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JOURNAL OFVIROLOGY, Dec. 2011, p. 12950–12961 Vol. 85, No. 24 0022-538X/11/$12.00 doi:10.1128/JVI.05384-11

Copyright © 2011, American Society for Microbiology. All Rights Reserved.

The Cargo-Binding Domain of Transportin 3 Is Required for

Lentivirus Nuclear Import

Eric C. Logue, Kayleigh T. Taylor, Peter H. Goff, and Nathaniel R. Landau*

Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, New York 10016

Received 13 June 2011/Accepted 23 September 2011

Lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic DNA. Several nuclear import and nuclear pore components have been

implicated as playing a role in nuclear import, including transportin 3 (TNPO3), a member of the importin-

family of nuclear import proteins. We demonstrated that TNPO3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) the most dependent and human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) the least. Analysis of HIV-1/SIVmac239 chimeric viruses showed that dependence on TNPO3 mapped to the SIVmac239 capsid. Mutation of a single amino acid, A76V in the SIVmac239 capsid, rendered the virus TNPO3 independent and resistant to mCPSF6-358, a truncated splicing factor that prevents HIV-1 nuclear import. Using a complementation assay based on 293T cells that express a TNPO3-targeted short hairpin RNA

(shRNA), we showed that theDrosophilaTNPO3 homologue can substitute for its human counterpart and that

it mapped a key functional domain of TNPO3 to the carboxy-terminal cargo-binding domain. Within the cargo-binding domain, two hydrophobic motifs were required for TNPO3-dependent infection. The mutated TNPO3 proteins maintained their ability to localize to the nucleus, suggesting that their inability to restore lentivirus infection resulted from an inability to bind to a host or viral cargo protein.

Lentiviruses are unique among the retroviruses in their abil-ity to infect nondividing cells (25, 40). While gammaretrovi-ruses, such as murine leukemia virus (MLV) and feline leuke-mia virus (FeLV), require breakdown of the nuclear envelope during mitosis to access the target cell chromosomal DNA (26, 35), lentiviruses transit through the nuclear pore, allowing them to infect terminally differentiated and nondividing cells, such as macrophages and dendritic cells (7). Early studies proposed that the ability of the lentivirus preintegration com-plex (PIC) to transit the nuclear pore was mediated by the concerted action of the virus matrix protein (MA) and the accessory proteins Vpr and Vpx (6, 14, 39). Further studies suggested that integrase (IN) (4, 9) and the central DNA flap (2, 34), a structure that forms during plus-strand synthesis as a result of initiation of reverse transcription primed by the cen-tral polypurine tract, also played a role. More recent studies have called into question the roles of each of these viral com-ponents in PIC nuclear import (12, 15, 17, 27, 33–35, 41, 42). Notably, Yamashita and Emerman showed that a panel of MLV/human immunodeficiency virus type 1 (HIV-1) chimeric viruses that lacked accessory proteins maintained the ability to infect nondividing cells and that the key viral determinant for nuclear import mapped to the capsid protein (CA) (41).

Recent genome-wide small interfering RNA (siRNA) screens to identify host factors required for the early events of HIV-1 infection identified several genes that encode proteins involved in

the nuclear import pathways of the cell. These include the nuclear pore complex (NPC) components Nup153, RanBP2/Nup358, and Nup155 and the nuclear import protein transportin 3 (TNPO3/ transportin-SR2) (5, 20). TNPO3 was also identified in a yeast two-hybrid screen for proteins that interact with HIV-1 IN (9). Further analyses implicated TNPO3 as playing a role in viral nuclear import. In newly infected cells, siRNA knockdown of TNPO3 reduced the number of HIV-1 2-long-terminal-repeat (2-LTR) circles, a marker for nuclear import, but had no effect on the number of 2-LTR circles in cells infected by MLV (5, 9). The requirement for TNPO3 was relieved in a mutant HIV-1 that contains an N74D mutation of CA. Lee et al. derived this mutant virus by selecting for an HIV-1 variant resistant to a dominant-negative CPSF6 (mCPSF6-358). The mCPSF6-358 fragment is a carboxy-terminally truncated variant of a cellular polyadenylation factor that blocks HIV-1 infection at nuclear import when it is expressed in the target cell (24). The connection between TNPO3 and CPSF6 is not yet understood, but the finding demonstrates that it is possible for HIV-1 to infect cells in a indepen-dent manner. However, it is important to note that TNPO3-dependent nuclear import does lead to the preferential integra-tion of the provirus into gene-rich regions of the genome (31).

TNPO3 is a member of the importin-␤family of the Ran-GTP-dependent nuclear import proteins. Its known cargo pro-teins include the serine/arginine-rich (SR) family of RNA-processing factors (19, 22, 23). Members of the importin-␤ family have three-dimensional structures consisting of stacked HEAT domain repeats that contain two antiparallel␣-helices linked by a flexible loop region (3, 8, 18, 38). These proteins are divided into three overlapping functional domains: a RanGTP binding amino-terminal domain, a nuclear pore bind-ing domain located in the core region of the protein, and a cargo-binding carboxy-terminal domain (36). The

carboxy-ter-* Corresponding author. Mailing address: Department of Microbi-ology, New York University School of Medicine, 550 First Avenue, New York, NY 10016. Phone: (212) 263-9197. Fax: (212) 263-9180. E-mail: nathaniel.landau@med.nyu.edu.

