Human CRM1 Augments Production of Infectious ... - Journal of Virology

Human CRM1 Augments Production of Infectious Human and Feline Immunodeficiency Viruses from Murine Cells Hila Elinav,a* Yuanfei Wu,a Ayse Coskun,a Katarzyna Hryckiewicz,a* Iris Kemler,b Yani Hu,a Hilary Rogers,a Bing Hao,c Choukri Ben Mamoun,a Eric Poeschla,b and Richard Suttona Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USAa; Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USAb; and Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut, USAc

Productive replication of human immunodeficiency virus type 1 (HIV-1) occurs efficiently only in humans. The posttranscriptional stages of the HIV-1 life cycle proceed poorly in mouse cells, with a resulting defect in viral assembly and release. Previous work has shown that the presence of human chromosome 2 increases HIV-1 production in mouse cells. Recent studies have shown that human chromosome region maintenance 1 (hCRM1) stimulates Gag release from rodent cells. Here we report that expressions of hCRM1 in murine cells resulted in marked increases in the production of infectious HIV-1 and feline immunodeficiency virus (FIV). HIV-1 production was also increased by hSRp40, and a combination of hCRM1 and hSRp40 resulted in a more-than-additive effect on HIV-1 release. In contrast, the overexpression of mouse CRM1 (mCRM1) minimally affected HIV-1 and FIV production and did not antagonize hCRM1. In the presence of hCRM1 there were large increases in the amounts of released capsid, which paralleled the increases in the infectious titers. Consistent with this finding, the ratios of unspliced to spliced HIV-1 mRNAs in mouse cells expressing hCRM1 and SRp40 became similar to those of human cells. Furthermore, imaging of intron-containing FIV RNA showed that hCRM1 increased RNA export to the cytoplasm.By testing chimeras between mCRM1 and hCRM1 and comparing those sequences to feline CRM1, we mapped the functional domain to HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats 4A to 9A and a triple point mutant in repeat 9A, which showed a loss of function. Structural analysis suggested that this region of hCRM1 may serve as a binding site for viral or cellular factors to facilitate lentiviral RNA nuclear export.

T

o date, no immunocompetent small-animal model permissive to human immunodeficiency virus type 1 (HIV-1) has been developed. HIV-1 encounters multiple blocks to its life cycle in mouse cells. Murine CD4 and CCR5 cannot function as HIV-1 entry receptors (4, 10, 28, 30, 45), and murine cyclin T1 does not support efficient transcription elongation secondary to a loss-offunction point mutation (5, 23, 61). While expression of human CD4, CCR5, and cyclin T1 in murine cells improves HIV-1 replication to the point of transcription, a potent block to virion assembly and release persists (36, 57, 58, 65). Thus, murine cells produce very low levels of infectious HIV-1 particles, and full viral replication cannot occur (3). The underlying molecular mechanisms of this barrier remain poorly understood and have not been fully characterized. Nuclear HIV-1 mRNAs have two potential fates: they may be fully spliced and exported from the nucleus by the canonical mRNA splicing and nuclear export machineries, or they may remain partially or fully unspliced and be exported from the nucleus in a distinct pathway (14, 62). Fully spliced HIV-1 mRNAs are exported to the cytoplasm in a Tip-associated protein (TAP)-dependent manner (12, 13), and those mRNAs are translated into Rev, Tat, and Nef proteins. Rev is then imported into the nucleus, where it multimerizes and binds to the Rev-responsive element (RRE) on RRE-containing mRNAs to facilitate their nuclear export (19, 20, 33–35, 44). The export process requires interaction of the Rev-RRE complex with CRM1 (6, 21, 24, 41) as well as with Ran-GTP (1), RanBP3 (17, 31, 40), and DDX3 (64). Following interaction of this complex with RanBP2/NUP358, the nuclear complexes shuttle through the nuclear pores (2, 25). Recently it was shown that the trimethyl capping enzyme protein PIMT may

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also be involved in the nuclear export of intron-containing HIV-1 RNAs (63). Once in the cytoplasm, the complex disassembles, a process assisted by RanBP1 and RAN GTPase-activating protein (RanGAP) (26, 29). Ran-GDP, CRM1, and Rev then shuttle back to the nucleus. Partially spliced and unspliced cytoplasmic HIV-1 mRNAs serve as the templates for the translation of other viral proteins (Gag, Pol, Env, Vpr, Vif, and Vpu), whereas the unspliced mRNAs may also be packaged into virions (53). Nearly a decade ago it was shown that HIV-1 mRNAs were overspliced in mouse cells, leading to markedly reduced amounts of cytoplasmic intron-containing HIV-1 mRNAs (3, 11, 54). The relatively low levels of unspliced mRNAs produced in mouse cells may impede the assembly of virions due to low levels of viral structural proteins and genomic RNA. Although this step in viral replication has been intensively investigated, the molecular mechanism has not been fully elucidated (18, 50, 51, 57). Several host factors have been implicated in the modulation of the ratio between spliced and unspliced HIV-1 mRNA (50). Overexpression

Received 30 July 2012 Accepted 18 August 2012 Published ahead of print 29 August 2012 Address correspondence to Richard Sutton, [email protected]. * Present address: Hila Elinav, Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; Katarzyna Hryckiewicz, Poznan University of Medical Sciences, Department of Infectious Diseases, Poznan, Poland. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.01970-12

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FIG 1 Human CRM1 and SR proteins increase infectious HIV release from mouse cells. (A) Vector constructs used here; the brackets indicate deletions, and ⌿ denotes packaging signals. The arrows indicate the locations of primers used for spliced/unspliced RNA analyses. Expressions of indicated proteins were confirmed by immunoblotting in 293T cells after transfection (B) and in B78 cells after nucleofection (C). Mock transfection lanes are indicated by “-.” Despite repeated attempts, the expression of PIMT was never observed; the band in this lane reflects spillover from the adjacent lane. In selected lanes, the asterisks indicate protein bands. (D through F) Resultant infectious HIV titers after nucleofection of B78.CIB cells with indicated cDNA expression plasmids, normalized to those of empty vectors by transfection efficiency as judged by eYFP epifluorescence and cell viability by light microscopy; panels D through F reflect the mean results of 4 independent experiments. *, P is ⬍0.05 by one-way analysis of variance (ANOVA).

of human p32 in mouse cells decreased splicing of HIV-1 mRNA and increased virion-associated capsid (CA) by inhibiting alternative splicing factor/splicing factor 2 (ASF/SF2) (66). Overexpression of both human and murine serine-rich (SR) proteins SRp40 and SRp55 in mouse cells altered the HIV-1 mRNA ratio in favor of unspliced mRNA and was associated with improved Gag translation and consequently increased CA production (55). Expression of the synthetic codon-optimized gag-pol construct in murine cells increased virallike particle production to levels similar to those of 293T cells without a concomitant increase in infectious viruses, possibly due to reduced levels of HIV RNA transcripts (15). We have previously demonstrated that the presence of human chromosome 2 in murine fibroblasts dramatically increased CA production and infectious HIV-1 release, but the genetic determinant was not identified (11). More recently, however, it was shown that expression in rat cells of human chromosome region mainte-

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nance 1 (hCRM1), which is encoded by human chromosome 2, resulted in slightly elevated levels of intracellular Gag but much greater increases in levels of extracellular CA (39). These effects were not observed when rat CRM1 was overexpressed. Sherer et al. demonstrated that expression of hCRM1 in mouse cells augments the nuclear export of partially spliced or unspliced RNAs and the production of Gag and the subsequent processing of CA (48). Although only small increases in cytosolic Gag levels were observed, as well as greater increases in unspliced than spliced HIV-1 mRNAs, there was a marked increase in the amount of CA released from the cells. This difference was attributed to cooperative, nonlinear multimerization of Gag due to its greater abundance, especially at the plasma membrane, and the effect was mapped to two of the HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats of hCRM1, 9A and 10A.

Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

FIG 2 Mouse CRM1 is a loss-of-function allele. (A through C) Increasing amounts of hCRM1 were nucleofected into B78.CIB cells, and the resultant HIV-1 titers were normalized to both transfection efficiency and cell viability as per Fig. 1. (D) hCRM1 and/or mCRM1 were similarly nucleofected into B78.CIB cells, and the resultant Bsdr, eYFP, and Purr titers were normalized to both transfection efficiency and cell viability; the results reflect a representative experiment performed in triplicate, and the experiment was repeated 4 times with similar results. *, P is ⬍0.05 by one-way ANOVA. The expressions of hCRM1 and mCRM1 were confirmed by immunoblotting in 293T cells (E) and B78 cells (F), with the mock-transfected lane indicated by “-.”

We have now extended those findings and report here that hCRM1 and SRp40 act in a more-than-additive manner to augment infectious HIV-1 release from mouse cells, and the effect of hCRM1 on feline immunodeficiency virus (FIV) production was even more pronounced. This effect was due in part to the modulation of unspliced versus spliced viral mRNA levels and the export of unspliced viral mRNA from the nucleus. We mapped the determinant first to several HEAT repeats and then to specific residues of hCRM1. The striking structural similarities between mCRM1 and hCRM1 suggest that specific residues in the HEAT repeats may serve as binding sites for either a viral protein or an additional currently unknown host cellular factor. MATERIALS AND METHODS Cell culture. Human and mouse cells were grown at 37°C in Dulbecco’s modified Eagle high-glucose medium (DMEM) containing L-glutamine and supplemented with 10% fetal bovine serum (Gibco), penicillin/streptomycin, and ciprofloxacin (10 ␮g/ml) in a 5% CO2 water-jacketed incubator. B78 (murine melanoma) and A9 (murine fibroblast) cells were transduced with a replication-defective HIV-1 vector encoding both truncated human cycT1 and blasticidin S deaminase (bsd), which confers resistance to blasticidin (pHIV-1-cycT1-IRES-bsd) (Fig. 1A) (11). The resulting cell lines B78.CIB and A9.CIB were maintained in DMEM supplemented with 10 ␮g/ml blasticidin (InVivogen). B78 cells that had been transduced with a murine leukemia virus (MLV) vector encoding both human cycT1 and puromycin acetyltransferase (B78.cycT1) were maintained in DMEM supplemented with 10 ␮g/ml puromycin (SigmaAldrich). Human osteosarcoma (HOS) cells transduced with the HIV-1Hygro vector were designated HOS-Hygro and maintained in DMEM with 0.5 mg/ml of hygromycin (Calbiochem). GM11686 cells transduced with HIV-1-cycT1-IRES-bsd (11) were maintained in DMEM supplemented with 10 ␮g/ml blasticidin and 0.5 mg/ml active G418 (Calbiochem). 293T and 3T6 (murine fibroblast) cells were transduced with an HIV-1 vector encoding both firefly luciferase (or enhanced firefly luciferase) and the blasticidin resistance marker. These cells were termed

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293T.LIB and 3T6.effluc-IB, respectively, and maintained in DMEM supplemented with 10 ␮g/ml blasticidin. Plasmids. Human Ran, RanBP1, and RanBP3 cDNAs were PCR amplified from a HeLa cell cDNA library, DNA sequenced, and subcloned into FLAG pcDNA3.1 between the EcoRI and BamHI sites. Of note, in comparison to the reference sequence, the penultimate amino acid of the cloned RanBP3 was arginine instead of alanine. RanBP3 cDNA cloned from several different cDNA libraries yielded the same substitution. The encoded proteins harbored an N-terminal FLAG epitope tag. We confirmed protein expression by transient transfection of the proteins into 293T cells and B78.CIB cells and separation by SDS-PAGE of detergenttreated cellular lysates, followed by immunoblotting using an anti-FLAG mouse monoclonal antibody (Sigma-Aldrich). hCRM1 and mCRM1 cDNAs were obtained from Open Biosystems and were PCR amplified, DNA sequenced, and cloned into the expression plasmid pCMV-HA (Clontech) between the XhoI and NotI sites such that the hemagglutinin (HA)-epitope tag was the amino terminus. Protein expression in transfected cells was confirmed as described above using an anti-HA monoclonal antibody (Covance). Chimeras between HA-tagged mCRM1 and hCRM1 were constructed by using convenient, conserved restriction endonuclease sites between the two cDNAs, and the expression of each chimera in transfected human or mouse cells was verified as described above. Human DDX3, RIP, and ei5FA, each subcloned into pCMV-HA, were kindly provided by J. Henao-Mejia (Yale University). Human SRp40 and SRp55, each T7 tagged in pCGT-T7, were gifts from A. R. Krainer (Cold Spring Harbor Laboratory) (8). The vesicular stomatitis virus (VSV) glycoprotein expression plasmid pME-VSV G, the HIV-1 packaging plasmid, and the HIV-1-cycT1-IRES-eYFP were as previously described (47) (Fig. 1A). HIV-puro G⫺P⫺E⫺F⫺V⫺ was derived from HIV-1-AP G⫺P⫺E⫺F⫺V⫺ (52) by replacing the 1.6-kb alkaline phosphatase cassette with a 1.1-kb cassette of simian virus 40 (SV40) ori promoter driving the puromycin acetyltransferase (PAC) gene (Fig. 1A). pSR␣-YU2 Env encodes a codon-optimized HIV-1 YU2 envelope glycoprotein downstream of the SR␣ promoter. The FIV packaging plasmid pFP93 and transfer vector pGINSIN were described previously (32, 46) (see Fig. 5C). The equine infectious anemia virus (EIAV) packaging plasmid pCEV53B

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TABLE 1 Recoverable HIV titers from mouse and human cell linesa

Cell line

Bsdr titer with empty vector

Bsdr titer with hCRM1

Purr titer with empty vector

Purr titer with hCRM1

eYFP titer with empty vector

eYFP titer with hCRM1

A9.CIB 3T6.efflucIB GM11686.CIB B78.CIB 293T.LIB

13 66 900 1,500 8.0 ⫻ 106

804 1,920 NDb 10,000 ND

120 4,685 12,000 3,200 1.5 ⫻ 106

5,033 35,333 ND 21,000 ND

⬍10 18,666 5,000 25,000 1.5 ⫻ 106

2,500 163,333 ND 150,000 ND

a Indicated cell lines were transfected (human) or nucleofected (mouse) with pME VSV G, HIV-CIY, HIV-puro G⫺P⫺E⫺F⫺V⫺ and either an empty vector or a plasmid encoding hCRM1. After 48 h, the supernatant titer was determined on HOS-Hygro cells as described in the text. Results shown are representative of one of three experiments. b ND, not done.

