging Functions of Rev

1 the particles, but not in the cytoplasm. Numbers inside the bars for the RNA copy numbers in

2 the particles give the mean of the p24 concentrations in the particle preparations.

4 Figure 3: Purity of fractions.

5 A) Western blot analyses of different fractions of cell lysates. Cell lysates of transfected cells

6 were normalized for total protein content and subjected to SDS-PAGE. Antibodies detecting

7 histone H2B, α-tubulin, and GFP were used, respectively.

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8 B) Cellular pre-mRNA of GAPDH was detected via RT-qPCR. The cytoplasmic RNA levels

9 are given as percentage of nuclear RNA levels. Results shown are the mean +SEM of 4

10 experiments.

11 C) Unspliced HIV-1 RNA was detected in cytoplasmic and nuclear fractions using RT-qPCR.

12 Samples were normalized for RNA content. The results shown are the mean +SEM of 3

13 experiments.

14

15 Figure 4: Effect of Rev on cytoplasmic RNA levels in infected lymphoid

16 cells.

17 T cells were infected at an MOI of 0.1 with VSV-G pseudotyped HIV-1 constructs only

18 differing in their Rev expression. Unspliced RNA copy numbers were determined in the

19 cytoplasmic fractions by RT-qPCR. Shown are the means +SEM of 3 independent

20 experiments. The fold-enhancement by Rev is given above the horizontal bars.

21

22 Figure 5: Functional replacement of Rev

23 A) Copy numbers of unspliced HIV-1 RNA in the cytoplasmic RNA fraction and in the RNA

24 extracted from viral particles of cells co-transfected with HIVRev-/4xMS2, HIVRev-, a Rev

24
 Nuclear RNA Export and Packaging Functions of Rev

1 expression plasmid and/or the MS2-TAP expression plasmid. The mean values +SEM of 2-4

2 independent experiments are shown. The fold-enhancement of unspliced HIV-1 RNA levels

3 in the cytoplasm and particles by MS2-hTAP is given. Statistical analysis revealed that the

4 difference between the HIV-1 RNA copy numbers in the presence or absence of MS2-TAP is

5 statistically significant (p < 0.05) in the particles, but not in the cytoplasm.

6 B) VSV-G pseudotyped HIVRev-/4xMS2 particles were prepared by cotransfection with or

7 without Hgpsyn and expression plasmids for Rev or MS2-TAP. The mean values +SEM of the

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8 infectious titers of 2 to 3 independent experiments are shown. * Mean p24 concentration in

9 the supernatant of cells co-transfected with HIVRev-/4xMS2 or cotransfected with HIVRev-

10 /4xMS2 and Rev or MS2-TAP.

11

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