† Supplemental material for this article may be found at http://jvi .asm.org/.

Published ahead of print on 5 October 2011.

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minal domain of TNPO3 was essential for the binding and nuclear transport of its cellular cargoes (22).

The molecular mechanism by which TNPO3 facilitates len-tiviral nuclear import remains to be determined. In light of its proposed interaction with IN, TNPO3 might serve as a chap-erone that associates with the PIC postentry to guide it through the nuclear pore. Although TNPO3 was found to interact with IN in a yeast two-hybrid screen and in a pulldown assay using recombinant HIV-1 IN (9), TNPO3 also associated with the IN of MLV, a virus that does not infect nondividing cells and is not dependent on TNPO3 for infection (21). Several more indirect mechanisms are also possible. In its role in cellular nuclear import, TNPO3 has various cargos, one class of which, the SR splicing factors, regulate the production of a large number of cellular mRNAs. The production of specific cellular mRNAs regulated by SR proteins might be needed for PIC nuclear import. Alternatively, one of TNPO3’s cellular cargos may need to be transported to the nucleus to mediate PIC nuclear import, or TNPO3 could serve to prevent the accumu-lation of a cellular protein in the cytoplasm that interferes with PIC nuclear import.

In this study, we have further investigated the mechanism of TNPO3-mediated lentivirus nuclear import. We show that TNPO3 is required for infection with several lentiviruses. Sim-ian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) were the most sensitive to TNPO3 knockdown. Analysis of HIV/SIV chimeric viruses showed that the sensitivity of SIVmac239 to TNPO3 knock-down mapped to CA but that in SIVmac239 a single A76V mutation in SIVmac239 CA rendered the virus TNPO3 inde-pendent. Using a complementation assay based on a cell line in which TNPO3 expression was reduced due to short hairpin RNA (shRNA) knockdown, we found that the function of TNPO3 that was necessary for lentiviral infection mapped to the carboxy-terminal cargo-binding domain. Mutational anal-ysis of this domain identified two hydrophobic motifs required for TNPO3-mediated infection. While the cargo-binding do-main mutants lacked the ability to mediate lentivirus infection, they maintained their ability to localize to the nucleus. Thus, these motifs within the cargo-binding domain represent poten-tial interaction sites for a cellular or viral protein that plays a role in lentivirus nuclear import.

MATERIALS AND METHODS

Cell culture.The 293T and HeLa cell lines were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. THP-1 cells were grown in RPMI 1640–10% FBS and antibiotics. THP-1 cells were differentiated for 4 days with 50 nM phorbol myristic acid.

Virus preparation.Viruses were produced in 293T cells cotransfected with reporter virus plasmid and vesicular stomatitis virus G (VSV-G) using Lipo-fectamine 2000 (Invitrogen). HIV-1 and SIVmac heat-stable antigen (HSA) reporter viruses, SIV-HSA, SIV-HSA (HCA-p2), and HIV-HSA, were pseu-dotyped with VSV-G by transfection at a mass ratio of 3:1. The EIAV reporter virus was produced with pONY3.2, EIAV-RFP (red fluorescent protein) re-porter vector, and VSV-G at a ratio of 2:2:1. Feline immunodeficiency virus (FIV) reporter virus was produced by transfection with pFP93, pGinSin (FIV-GFP [green fluorescent protein]), and VSV-G at a mass ratio of 2.8:2.8:1. MLV-GFP reporter virus was produced by transfection with pMx-GFP, pHIT-60, and VSV-G at a mass ratio of 4:2:1. HIV-SIV capsid-dsred and the control wild-type 1 were made by cotransfection with a wild-type 1 or HIV-SCA packaging construct, HIV-1-dsred reporter construct, and VSV-G at a mass ratio of 5:5:1. The culture medium was changed 6 h posttransfection, and the

supernatant was harvested after 48 h. The supernatants were passed through a 0.45-␮m filter and frozen in aliquots at⫺80°C. To determine the virus titer, thawed virus-containing supernatant was added to 2.5⫻105293T cells in a 6-well plate. For HSA reporter viruses, the cells were infected and, after 72 h, stained with phycoerythrin (PE)-conjugated rat anti-mouse CD24 (BD Pharmingen) and fixed in 1% paraformaldehyde for at least 1 h at 4°C. The cells were analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences), and the data were analyzed with Flojo software (Treestar).