(42) and the transfer vector SIN6.1CeGFPW (37) were kindly provided by J. Olsen (University of North Carolina, Chapel Hill), and the SIVmac239 (SIVmac)-packaging vector and the transfer vector were from Hung Fan (University of California, Irvine) (60). The murine leukemia virus (MLV)

packaging plasmid pHIT60 was as described previously (49), and pQCXIP (Clontech) is an MLV transfer vector. Single and multiple point mutants of hCRM1 were produced by a modification of standard site-directed QuikChange mutagenesis (Stratagene) using a TaKaRa high-fidelity polymerase enzyme. For each mutant CRM1, the DNA sequence of the entire 3.2-kb insert was confirmed by automated DNA sequencing. Cell transfection and virus production. Retroviral and lentiviral pseudotyped particles were produced using Lipofectamine 2000 reagent (Invitrogen) by transient transfection of 293T cells using the indicated plasmids according to the manufacturer’s instructions. For virus production by transfection using nucleofection of mouse cells, the MEF1 reagent and an Amaxa Nucleofector machine were used with the indicated amounts of various plasmids according to the manufacturer’s instructions. For transfections, 293T cells in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco) were plated at 30% confluence in 6-well plates 24 h prior to transfection. When cells were transfected using the Lipofectamine 2000 reagent, the medium was changed 5 h posttransfection. Viability and transfection efficiency were assessed 24 h after transfection. Forty-eight hours after transfection, the medium was harvested

FIG 3 SRp40 and hCRM1 have more-than-additive effects on HIV-1 titers. B78.CIB cells were nucleofected with the indicated amounts of plasmids, and the resultant Bsdr (A), eYFP (B), and Purr (C and D) titers are shown, normalized to empty vector, transfection efficiency, and cell viability. Note that the more-than-additive effect on Purr titers was reproducibly observed using only smaller amounts of plasmid. The data shown reflect the mean results of 5 independent experiments.

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Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

FIG 4 SRp40 and SRp55 do not have a more-than-additive effect on HIV-1 titers. B78.CIB cells were nucleofected with the indicated amounts of plasmids, and the resultant Bsdr (A), eYFP (B), and Purr (C) titers are shown, normalized to empty vector, transfection efficiency, and cell viability. The data shown reflect results of two independent experiments.

by centrifugation at 1,800 ⫻ g for 10 min. For immunoblotting analyses, transfected cells were lysed in 150 mM NaCl, 50 mM Tris-Cl (pH 7.5), 10% glycerol, 1 mM dithiothreitol (DTT), and 1% Triton X-100, supplemented with a protease-inhibitor cocktail (catalog no. 1015750; Roche Diagnostics, Germany). For RNA preparation, cells were lysed using RLT buffer (Qiagen) supplemented with ␤-mercaptoethanol (Sigma-Aldrich) according to the manufacturer’s instructions. For nucleofection, murine cells were preincubated for 48 h until they reached 80 to 90% confluence. Cells were nucleofected using the MEF1 reagent (Lonza) and an Amaxa Nucleofector (program A-23) according to the manufacturer’s instructions. Viability and transfection efficiency were assessed at 24 h posttransfection using light and inverted fluorescence microscopy. Forty-eight hours after nucleofection, the cells were pelleted by centrifugation (1,800 ⫻ g for 10 min), and the culture supernatant was harvested. Nucleofected cells were lysed as described above for immunoblotting or RNA extraction. Infectivity assay. HOS-Hygro cells were plated in 12-well tissue culture plates 24 h prior to transduction such that they were 40 to 50% confluent at the time of transduction. The cell supernatant harvested from transfected cells was centrifuged at 1,800 ⫻ g for 10 min, and the titers were determined by limiting the dilution on the HOS-Hygro target cells.

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Forty-eight hours posttransduction, enhanced yellow fluorescent protein-positive (eYFP⫹) target cells were visualized by inverted fluorescence microscopy. Target cells were then trypsinized and transferred to DMEM supplemented with hygromycin (1.0 mg/ml) and blasticidin (10 ␮g/ml) or hygromycin (1.0 mg/ml) and puromycin (10 ␮g/ml). Approximately 7 days later, the cell colonies were visualized by fixation using methanol, acetic acid, and crystal violet. To determine the infectious viral titers, colonies were enumerated and normalized to both cell viability and transfection efficiency as measured 24 h posttransfection. For the FIV, EIAV, and SIVmac vectors 48 to 72 h after cellular transduction, target cells were analyzed by flow cytometry (BD LSRII), with collection of 50,000 events and gating on enhanced green fluorescent protein-positive (eGFP⫹) cells in the eGFP channel. Immunoblotting and protein analyses. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes using a semidry apparatus (Bio-Rad). The membranes were blocked for 1 h using Trisbuffered saline in the presence of 0.05% Tween 20 and 5% nonfat dried milk (TBS-T plus NFDM), probed using a primary antibody as specified below, washed extensively in TBS-T, probed using a secondary antibody conjugated to horseradish peroxidase (HRP) at a 1:10,000 dilution in TBS-T plus NFDM for 1 h, washed again, and developed using enhanced

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FIG 5 Human CRM1 increases infectious FIV release from mouse cells. (A) B78.CIB cells were nucleofected with the indicated plasmids, and resultant HIV-1 titers are shown, normalized to empty vector, transfection efficiency, and cell viability. YU2 denotes the expression plasmid for the M-tropic envelope. (B) B78.cycT1 cells were nucleofected with VSV G and the transfer and packaging vectors for FIV, SIV, and EIAV, and the resultant eGFP titers for each were quantified by flow cytometry. The titers were normalized to transfection efficiency and cell viability. (C) FIV vector constructs used here. (D) B78.cycT1 cells were nucleofected with FIV packaging and transfer vectors and indicated plasmids, and the resultant FIV titers were quantified by flow cytometry and normalized to transfection efficiency and cell viability. * and **, P is ⬍0.05 by one-way ANOVA. The data shown are the mean results of three independent experiments.

chemiluminescence. ImageJ software was used for analyses of the immunoblotting results. HIV-1 Gag and other viral gene products were detected using a 1:1,000 dilution of human anti-HIV-1 antibody from the AIDS Research and Reference Reagent Program (no. 1984) (59); CA was detected using anti-CA mouse monoclonal antibody from the AIDS Research and Reference Reagent Program (no. 3537) (9, 56) at a dilution of 1:1,000; the HA epitope was detected using an anti-HA mouse monoclonal antibody (Covance) at a dilution of 1:1,000, the FLAG epitope was detected using an anti-FLAG M2 mouse monoclonal antibody (SigmaAldrich) at a dilution of 1:1,000, the T7 epitope was detected using an anti-T7 epitope mouse monoclonal antibody (Novagen) at a dilution of 1:7,500, and tubulin was detected using an anti-tubulin mouse monoclonal antibody (Sigma-Aldrich) at a dilution of 1:1,000. CA in the cell lysates and culture supernatants was quantified by a commercial enzyme-linked immunoassay (ELISA) from Advanced Bioscience Laboratories. RNA imaging and analysis. B78 cells were nucleofected with a previously described MS2-based system that enables lentiviral RNAs to be

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tracked by fluorescent protein labeling in cells (27). Adapted from the RNA-labeling method of Singer and colleagues (22), it utilizes a plasmid encoding a transfer vector RNA containing 24 MS2 protein-binding RNA stem-loops (pFIVpsi-24), a plasmid that encodes a fusion of the phage MS2 protein to mCherry (pMS2Cherry), and the FIV packaging plasmid pFP93 (27). These were conucleofected with either pCMV-HA (an empty plasmid) or a plasmid encoding hCRM1. Cell images were obtained on an LSM 780 laser-scanning confocal microscope using a Plan Apochromat at a magnification of ⫻100 with an (1.4 numerical aperture) oil-immersion objective. mCherry was excited with a 561-nm laser, and emission was imaged between 569 nm and 740 nm. Total RNA was extracted from cells using a Qiagen RNeasy kit according to the manufacturer’s instructions. First-strand cDNA was produced using Superscript II reverse transcriptase (Invitrogen) and 2 ␮g of total RNA by following the manufacturer’s protocol. DNA PCR for both mouse and human ␤-actin was performed using PrimeSTAR HS DNA polymerase (TaKaRa) and the primers 5=-CTGCGCAAGTTAGGTTTTG TCA-3= and 5=-TCATTGCTCCTCCTGAGCG-3=. Amplification condi-

Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

tions were 98°C for 2 min followed by 27 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 5 s, and elongation at 72°C for 60 s. PCR products were visualized by agarose gel electrophoresis and ethidium bromide (EtBr) staining. For HIV-1 RNA, primers spanning the tat-rev intron in order to detect both spliced and unspliced RNAs were as described previously (11), and amplification conditions were 98°C for 2 min followed by 28 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 5 s, and elongation at 72°C for 90 s using PrimeSTAR HS DNA polymerase and the equivalent of 15, 45, and 145 ng of total RNA from the first-strand reaction. DNA products of 1,250 and 350 bp, corresponding to unspliced and spliced viral mRNAs, respectively, were visualized by agarose gel electrophoresis and EtBr staining. As a negative control, reverse transcriptase was omitted from the first-strand reaction. Total RNA prepared from 293T.LIB cells was used as a positive control for both spliced and unspliced viral RNAs. The ratio between unspliced and spliced products was calculated using ImageJ software (NIH). Flow cytometry. Transduced target HOS-Hygro cells were analyzed by flow cytometry using an LSR II Instrument (BD). Nonviable cells were excluded by 4=,6-diamidino-2-phenylindole (DAPI) staining, and eYFP⫹ cells were recorded in the eYFP channel. Mock-transduced cells were used to establish gates, and 50,000 events were collected and analyzed for each sample.

RESULTS

Human CRM1, SRp40, and SRp55 increase viral production from murine cells. There are multiple blocks to HIV-1 replication in murine cells, with a major impediment occurring at a posttranscriptional or late stage of virus assembly and release. In order to identify factors that can improve HIV-1 assembly and release, we developed an experimental system that allows quantification of infectious virus production by murine cells. This system is based on B78.CIB murine melanoma cells, which have a replicationdefective HIV-1 integrated in their genome, encoding Gag, Pol, Tat, and Rev and a truncated form of the Tat cofactor human cyclin T1 (cycT1) and the positive selectable marker Bsd (11) (Fig. 1A). Expression of cycT1 allows high-level transcription of the long terminal repeat (LTR) and the generation of pseudotyped particles when cells are cotransfected with a plasmid encoding an envelope glycoprotein. These cells were nucleofected with pMEVSVG and two additional HIV-1 transfer vectors, pHIV-CIY and pHIV-puro G⫺P⫺E⫺F⫺V⫺ (Fig. 1A). In addition, plasmids encoding full-length cDNAs of Ran, RanBP1, RanBP3, DDX3, hCRM1, SRp40, SRp55, hRIP, and ei5F␣ were separately cotransfected with an HA-tagged empty vector as a negative control. Cell culture supernatants were harvested 48 h posttransfection, and titers were determined on HOS-Hygro target cells. All of the cDNAs encoded proteins of the expected size, as confirmed by immunoblotting using the appropriate anti-epitope tag antibody, indicating full-length expression in both human (Fig. 1B) and murine cells (Fig. 1C). Of the cDNAs tested, only three resulted in significantly increased infectious HIV-1 production: hCRM1, SRp40, and SRp55 (Fig. 1D to F). Increases by 8- to 12-fold in HIV-1 titers in the presence of hCRM1 or SRp40 using Bsdr readouts and 5- to 7-fold increases using eYFP readouts were observed, whereas increases in the Purr titers (where “Purr titer” reflects the number of puromycin-resistant cell colonies per ml of tissue culture supernatant arising after treatment of target cells with puromycin) were not as pronounced (Fig. 1F). SRp55 significantly increased the Bsdr titers (where “Bsdr titer” reflects the number of blasticidin-resistant cell colonies per ml of tissue culture supernatant arising after treatment of target cells with blasti-

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TABLE 2 Retroviral production inhibition by SRp40 Vectora

SRp40/SRp55/empty plasmidb

Titer (% ⫾ SD)c

EIAV EIAV SIV SIV FIV FIV HIV HIV HIV MLV MLV

Empty SRp40 Empty SRp40 Empty SRp40 Empty SRp40 SRp55 Empty SRp40

100 ⫾ 2.9 1.6 ⫾ 0.26 100 ⫾ 15 1.3 ⫾ 0.61 100 ⫾ 9.8 4.2 ⫾ 1.9 100 ⫾ 7 11.2 ⫾ 0.40 0.16 ⫾ 0.011 100 ⫾ 11 1.45 ⫾ 0.05

P valued 0.00015 0.0038 0.0023 0.00093 6.5E⫺05 0.0022

a

All vectors were pseudotyped with VSV G. EIAV, SIV, and FIV were produced as described in Materials and Methods; in this case, HIV was based upon a thirdgeneration vector, FG12, and the MLV was based upon pQXIP (Clontech), but Bsdr had replaced Purr. The targets were Hos cells and were either subjected to flow cytometry 48 to 72 h posttransduction or passaged into a selective medium in order to quantify cell colonies 5 to 7 days later. b Either a CMV-driving expression vector encoding SRp40 or SRp55 or a corresponding empty plasmid was added to the transfection. c The titer was normalized at 100% in the absence of SR protein for each of the retroviral vectors; the titers were 98,000 IU/ml for EIAV, 33,000 IU/ml for SIV, 108,000 IU/ml for FIV, 7.0 ⫻ 106 IU/ml for HIV, and 1.1 ⫻ 105 IU/ml for MLV. d One-sided paired Student’s t test results, compared to those of the corresponding empty vector control.