Plasmids.Hemagglutinin (HA)-tagged TNPO3 constructs were generated by PCR using Phusion Hot Start II high-fidelity polymerase (Finnzymes) and prim-ers containing EcoRI and XhoI sites. The amplicons were cleaved with EcoRI and XhoI and then ligated into EcoRI- and XhoI-digested pcDNA6 A/myc-his (Invitrogen). The human TNPO3 open reading frame was amplified from the cDNA clone IMAGE:5301701 (GeneCopoeia) using primers TNPO3-F and TNPO3-R. The cDNA was found to contain a premature stop codon after amino acid 605. This was corrected by overlapping PCR using primers TNPO3.correct-F and TNPO3.correct-R. An shRNA-resistant TNPO3 was gen-erated by overlapping PCR using the primers TNPO3.sh32mut-F and TNPO3.sh32mut-R. TheDrosophilaTNPO3 open reading frame was amplified from the cDNA clone LD21546 (Berkeley Drosophila Genome Project) using the primers drTNPO3-F and drTNPO3-R. Truncated TNPO3 fragments were generated using the shRNA-resistant TNPO3 construct as a template with the primers TNPO3-F and TNPO3.⌬Cargo-R for the TNPO3⌬Cargo construct and the primers TNPO3-F and TNPO3.⌬C18-R for the TNPO3⌬C18 construct. Point mutations were introduced into the TNPO3 cargo-binding domain by overlapping PCR using the shRNA-resistant TNPO3 construct as a template and specific primers. GFP-tagged constructs were generated by PCR amplification from the corresponding pcDNA6-TNPO3 construct, digested with EcoRI and BamHI, and ligated into an EcoRI- and BamHI-digested pEGFP-C1 vector (Clontech). Full-length TNPO3 and TNPO3 point mutants were amplified with primers TNPO3-F and Bam.TNPO3-R. The TNPO3⌬Cargo truncation mutant was amplified with primers TNPO3-F and Bam.TNPO3.⌬Cargo-R. The SIV-HSA (HCA-p2) vector was constructed with an HIV-1 CAp2-containing frag-ment from an SfoI/SbfI-digested p239SpSp5⬘(HIV-CA) vector ligated to an SfoI/SbfI-digested SIV-HSA reporter virus. All plasmids were confirmed by nucleotide sequencing, and their expression was verified on an immunoblot.

siRNA knockdown.HeLa cells (2.5⫻105

) were transfected with 100 pmol of On-Targetplus siRNAs using Lipofectamine 2000 (Invitrogen). After 48 h, the cells were transferred to a 6-well culture dish for infection at 2.5⫻105

cells per well. The cells were infected 72 h posttransfection with HIV-1-HSA or SIVmac-HSA. The infected cell cultures were stained with PE-conjugated rat anti-mouse CD24 (BD Pharmingen) and fixed in 1% paraformaldehyde for at least 1 h at 4°C prior to analysis by flow cytometry.

Stable TNPO3 knockdown cell lines.An shRNA lentiviral vector that targeted nucleotides 1956 to 1977 of TNPO3 was constructed using the complementary primers 5⬘-CGTGCAGACAACCGCTCGTAGGCGTTGTTGCAGGTGCCT G-3⬘ and 5⬘-CGATGCGGTTGTCTGCACGGTGCTTATTTAGAGTCTCGG ATAAAAC-3⬘cloned into the AgeI and EcoRI sites of lentiviral vector pLKO.1-puro. VSV-G-pseudotyped virus was produced by transfection of 293T cells and used to transduce 293T, HeLa, and undifferentiated THP-1 cells. The transduced cells were isolated by puromycin drug selection. Single-cell 293T and HeLa clones were derived, and the extent of TNPO3 knockdown was determined by immunoblot analysis.

Quantitative real-time PCR. HeLa cells were infected at a multiplicity of infection (MOI) of 5 with virus stock that had been treated for 1 h with 50 U/ml of Benzonase (Invitrogen) to remove contaminating plasmid. To control for residual plasmid DNA, 25␮M zidovudine (AZT) was added to one well 14 h prior to infection. DNA was isolated 2, 10, 24, and 48 h postinfection using a DNeasy kit (Qiagen). Reverse transcripts in 250 ng of DNA were quantitated in triplicate by quantitative PCR (qPCR) using an ABI Prism 7300 and SYBR green (Applied Biosystems). Primer pairs were used to specifically measure HIV-1 late reverse transcription products (5⬘-TGTGTGCCCGTCTGTTGTG T-3⬘and 5⬘-GAGTCCTGCGTCGAGAGAGC-3⬘), HIV-1 2-LTR circles (5⬘-A ACTAGGGAACCCACTGCTTAAG-3⬘and 5⬘-TCCACAGATCAAGGATAT CTTGTC-3⬘), and SIVmac 2-LTR circles (5⬘-CTTGCACTGTAATAAATCC C-3⬘ and 5⬘-CCTTTCTGCTTTGGGAAACCG-3⬘). Standard curves were generated by amplification of serially diluted proviral and 2-LTR plasmids.