cidin) only, whereas there was a trend toward increased eYFP titers (where “eYFP titer” reflects the number of eYFP-positive cells per ml of tissue culture supernatant, as measured by fluorescence microscopy or flow cytometry) and Purr titers. To further evaluate the role of hCRM1 in augmenting infectious HIV-1 production, increasing amounts of hCRM1, from 0.25 to 24.0 ␮g, were used. Clear dose-response effects were observed in the range of 0.25 to 8.0 ␮g for Bsdr and eYFP titers, with increases of 10- to 12-fold, and between 0.25 and 4.0 ␮g for Purr titers, with increases of ⬃6-fold (Fig. 2A to C). While Purr and eYFP titers declined at the higher doses of hCRM1, the Bsdr titers plateaued at those amounts (Fig. 2A to C). To exclude the possibility of an overexpression artifact, we compared the results of transfection using 4 ␮g of hCRM1, 4 ␮g mCRM1, and 4 ␮g of hCRM1 together with 4 ␮g mCRM1 in mouse cells. eYFP, Bsdr, and Purr titers increased significantly in the presence of hCRM1 but not mCRM1 (Fig. 2D), although each was expressed at equivalent levels in human and murine cells as judged by immunoblotting (Fig. 2E and F, respectively). Cotransfected mCRM1 did not inhibit or reduce the positive effects of hCRM1, suggesting that mCRM1 is a loss-of-function allele, without having a dominant negative effect (Fig. 2D). To determine whether the effect of hCRM1 could be seen in other mouse cells, we performed the same experiment using 3T6.cycT1 mouse fibroblasts that had been transduced previously with an HIV-1 vector that encodes both enhanced firefly luciferase and Bsd (3T6.effluc-IB cells). Although the lowered viability of 3T6 cells after nucleofection may have interfered with virus production, recoverable titers of Bsdr, eYFP, and Purr all increased in the presence of hCRM1, relative to the empty vector control (Table 1). Cotransfection with SRp55 yielded a significant increase in Bsdr titer but not in the other titers, and cotransfection with SRp40 did not yield any significant increase in titers (data not shown).

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FIG 6 Human CRM1 increases HIV-1 Gag production in mouse cells. (A) B78.CIB cells were nucleofected with the indicated cDNAs, and the cell lysates were immunoblotted using anti-HIV-1 antiserum (upper panel), with tubulin immunoblot serving as the loading control (lower panel). (B) B78.CIB cells were nucleofected with the indicated plasmids and incubated in media containing lopinavir. The cell lysates were immunoblotted as described for panel A, showing neat and 1:10 dilutions of cell lysates. (C and D) Similar to that shown in panel B, both cell lysates and supernatants were immunoblotted; the latter were concentrated by ultracentrifugation (shown at a 1:15 dilution in panel D). p55 Gag bands were quantified using ImageJ software, with the intensity of the empty vector control band set at 1.0. (A) Results of one of three representative experiments; (B through D) results of one of two representative experiments. In each of these experiments, each of the 5 transfections was performed in quadruplicate, and the supernatants of identical samples were pooled in order to obtain 10 ml of supernatant for ultracentrifugation.

Similar experiments were performed using A9 mouse fibroblasts that had been transduced with an HIV-1 vector that encodes both cycT1 and Bsd (A9.CIB cells). An increase in Bsdr titer from 13 IU/ml (range 0 to 40 IU/ml) to 804 IU/ml (range, 350 to 1,400 IU/ml) was observed in the presence of hCRM1, relative to the empty vector control (Table 1). Similarly, Purr and eYFP titers increased from 120 and 0 IU/ml (empty vector) to 5,033 and 2,500 IU/ml (hCRM1), respectively. GM11686.CIB cells, which harbor human chromosome 2 and express hCRM1 mRNA as judged by real-time PCR, were nucleofected with pME-VSVG, pHIV-CIY,

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and pHIV-puro G⫺P⫺E⫺F⫺V⫺. In the absence of additional hCRM1, resultant titers were 900 IU/ml Bsdr, 12,000 IU/ml Purr, and 5,000 IU/ml eYFP, approximately twice those of the parental A9.CIB cells nucleofected with hCRM1 (Table 1). Results obtained using B78.CIB cells transfected with hCRM1 and 293T cells are also shown as comparators in Table 1. These results underscore the fact that recoverable HIV titers from murine cells were approximately 1% of those from 293T cells, although there was marked variability depending upon the mouse cell line and the HIV vector being recovered.

Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

SRp40 combined with human CRM1 has a more-than-additive effect upon infectious HIV-1 release. We next investigated the effects of combining both SRp40 and hCRM1 on infectious HIV-1 production in mouse cells. To do so, we used increasing amounts of hCRM1 (0 to 8.0 ␮g) with increasing amounts of SRp40 (0 to 6.8 ␮g). For most combinations, the increases observed in both eYFP and Bsdr titers were much greater than those observed when we used each cDNA alone, consistent with morethan-additive effects (Fig. 3A and B). For example, the addition of 4.0 ␮g of hCRM1 resulted in a 14-fold increase in the Bsdr titer, and 6.8 ␮g of SRp40 resulted in an increase of 28.4-fold relative to the empty vector. When hCRM1 and SRp40 were combined, the Bsdr titers increased 232-fold. Thus, the combined effect was clearly more than additive but less than multiplicative (Fig. 3A and B). Although Purr titers appeared to be inhibited by the combination of SRp40 and hCRM1 when used at higher doses (Fig. 3C and D), when lower doses of both hCRM1 (1.0 ␮g) and SRp40 (0 to 1.7 ␮g) were used, a similar more-than-additive enhancement of infectious titers was observed (Fig. 3D). At higher doses of hCRM1 (2.0 ␮g), Purr titers were reduced (Fig. 3C and D). The reason for this decrement is not clear but may be due to the fact that the HIV-puro G⫺P⫺E⫺F⫺V⫺ vector differs from the other two HIV transfer vectors in that Gag-Pol is not encoded in cis (Fig. 1A). We then performed the same experiment using increasing doses of SRp40 (0 to 1.5 ␮g) and SRp55 (0 to 1.6 ␮g), alone or in combination. Each of these cDNAs alone resulted in an increase in the infectious HIV-1 titers from ⬃2- to ⬃7-fold, but in combination, enhancement beyond an additive effect was not observed (Fig. 4A to C). In fact, for most combinations of SRp40 and SRp55, even an additive effect was not seen. Taken together, the morethan-additive effects seen for hCRM1 and SRp40 in combination suggest that these two proteins increase viral production independently and that their modes of action are likely distinct, whereas SRp40 and SRp55 likely act in similar manners mechanistically. To determine whether the effects of hCRM1 are independent of the envelope glycoprotein used for pseudotyping, we replaced VSV G with the M-tropic YU2 Env in the cotransfection. This produced a 10- to 15-fold increase in eYFP, Bsdr, and Purr titers, normalized to the empty vector control, similar to what was observed using VSV G (Fig. 5A). These results suggest that the observed effect of hCRM1 is independent of the envelope or glycoprotein used. Human CRM1 enhanced FIV production in murine cells. Next, the effects of hCRM1 on other retroviruses were investigated. For each virus tested, VSV G, an appropriate packaging vector, and a transfer vector were nucleofected into the mouse cells, in the presence and absence of hCRM1. No increases in MLV infectious titers from mouse cells in the presence of hCRM1 compared to empty vectors were detected (data not shown). Infectious titers of EIAV and SIV produced from 293T cells were 1.0 ⫻ 105 IU/ml and 5.0 ⫻ 104 IU/ml, respectively, while titers of EIAV and SIVmac from murine cells were 300 IU/ml and 1,500 IU/ml, respectively. Unexpectedly, the titers in murine cells did not increase significantly with the addition of hCRM1 (Fig. 5B). Interestingly, in the case of the FIV baseline, eGFP⫹ titers of ⬃300 IU/ml increased approximately 140-fold, to ⬃46,000 IU/ml, in the presence of hCRM1 (Fig. 5B). mCRM1, SRp40, and SRp55 increased FIV titers 5- to 7-fold (data not shown), although these effects were minimal in comparison to the 2-log10-increased effect of

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FIG 7 SRp40 and hCRM1 have more-than-additive effects on HIV-1 CA production. (A) B78.CIB cells were nucleofected with the indicated amounts of SRp40 and hCRM1, and released HIV-1 CA was quantified by ELISA. (B) Similar to the experiment described for panel A, but specific infectivities were calculated by dividing the total number of IU (arithmetic sum of Bsdr, eYFP, and Purr titers) by the CA amounts (pg). These are representative results of one of three independent experiments.

hCRM1. Surprisingly, when hCRM1 and SRp40 were used in combination, the increase in the FIV titer was less than with hCRM1 alone (Fig. 5D). This finding highlights the idea that the SR proteins are acting to boost the infectious viral titer in a mechanism that is fundamentally different from that of hCRM1. To underscore these mechanistic differences between CRM1 and the SR proteins, we tested the effects of overexpression of both in human cells. Transient overexpression of hCRM1 or mCRM1 did not affect retroviral or lentiviral vector titers obtained from 293T cells (not shown), but the SR proteins were significantly inhibitory to the production of VSV G-pseudotyped HIV, SIV, EIAV, FIV, and MLV particles (Table 2). For some of the vectors (notably SIV and FIV), transfection efficiency was substantially reduced in the presence of the SR proteins, whereas for HIV and

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FIG 8 SRp40 and hCRM1 increase the amounts of unspliced HIV-1 RNAs. (A) B78.CIB cells were nucleofected with SRp40 and/or hCRM1, total RNA was prepared, and the cells were subjected to RT-PCR. Spliced and unspliced products were PCR amplified using primers flanking the tat-rev intron. Indicated are the expected PCR products from spliced (350-bp) and unspliced (1,250-bp) RNAs. The wedges indicate decreasing amounts of cDNA used (3-fold dilutions), and the thicknesses of the bars indicate the amounts of SRp40 and CRM1 nucleofected. For comparison, the results from using 293T.LIB cells are shown at the far right. Noted above each lane is the ratio of the unspliced to spliced products. These data reflect the results of one of two representative experiments. (B) Unspliced/spliced RNA ratios as described for panel A, including the standard deviations (SD).

EIAV, there was no apparent effect, as judged by eGFP/eYFP autofluorescence of the 293T producers (not shown). The mechanism by which the SR proteins are inhibitory to retroviral production in human cells remains to be explored. Human CRM1 and SRp40 markedly increase production of HIV-1 Gag. We next examined the effects of the various cDNAs on cell-associated Gag levels and processing (Fig. 6). Nucleofection of hCRM1, SRp40, and SRp55 demonstrated a small but reproducible increase in Gag p55 levels in murine cell lysates, as assessed by immunoblotting, whereas hRIP, DDX3, ei5f␣, Ran, RanBP1, and RanBP3 did not. This increase was associated with visible increases in CA, MA, and the processing intermediate p41 (Fig. 6A). When hCRM1, mCRM1, and SRp40 were nucleofected either alone or in combination in the presence of the protease inhibitor lopinavir, there was a slight increase in amount of cellassociated p55 Gag in all cases (Fig. 6B and C). However, the amount of Gag released was increased in the presence of hCRM1 and was markedly greater in the presence of hCRM1 plus SRp40 (Fig. 6C and D). The observed increase in secreted Gag paralleled the higher titers observed when hCRM1 and/or SRp40 was cotransfected into murine cells. To quantify these results, ELISA was used to measure the amounts of CA production in the presence of nucleofected hCRM1 and SRp40 (Fig. 7). Increasing amounts of hCRM1 or SRp40 resulted in greater amounts of released CA, and together the effects were more than additive but less than multiplicative (Fig. 7A). Indeed, the amounts of CA detected by ELISA in nucleofected cell culture supernatants paralleled the increase in infectious titers such that the specific infectivities (pg CA/IU) of parti-

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cles varied only 3-fold (Fig. 7B). Of note, the observed effect was Rev dependent, since when a codon-optimized, cytomegalovirus (CMV)-driven gag-pol plasmid was used, no increase in CA release from B78 cells was observed in the presence of hCRM1. Overall, these results suggest that in mouse cells in the presence of hCRM1 and SR proteins, Gag was produced in greater amounts without accumulating and instead was released from the cells and then processed to CA. Human CRM1 and SRp40 invert the ratio between spliced and unspliced HIV-1 RNA in murine cells. As CRM1 has a central role in unspliced/partially spliced HIV-1 mRNA export, we investigated the effects of hCRM1 on levels of spliced and unspliced HIV-1 mRNA in murine cells using our experimental system. Forty-eight hours after transfection of B78.CIB cells with VSV G, pHIV-1-CycT1-IRES-eYFP, pHIV-1-puro G⫺P⫺E⫺F⫺V⫺, hCRM1, and SRp40, total RNA was prepared and first-strand cDNA synthesized. PCR analysis was performed using primers flanking the tat-rev intron to amplify an unspliced 1,250-bp and a spliced 350-bp cDNA (corresponding to mRNA). We compared both the amounts and the ratios between unspliced and spliced mRNAs in the presence of hCRM1 and SRp40 (Fig. 8). Consistent with previous reports, 293T.LIB cells produced much greater amounts of viral RNA with a clear abundance of unspliced compared to spliced mRNAs, whereas in mouse cells the total amounts of viral RNAs were reduced, with spliced RNAs predominant over unspliced mRNAs (Fig. 8). The expression of either SRp40 or hCRM1 resulted in approximately equal amounts of unspliced and spliced HIV-1 RNAs. When used in combination, expressions of both hCRM1 and SRp40 inverted the ratio of

Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

FIG 9 Human CRM1 increases nuclear export of unspliced FIV RNA in mouse cells. (A) B78.CIB cells were nucleofected with different combinations of expression plasmids encoding the FIVpsi-24 transfer vector RNA, MS2-Cherry, pFP93 packaging plasmid (encodes FIV Rev and Gag-Pol), and either hCRM1 or empty vector (CMV-HA); on the left are representative fluorescent images, and on the right are superimposed bright-field images. Bars indicate 10 ␮m. (A) MS2-Cherry and hCRM1; (B) MS2-Cherry, FIVpsi-24, pFP93, and CMV-HA; (C) MS2-Cherry, FIVpsi-24, pFP93, and CMV-hCRM1. (D) Quantitation of cells having RNA aggregates in cytosol in the presence and absence of hCRM1. For the empty vector, 10 of 52 cells analyzed had cytoplasmic aggregates (19%), while for hCRM1, 42 of the 72 cells analyzed had cytoplasmic aggregates (58%).