TNPO3 complementation assay.293T shRNA TNPO3 knockdown cells (5.0⫻

105

) were transfected with 4␮g of wild-type or mutant shRNA-resistant TNPO3 plasmid DNA using Lipofectamine 2000. After 24 h, the cells (2.5⫻105) were transferred to a 6-well dish. The next day, the transfected cells were infected with VSV-G-pseudotyped single-cycle HIV-1, EIAV, and SIV-HSA reporter virus and 3 days later analyzed by flow cytometry.

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Immunoblot analysis.Transfected cells were lysed in RIPA buffer containing Halt Protease Inhibitor (ThermoScientific) and normalized for protein content. Lysate containing 10␮g of protein was separated on a 4 to 12% Bis-Tris SDS polyacrylamide gradient gel in MES (morpholineethanesulfonic acid) running buffer. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then probed with anti-HA epitope-tagged monoclonal antibody (MAb) HA.11 (Covance) or anti-TNPO3 MAb, clone 3152C2a (Abcam). The filters were then hybridized to anti-mouse IgG biotin-conjugated secondary an-tibody (ThermoScientific Pierce) and a DyLight 800 Streptavidin Conjugate (Thermo Scientific Pierce) and imaged on an Odyssey imaging system (Li-Cor).

Fluorescence microscopy.HeLa cells (5.0⫻105

) were transfected with 4␮g of TNPO3-GFP fusion expression vector in a 6-well dish and then transferred to a 35-mm glass bottom culture dish (MatTek). The following day, the cells were fixed with 4% paraformaldehyde. The cells were counterstained with Alexa Fluor 594 wheat germ agglutinin and Hoechst 33342 (Molecular Probes) according to the manufacturer’s instructions to visualize the plasma membrane and nucleus, respectively. The cells were imaged on an LSM 710 laser scanning microscope (Zeiss), and the images were analyzed using ImageJ software.

RESULTS

Dependence of lentiviruses on TNPO3 for nuclear import. The ability to infect nondividing cells is a property shared by lentiviruses, but not gammaretroviruses. To determine the rel-ative requirements of different lentiviruses for TNPO3

depen-dence, we knocked down TNPO3 in HeLa cells and then in-fected them with single-cycle reporter viruses. HIV-1 infection was reduced 33% by knockdown of TNPO3 and Nup153 (Fig. 1A). SIVmac239 infection was reduced 70% by knockdown of TNPO3 and 30% by knockdown of Nup153. The findings sug-gest that the two viruses have similar requirements for Nup153 but that SIVmac239 is more dependent on TNPO3 than is HIV-1.

[image:3.585.49.541.67.412.2]

To show that the effect of TNPO3 knockdown was at the level of nuclear import and to confirm the increased depen-dence of SIVmac239, we determined the numbers of late re-verse transcripts and 2-LTR circles generated in newly infected cells by using quantitative real-time PCR (qRT-PCR). For HIV-1, the number of late reverse transcripts was not signifi-cantly affected by the TNPO3 knockdown using two different siRNAs over the 48-h time course. Analysis of the 2-LTR circles showed that they were not affected at 24 h but at 48 h postinfection were reduced 4- to 8-fold (Fig. 1B). These data demonstrate that the knockdown specifically affected nuclear import and not the ability to complete reverse transcription. For SIVmac239, TNPO3 knockdown reduced the number of

FIG. 1. TNPO3 knockdown inhibits HIV-1 and SIVmac239 nuclear import. (A) HeLa cells were transfected with nontarget control (NTC), TNPO3, Nup153, or mock siRNA and then infected with HIV-1 and SIVmac239 reporter virus. (B) HeLa cells were transfected with NTC and two different TNPO3 siRNAs (TNPO3.9 and TNPO3.12) or were mock transfected. The cells were then infected with HIV-1 or SIVmac239 reporter virus. AZT was added to one sample to control for intravirion reverse transcripts. DNA was harvested from infected cells at 2, 10, 24, and 48 h postinfection, and viral late-reverse transcription (RT) products and 2-LTR circles were measured by qRT-PCR. Each point represents the mean of triplicate samples, with the error bars representing the standard deviations.

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2-LTR circles 3- to 6-fold at 24 h postinfection and 10- to 20-fold at 48 h. SIVmac239 late reverse transcripts could not be quantified because of the presence of high input DNA signal, possibly caused by intravirion reverse transcription. These results further support an increased dependence of SIVmac239 on TNPO3 compared to HIV-1. We also noted that the effect of TNPO3 knockdown on the 2-LTR circles was more pronounced than the effect on infectivity as determined by flow cytometry.