unspliced to spliced RNAs in a manner that was qualitatively similar to what is observed in human cells. This reversed ratio was due to an increase in the amounts of unspliced RNAs, not a decrease in the amounts of spliced RNAs (Fig. 8). To directly visualize intron-containing lentiviral RNAs in mouse cells in the presence of hCRM1, we employed a recently described system (27) in which the FIV transfer vector has multiple MS2 protein-binding sites and the RNAs can be imaged by confocal microcopy using MS2-NLS-autofluorescent protein fusions, such as mCherry. In the absence of FIV transfer and packaging vectors, mCherry fluorescence was predominantly nuclear (Fig. 9A). When these constructs were nucleofected into B78 cells in the absence of hCRM1, the FIV RNA remained mostly nuclear, as assessed by localization of mCherry signals (Fig. 9B). In the presence of hCRM1, however, the amounts of exported RNAs, observed as cytosolic speckles, increased, similar to what is observed in human or cat cells, which are more permissive to FIV replication (Fig. 9C). Overall, there was a 3-fold increase in the percentage of cells exhibiting cytosolic mCherry speckles in the presence of hCRM1 (Fig. 9D). This is consistent with the canonical role of CRM1 in increasing the nuclear export of intron-containing viral RNA. Functionality of hCRM1 mapped using chimeras and point mutants. hCRM1 and mCRM1 are both 1,071 amino acids in length with 21 residues that are different throughout the length of the proteins. Because mCRM1 appeared to be a loss-of-function and not a dominant-negative allele, we decided to map the functional domain(s) of hCRM1 by making chimeras using conserved

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restriction sites between the two genes (Fig. 10A). Immunoblotting assays showed similar levels of expression of the various chimeric proteins in 293T (Fig. 10B) and B78.CIB cells, with the exception of h675m, which showed slightly reduced expression (Fig. 10C). For each transfected chimera, the effects on infectious HIV-1 titers were measured as described above. For each of the three HIV vector readouts, there was a trend or a statistically significant increase in titers for chimeras that included the region of hCRM1 from residues 146 to 444, whereas for those chimeras that did not include that region of CRM1, all three titers were close to those of mCRM1 (Fig. 10D to F). These results suggested that an important functional determinant of hCRM1 lay between amino acid residues 146 and 444. To further clarify these results, we performed two experiments with an improved dynamic range of viral production. First, we compared the Bsdr titers of each chimera combined with SRp40 to SRp40 combined with an empty vector, mCRM1, or hCRM1. As anticipated, all of the chimeras that contained the region between residues 146 and 444 produced significant increases in Bsdr titers compared to SRp40 plus an empty vector or mCRM1 (Fig. 10G). We next repeated this experiment using the FIV packaging and transfer vectors and obtained similar results (Fig. 10H). For all of the chimeras that contained this region of hCRM1, the results were significant elevations in infectious FIV titers, between 28and 39-fold higher than those of mCRM1. The only exception was h675, which had lower expressions as judged by immunoblotting (Fig. 10C). This effect was most apparent when we compared the results from m146h444 and h146m444 chimeras. Of note,

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FIG 10 Mouse-human chimeras localize functional elements of CRM1. (A) Schematic of chimeras tested, with NES binding sites bracketed. Filled and open boxes represent mouse and human CRM1 sequences, respectively, with numbers indicating amino acid transitions. Expressions of chimeras confirmed by immunoblotting in 293T (B) and B78 cells (C); “-” denotes empty vectors. (D through F) Resultant Bsdr, eYFP, and Purr HIV-1 titers after nucleofection of B78.CIB cells with the indicated chimeras. (G) Resultant Bsdr titers after nucleofection with SRp40 and the indicated chimeras; shown are the results of one of three representative experiments. (H) GFP titers after nucleofection of B78.CycT1 cells with the indicated chimeras and FIV packaging and transfer vectors; *, P is ⬍0.05 by one-way ANOVA compared to empty vector. The means of the results of three independent experiments are shown. Panel H reflects the results of one of three independent experiments.

mCRM1 did have a 4-fold-increased effect compared to the empty vector, which suggests that mCRM1 is not a completely null allele. Given the observed variability in FIV titers and the reduced dynamic range in the case of HIV-1, we cannot, however, entirely exclude the possibility that there are critical determinants outside this region that may contribute to the functionality of hCRM1.

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We then constructed more than 10 single and multiple point mutants of hCRM1, converting the human to the corresponding mouse residues by site-directed mutagenesis. These included the S191A, V284E, D334G, D334G⫹I337L, V402I, P411T, M412V, F414S, P411T⫹M412V⫹F414S, R474I, E478K, H481Q, R474I⫹ E478K, and R474I⫹E478K⫹H481Q mutants. Expression of each

Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

FIG 11 Structures of mCRM1 and hCRM1. (A) Superimposition of mCRM1 (Protein Data Bank [PDB] code 3GJX) and hCRM1 (PDB code 3GB8) structures,

with HEAT repeats labeled H1 through H21. (B) An enlargement of the region between H4A and H9A, rotated ⬃90° along the x axis. The amino acid residues that are different between the human and mouse CRM1s are labeled in pairs, and side chains are presented as stick models. (C) Resultant normalized eYFP titers (⫾SD) after nucleofection of B78.CIB cells with the indicated hCRM1 mutants along with pME VSV G and HIV-CIY. (D) Resultant normalized eGFP titers (⫾SD) after nucleofection of B78.cycT1 cells with the indicated hCRM1 mutants along with pME VSV G and FIV transfer and packaging vectors. *, P is ⬍0.05 by one-way ANOVA compared to mCRM1. (E) Expression of different mutants in B78.CIB cells.

mutant was confirmed by immunoblotting using anti-HA antisera after Lipofectamine 2000-mediated transient transfection in 293T cells or by nucleofection of B78 cells (Fig. 11E). These were each individually conucleofected into B78.CIB cells, along with pME VSV G and HIV-CIY, and recovered supernatant with the titer determined on HOS-Hygro targets. Results, shown in Fig. 11C, indicate that only the P411T⫹M412V⫹F414S triple mutant showed loss of function in that titers were not statistically significantly different from those resulting from mCRM1 nucleofection. Because the dynamic range was poor in the case of HIV, we repeated the experiment but instead nucleofected the FIV transfer and packaging vectors (Fig. 11D). Again, only the same triple

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point mutant showed a clear loss of function. These results indicate that the three residues P411, M412, and F414 are critical for hCRM1 function. DISCUSSION

A nonprimate small-animal model for HIV-1 replication could expedite the search for novel HIV-1 therapeutics and vaccines and also provide a better understanding of HIV-1 pathogenesis in vivo. Although great strides have been made using immunodeficient mice transplanted with human lymphoid and bone marrow tissues, the anti-HIV-1 immune responses are typically blunted in those chimeric animals (3, 7, 36). Unfortunately, there are multi-