To further determine the dependence on TNPO3 knock-down, we generated a TNPO3-targeted shRNA lentiviral vec-tor and used it to establish 293T, HeLa, and THP-1 cell lines

[image:4.585.44.539.69.466.2]

in which the levels of TNPO3 were reduced. The transduced clones with the largest TNPO3 reduction grew more slowly (data not shown), suggesting that TNPO3 is essential for via-bility and is not redundant with other importin-␤family mem-bers. We chose for further use cell clones with an intermediate level of knockdown that had only a minimal reduction in growth rate. To test the relative TNPO3 dependence of the lentiviruses, we infected the 293T knockdown cell line with single-cycle FIV, EIAV, SIVmac239, HIV-1, and MLV. We found that the TNPO3 knockdown had a minor effect on MLV and FIV, an intermediate effect on HIV-1, and the largest effect on SIVmac239 and EIAV (Fig. 2A). The results using

FIG. 2. TNPO3 knockdown in dividing and nondividing cells blocks infection with multiple lentiviruses. (A) Empty-vector-transduced 293T cells and 293T cells that stably express a TNPO3-targeted shRNA were infected with MLV-GFP, FIV-GFP, EIAV-RFP, SIVmac-HSA, and HIV-1-HSA reporter virus at the indicted MOI. (B) Empty-vector-transduced HeLa cells and HeLa cells that stably express a TNPO3-targeted shRNA were infected with MLV-GFP, FIV-GFP, EIAV-RFP, SIVmac-HSA, and HIV-1-HSA reporter virus at an MOI of 0.3. (C) PMA-differentiated wild-type and stable TNPO3 knockdown THP-1 cells were infected with SIVmac239 or HIV-1 reporter virus. (D) Control and TNPO3 knockdown 293T cells were infected with wild-type HIV-1, wild-type SIVmac239, SIVmac239, SIVmac with HIV-1 capsid and p2 (SIV-HCAp2), or HIV-1 with SIVmac239 capsid (HIV-SCA) reporter virus. (E) Control and TNPO3 knockdown 293T cells were infected with HIV-SCA reporter virus containing a wild-type SIVmac239 CA or an SIVmac239 CA with an A76V mutation. (F) Control and mCPSF6-358-expressing HeLa cells treated with DMSO or 2␮g/ml aphidicolin overnight were infected with HIV-SCA reporter virus containing a wild-type SIVmac239 CA or an SIVmac239 CA with an A76V mutation. These experiments were done in triplicate, and the error bars indicate the standard deviations of the mean.

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the TNPO3 knockdown HeLa cell line were similar (Fig. 2B). THP-1 cells differentiated with phorbol myristate acetate (PMA) become macrophage-like cells and cease to divide. Differentiated TNPO3 knockdown THP-1 cells showed a re-duction in HIV-1 and SIVmac239 that was similar to that in the 293T and HeLa cells (Fig. 2C). Thus, TNPO3 is required to similar extents in dividing and nondividing cells. These findings also further confirm the increased sensitivity of SIVmac239 to TNPO3 levels.

The role of CA in enhanced SIVmac239 sensitivity to

TNPO3 knockdown.The ability of HIV-1 to infect nondividing

cells has been mapped to CA by using HIV/MLV chimeric viruses (41), and a single amino acid change in HIV-1 CA, N74D, causes the virus to become TNPO3 independent (24). Therefore, we considered whether the difference in the TNPO3 dependences of the different lentiviruses could be determined by differences in the CA sequence. To test this hypothesis, we determined the TNPO3 dependence of chime-ric HIV-1/SIVmac239 viruses in which the CA coding regions had been swapped (11, 32). We found decreased sensitivity to TNPO3 knockdown for an SIVmac239 virus containing an HIV-1 CA-p2 region (SIVmac239 HCAp2) (Fig. 2D), indicat-ing that the SIVmac239 CA was necessary for the enhanced sensitivity of SIVmac239 to TNPO3 knockdown. Reciprocally, an HIV-1 chimera with SIVmac239 CA 1-204 (HIV-1 SCA) was used to determine if this SIVmac239 CA fragment was

sufficient to confer on HIV-1 an increased sensitivity to TNPO3 knockdown. We did not observe a difference in sensi-tivity between HIV-1 SCA and wild-type HIV-1. This suggests that SIVmac239 CA was not sufficient to confer increased sensitivity to TNPO3 knockdown.

We also tested an HIV-1 SCA chimeric virus that had been passaged in human cells to adapt it for growth in human cells (16). The resulting virus had accumulated several mutations within Gag, one of which was an A76V mutation in SIVmac239 CA. This position was close to the analogous N74D in HIV-1 CA that had been identified by Lee et al. as providing TNPO3 independence (24). To determine whether the A76V mutation was responsible for TNPO3 independence in the virus, we introduced the A76V point mutation into SIVmac239. Analysis of the mutated virus showed that it had become TNPO3 inde-pendent (Fig. 2E).

[image:5.585.46.535.72.362.2]

Expression of mCPSF6-358, a truncated CPSF6 that fails to localize to the nucleus, blocks HIV-1 nuclear import (24). The N74D point mutation in HIV-1 CA allows the virus to evade the mCPSF6-358 block to infection. To determine whether the TNPO3-independent HIV-1 SCA chimeric virus similarly be-comes resistant to mCPSF6-358, we tested the ability of A76V CA-mutated HIV-1 SCA chimeric virus to infect cells that express mCPSF6-358. We found that the A76V CA mutant virus was largely resistant to mCPSF6-358. Analysis of cells arrested in the cell cycle with aphidicolin showed that the

FIG. 3. The carboxy-terminal cargo-binding domain of TNPO3 is required for SIVmac239 infection. (A) The shRNA-target site in TNPO3 (red) was mutated (black) to generate an shRNA-resistant TNPO3 expression vector. (B) TNPO3 knockdown 293T cells were transfected with expression vector encoding shRNA-resistant TNPO3, importin-13 (Imp13), and empty vector. The transfected cells and wild-type 293T cells (cntrl cells) were then infected with SIVmac239 reporter virus. (C) TNPO3 knockdown 293T cells were transfected with wild-type,⌬Cargo, or TNPO3 with the carboxy-terminal 18 amino acids deleted (⌬C18).