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ple obstacles to HIV-1 replication in all mice and murine cells tested, chief among them being a block to infectious particle production posttranscription. Using somatic-cell mouse-human hybrids, we previously identified a determinant on human chromosome 2 that increased virus production by several orders of magnitude from mouse cells, but the causative gene was not identified (11). Most recently, two groups have independently shown that hCRM1, which is encoded on human chromosome 2, when expressed in rodent cells is capable of increasing Gag, VLP, and infectious virus production (39, 48). One study showed that the effects were mainly posttranscriptional (39), whereas the other showed a 4-fold increase in unspliced mRNA levels (48). In each case, functionality was mapped to a specific region of hCRM1 using rodent-human chimeric proteins. Our work here shows that nucleofecting hCRM1 can act with SRp40 to more than additively increase infectious HIV-1 release from mouse cells. Although we had tested hCrm1 previously using cationic lipids, including Lipofectamine 2000, to transfect mouse cells, for uncertain reasons the transfection efficiency or the amount of protein expressed was insufficient to allow us to observe a clear effect on infectious HIV production. The effects of nucleofected hCRM1 were even more pronounced on FIV release from mouse cells. Interestingly, SRp40 was found to inhibit the hCRM1-induced increase in FIV production in mouse cells and the production of all tested retroviruses in human cells. This suggests that hCRM1 and SRp40 have differing mechanisms of action in increasing lentiviral titers in murine cells and that the positive effects of the SR proteins are for unknown reasons limited to HIV in mouse cells. Our studies also showed that hCRM1 acts independently of the pseudotyping envelope glycoprotein and is functional in several different mouse cell lines, and its stimulatory effect is not antagonized by overexpression of mCRM1. The hCRM1-induced increase in the infectious HIV-1 titer was only 50% of that observed in human chromosome 2-containing murine cell lines, suggesting that although hCRM1 is likely the major determinant missing from mouse cells, other unknown factors may play a role. Unfortunately, because of low transfection rates, attempts to efficiently knock down hCRM1 in any of the chromosome 2-containing cell lines have thus far been unsuccessful. An often observed difference between mouse and human cells is in the levels of unspliced and spliced HIV-1 mRNA (11, 54). In mouse cells, the ratio of unspliced to spliced HIV-1 mRNA is typically low, whereas in human cells it is inverted or high. Previously we had observed that the presence of human chromosome 2 in mouse cells had variable effects on this ratio, although the amounts of Gag and CA production were consistently high (11). Here, using real-time PCR (RT-PCR) to examine both spliced and unspliced HIV-1 mRNAs simultaneously, we observed that hCRM1 increased the amount of unspliced relative to spliced RNA, and this ratio was inverted with the addition of SR proteins. The RT-PCR data were further supported by fluorescence imaging using an MS2-Cherry FIV RNA construct (27). A marked increase in cytoplasmic, intron-containing FIV RNA in a speckled pattern very similar to that observed in permissive cell types was detected in these cells. Together these data suggest that hCRM1, but not mCRM1, acts in a canonical fashion to export unspliced viral RNAs from the nucleus into the cytoplasm, directed away from or in evasion of the splicing pathway.

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The effects on the ratio of unspliced to spliced RNAs in the presence of hCRM1 and the SR proteins are relatively modest. At the same time, the amount of HIV-1 Gag or CA secreted from cells is markedly increased, more or less paralleling the recoverable infectious titer, such that the specific infectivity of particles is not substantially altered in the presence of these factors. This also suggests that the amount of Gag, not genomic RNA, is limiting in mouse cells and that hCRM1 could also act to enhance the translation of Gag-encoding RNA. It is possible that this effect is mediated through cytosolic trafficking of the mRNA to polyribosomes or is due to increased mRNA stability. Cooperative, nonlinear assembly of viral particles at the plasma membrane, accelerated through highly concentration-dependent Gag-Gag interactions, could also contribute to these effects. By making chimeras between mCRM1 and hCRM1, Sherer et al. mapped the functional determinant of hCRM1 to HEAT repeats 9A and 10A (48). Our results with the HIV-1 chimeras were largely in agreement with those, although the results with some chimeras were somewhat ambiguous, especially in the absence of SRp40. In the presence of SRp40, HIV-1 had a greater dynamic range in the presence of hCRM1, and we were able to map the determinant more definitively to HEAT repeats 4A to 9A. In a comparison of mCRM1 to hCRM1, in this region there are nine amino acid residue differences that are on surface-exposed helical loops, far from the nuclear export sequence (NES) binding site or the binding sites of other host factors (Fig. 11). Importantly, the overall fold of hCRM1 (PDB code 3GB8) in this region is essentially the same as that of mCRM1 (PDB code 3GJX); the two structures can be superimposed with a root mean square deviation of 0.93 Å for the 236 C␣ atoms (Fig. 11A and B) (16, 38, 43). When we tested single and multiple point mutants of hCRM1, only the P411T⫹M412V⫹F414S triple mutant showed a clear loss of function for production of both HIV and FIV. The fact that these residues are nearly contiguous and surface exposed suggests that either a cellular or viral factor binds specifically to that region of hCRM1 and not to mCRM1. These findings are largely in agreement with the recently reported study of Sherer et al., which showed that some of these residues are important for the functional activity of hCRM1 (48). These analyses also suggest that the observed functional differences between hCRM1 and mCRM1 are not the results of allosteric or change-in-structure effects but instead may be due to altered affinities of either a viral (namely Rev) host factor or unknown host factors. It is conceivable that in murine cells an inhibitor interacts with the HEAT repeats 4A to 9A of mCRM1 but not hCRM1, which then prevents the binding of Rev (Fig. 12A). Another explanation for the functional disparity is that an activator similarly binds to HEAT repeats 4A to 9A of hCRM1 but not mCRM1, thus improving the affinity of Rev for hCRM1 (Fig. 12B). A third possibility is that in addition to binding at the NES site of CRM1 multimerized Rev interacts with the HEAT repeats 4A to 9A of hCRM1 but not mCRM1, which enhances the binding affinity of Rev to hCRM1 (Fig. 12C). Of note, FIV Rev is much larger than HIV-1 Rev (circa 150 versus 116 amino acid residues), suggesting that it could interact with other domains of hCRM1, outside the NES motif, and have different affinities for mouse and human CRM1 and consequent functional effects. In summary, we show here that hCRM1 increases infectious HIV-1 and FIV production from mouse cells, and it appears that the SR proteins act in a mechanistically distinct fashion from

Journal of Virology

CRM1 Increases HIV and FIV Production from Mouse Cells

This work was supported by NIH grant AI067034 and the Marom program of Hadassah-Hebrew University Medical Center.

REFERENCES

FIG 12 Schematic of possible mechanisms of action of CRM1. (A) Inhibitor present in cells that binds to mCRM1 and interferes with Rev function but does not bind to hCRM1; (B) activator present in cells that does not bind mCRM1 but binds hCRM1, allowing for Rev function; (C) a portion of Rev (activator module) binding to a second region of hCRM1 but not mCRM1, outside the NES, allowing for Rev function. The indentations in the surface of CRM1 are meant to denote changes in amino acid residues, not changes in structure.

hCRM1 to increase HIV-1 release. The effects of hCRM1 are at least in part due to an increase in unspliced viral RNA and export of this unspliced RNA from the nucleus to the cytosol. There may also be translational or posttranslational effects, since Gag synthesis is increased disproportionately to the amount of unspliced RNA. mCRM1 appears to be a simple loss-of-function allele, although more complicated scenarios may be envisioned. The functional domain of hCRM1 was mapped to HEAT repeats 4A to 9A and the specific residues therein. That the structure of CRM1 in this region is highly conserved between the mouse and human versions suggests that either a viral or cellular host factor is binding to one of the surface-exposed helices to facilitate lentivirus production. ACKNOWLEDGMENTS We thank Jorge Henao-Mejia, John Olsen, Adrian Krainer, and Hung Fan for generously providing reagents, Sarah Sutton for technical support, Eran Elinav for fruitful discussions, and Nathan Sherer and Mike Malim for sharing unpublished results. The following reagents were obtained through the NIH AIDS Research and Reference Reagent program, Division of AIDS, NIAID: HIV-1 p24 monoclonal antibody (183-H12-5C), from Bruce Chesebro and Kathy Wehrly, and HIV-1 neutralizing serum, from Luba Vujcic, FDA, Center for Biologics Evaluation and Research.

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