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mutant virus was resistant to mCPSF6-358 in dividing and nondividing cells (Fig. 2F).

The cargo-binding domain is required for lentivirus

infec-tion. To understand the role of TNPO3 in lentiviral nuclear

import, we were interested in testing the function of mutated forms of TNPO3. However, such an analysis is complicated by the presence of endogenous TNPO3, which is required for cell viability. We therefore established a complementation assay in which the TNPO3 shRNA 293T cells were complemented by transfection with shRNA-resistant TNPO3 expression vectors that contained silent mutations in the shRNA-targeted se-quence (Fig. 3A) and then challenged with SIVmac239 re-porter virus. To validate the assay, we complemented the cells by transfection with expression vectors for wild-type TNPO3 and the related protein importin-13 (30). Expression of impor-tin-13 and TNPO3 in the transfected cells was confirmed by immunoblot analysis (Fig. 3B). The knockdown cells were in-fected to an extent that was 23% that of the control cells. Complementation with wild-type TNPO3 restored the infec-tion to 77% that of the control. Importin-13 did not restore SIVmac239 infection, showing that the protein could not sub-stitute for TNPO3 (Fig. 3B).

Importin-␤family members contain a cargo-binding domain

at their carboxy termini. If TNPO3 mediates virus nuclear import by interacting with a viral or cellular protein, the cargo-binding domain would be expected to be required for function. To determine the importance of the cargo-binding domain for lentiviral infection, we generated TNPO3 expression vectors that encoded proteins with the cargo-binding domain or the final 18 amino acids of the domain deleted and tested them in the complementation assay. Both proteins were stably ex-pressed in the transfected cells, and neither was active in the complementation assay for SIVmac239 infection (Fig. 3C).

Drosophila TNPO3 is active for lentiviral nuclear import. The TNPO3 gene has been highly conserved throughout evo-lutionary history. Human TNPO3 shares 56% amino acid iden-tity with the Drosophila melanogaster homologue (Fig. 4A). This conservation allows theDrosophilaTNPO3 (drTNPO3) to mediate the nuclear import of SR proteins in human cells (1). To determine if drTNPO3 could substitute for human TNPO3 in order to rescue SIVmac239 infection, we tested it in our complementation assay. The results showed that drTNPO3 restored the ability of SIVmac239 to infect the TNPO3-defi-cient cells (Fig. 4B).

Identification of functional amino acid motifs in the

cargo-binding domain. To further map residues within the

cargo-FIG. 4.DrosophilaTNPO3 facilitates SIVmac239 infection. (A) The amino acid sequence of DrosophilaTNPO3 was aligned with human TNPO3 using ClustalW. Identical amino acids are shaded dark gray, and conservative changes are shaded light gray. (B) TNPO3 knockdown cells were complemented with wild-type human TNPO3,⌬C18, and drTNPO3 and then challenged with SIVmac239 reporter virus.

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binding domain of TNPO3, we targeted the carboxy-terminal 18 amino acids that were conserved between the human and

Drosophila proteins, changing individual residues to alanine

(Fig. 5A). Analysis of the mutated proteins in the complemen-tation assay showed that most retained function but that the F918A/F922A double mutant was almost completely inactive (Fig. 5B). The single mutants, F918 and F922A, maintained their function (Fig. 5C), indicating that both residues needed to be changed to inactivate the protein. The stability of the mutated proteins was confirmed by immunoblot anal-ysis (Fig. 5D).

To further probe the function of the cargo-binding domain, we used secondary-structure prediction software from the PSIPRED protein structure prediction server (Bloomsbury Centre for Bioinformatics, London, United Kingdom) and the PredictProtein server (Techniche Universita¨t, Munich, Ger-many). The analysis identified potential ␣-helical and loop regions in the cargo-binding domain (Fig. 6A). We focused on the loop regions, mutating groups of residues that were

con-served between human and drTNPO3 to alanine (Fig. 6A), because of the likelihood that the loops might serve as protein-protein interaction sites and were less likely to affect the sta-bility of the protein. Analysis of the functions of these mutated proteins showed that all were active, with the exception of the LLRS767-70 mutant (Fig. 6B). Single-amino-acid dis-section of these 4 residues showed that it was the LL767-8 dileucine motif that was necessary for function (Fig. 6C). The stability of the mutated proteins was confirmed by im-munoblot analysis (Fig. 6D).

[image:7.585.43.535.66.441.2]

To determine whether the TNPO3 motifs identified for their importance in mediating SIVmac239 infection were required by other lentiviruses, we tested the functions of the mutated TNPO3 proteins on EIAV and HIV-1 infection in the complementation assay. The results showed that wild-type TNPO3 was active on SIVmac239, HIV-1, and EIAV. The EIAV reporter virus system was not as robust as the other viruses, yet the amount of infection was sufficient to obtain significant results that were reproducible in multiple

FIG. 5. F918 and F922 in the cargo-binding domain of TNPO3 are required to facilitate SIVmac239 infection. (A) Specific amino acid residues in the carboxy-terminal 18 amino acids of TNPO3 were mutated to alanine (asterisks). (B) TNPO3 knockdown cells were complemented with wild-type,⌬Cargo, or mutated TNPO3 expression vector and then infected with SIVmac239 reporter virus. (C) TNPO3 knockdown cells were complemented with wild-type,⌬Cargo, F918A, F922A, or double F918A/F922A point mutated TNPO3 expression vector and then infected with SIVmac239 reporter virus. (D) Mutant TNPO3 proteins were visualized by immunoblot analysis.

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independent repetitions of the experiment. Analysis of the

⌬Cargo, F918A/F922A, and LL767-8 TNPO3 mutants showed that they were unable to rescue HIV-1 and EIAV infection, consistent with their lack of activity on SIV-mac239 infection (Fig. 7).

TNPO3 mutants retain the ability to localize to the nucleus. The lack of function of the mutated TNPO3 proteins could have been caused by a failure to interact with its cargo, by a failure to traffic through the nuclear pore, or by misfolding of the proteins. To distinguish between these possibilities, we generated vectors that expressed the mutated TNPO3 fused to enhanced green fluorescent protein (EGFP) and then determined their cellular localization in transfected HeLa cells by fluorescence microscopy. To enhance the vi-sualization, the nuclei were stained with Hoechst dye and the cell membrane was stained with wheat germ agglutinin (WGA). To determine whether the wild-type TNPO3-GFP

[image:8.585.42.536.70.460.2]

fusion was functional, we tested it in the complementation assay with SIVmac239. The analysis showed that the wild-type GFP fusion protein was active (Fig. 8A). Visualization of the proteins by confocal fluorescence microscopy showed that the TNPO3-GFP fusion protein localized to the nu-cleus. The ⌬Cargo, F918A/F922A, and LLRS767-70 mu-tants retained the ability to localize to the nucleus (Fig. 8B). We also visualized the localization of HA-tagged TNPO3 proteins to confirm these data. The HA-tagged TNPO3 pro-teins were localized to the nucleus and cytoplasm. This localization was not disrupted by the cargo-binding domain mutations (see Fig. S1 in the supplemental material). These results suggest that the proteins had folded properly and maintained their ability to interact with the nuclear import machinery of the cell. Thus, the likely explanation for their failure to mediate virus infection is an inability to bind to a cellular or virus cargo.

FIG. 6. A dileucine motif in the cargo-binding domain of TNPO3 is required to facilitate SIVmac239 infection. (A) Specific amino acid residues in the cargo-binding domain were mutated to alanine (asterisks). Individual loop mutants are boxed in black. (B) TNPO3 knockdown cells were complemented with a wild-type,⌬Cargo, or mutated TNPO3 expression vector. (C) TNPO3 knockdown cells were complemented with a wild-type,

⌬Cargo, and TNPO3 expression vector containing single or multiple point mutations of amino acid residues L767, L766, R769, and S770. (D) The mutated TNPO3 proteins were visualized by immunoblot analysis.

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DISCUSSION

Analysis of mutated TNPO3 proteins showed that the cargo-binding domain is required for lentivirus infection. Although the proteins with mutated cargo-binding domains had lost their ability to mediate lentivirus infection, they retained the ability to localize to the nucleus. Taken together, these findings sug-gest that the TNPO3 proteins with mutated cargo-binding do-mains retain the ability to interact with nuclear pore compo-nents but fail to interact with a host or virus protein that is required for virus nuclear import. The identity of this protein remains unknown.

The identification of the two hydrophobic motifs required for TNPO3-dependent SIVmac infection represents the first detailed mapping of the TNPO3 carboxy-terminal cargo-bind-ing domain. These motifs are a likely interaction site for the postulated cargo protein that influences virus nuclear import. The LL767-8 residues appear to be more important than the F918/F922 residues, since LL767-8AA mutants are inactive in the SIVmac239 complementation assay while the F918A/ F922A mutant remained partially active. The 150 amino acids separating these two motifs suggest that the cargo-binding domain offers a wide surface for interaction with its cargo. This is consistent with the crystal structures of transportin 1 (TNPO1) in complex with cargo protein nuclear localization signals (NLSs), which showed a wide cargo interaction surface in the TNPO1 carboxy-terminal and core domains (18). The large number of amino acids in TNPO1 that interact with the target NLSs and the fact that this interaction occurs over the span of 10 HEAT repeats (20␣-helices) indicates that TNPO3 is likely to interact with its cargo at several sites in addition to the hydrophobic motifs that we identified.

The putative cargo protein that interacts with TNPO3 to

mediate nuclear import of the PIC could act directly on nu-clear import by interacting with both the PIC and TNPO3. IN, a PIC component, was proposed to be the target of TNPO3 that mediates nuclear import. In support of this model, TNPO3 was initially identified in a yeast two-hybrid screen for IN-interacting cellular proteins (9). However, evidence argues against a role for IN as the major target of TNPO3. First, although TNPO3 was found to associate with recombinant HIV-1 IN in anin vitropulldown assay, the IN from MLV, a virus that is not transported through the nuclear pore and is independent of TNPO3, also associated with recombinant TNPO3 (21). Second, the requirement for the carboxy-termi-nal cargo-binding domain of TNPO3 is inconsistent with the previous finding that IN interacts with the amino-terminal do-main of TNPO3 (9). Third, we failed in repeated attempts to detect the interaction of TNPO3 with IN by coimmunoprecipi-tation from transiently transfected 293T cells that expressed both proteins at a high level (data not shown). Taken to-gether, these findings are inconsistent with a role for IN as the primary target of TNPO3 that mediates virus nuclear import, although a secondary role for IN in which it binds to the amino-terminal domain of TNPO3 to assist in nuclear import cannot be ruled out.

Conversely, the TNPO3 knockdown could act indirectly by altering the normal cellular environment. In this case, the unidentified cargo would be a cellular protein that is important for the correct splicing of a host that is directly involved in viral nuclear import or that gains an inhibitory activity for virus nuclear import when it is mislocalized to the cytoplasm. CPSF6 itself is a potential candidate, as it is a member of the SR family of RNA-processing proteins and therefore a potential TNPO3 cargo. Knockdown of TNPO3 could lead to the cytoplasmic

FIG. 7. The cargo-binding domain is required for TNPO3-dependent infection by several lentiviruses. TNPO3 knockdown cells were comple-mented with shRNA-resistant wild-type,⌬Cargo, F918A/F922A, and TNPO3 LL767-8AA expression vector or with the empty vector. The cells were then infected with SIVmac239, HIV-1, or EIAV reporter virus.

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accumulation of CPSF6, which could then bind to the viral PIC and interfere with uncoating or docking at the nuclear pore. Lee et al. showed that the mCPSF6-358 fragment, which lacks its carboxy-terminal RS domain, accumulates in the cytoplasm and that the mCPSF6-358 fragment binds to wild-type CA but not to N74D CA in core complexes (24). They speculated that when the protein accumulates in the cytoplasm this interaction with CA could prevent infection by interfering with interac-tions that are important for uncoating or nuclear transport. Moreover, single point mutations in HIV-1 or SIV CA caused the virus to become TNPO3 independent and resistant to mCPSF6-358, a finding that links TNPO3 and CPSF6 function-ally. Whether CPSF6 is, in fact, a cargo of TNPO3 and accu-mulates in the cytoplasm following TNPO3 knockdown re-mains to be determined.

Although we found that several lentiviruses were TNPO3 de-pendent, the viruses differed in their relative sensitivities to TNPO3 knockdown. SIVmac239 and EIAV were the most sen-sitive, while HIV-1 was less sensitive and FIV was only slightly affected by TNPO3 knockdown. The sensitivity of SIVmac239 to TNPO3 knockdown was confirmed in the infectivity assay and by

2-LTR circle quantification. These results are similar to those of Krishnan et al. (21), except that we found EIAV and SIVmac239 to be similarly sensitive to TNPO3 knockdown. The cause of this difference in sensitivity to TNPO3 knockdown is not clear but could result from differences in the extent of knockdown, the cell type, or the EIAV reporter system.

The CA protein appears to be a viral determinant that is important in determining sensitivity to TNPO3 knockdown. Analysis of SIVmac239/HIV-1 chimeras showed that CA was a necessary but not a sufficient determinant of TNPO3 dependence. Replacement of SIVmac239 CA-p2 with that of HIV-1 resulted in a virus with HIV-1-like sensitivity to TNPO3 knockdown, while the reciprocal swap of SIV-mac239 CA into HIV-1 did not confer an increased sensi-tivity to TNPO3 knockdown. These findings suggest that viral components in addition to CA play a role in determin-ing sensitivity to TNPO3 knockdown.

How CA regulates the nuclear import of lentiviruses is un-clear, since there is a lack of evidence for a direct interaction of CA with TNPO3 (13, 29). It is possible that CA affects nuclear import by controlling the trafficking of the viral particle

FIG. 8. Nonfunctional mutated TNPO3 proteins localize to the nucleus. (A) TNPO3 knockdown cells were complemented with a wild-type TNPO3-GFP, mutated TNPO3-GFP, or GFP expression vector. The cells were gated for GFP fluorescence, and the percentages of infected GFP⫺ and GFP⫹cells were determined. (B) HeLa cells transfected with GFP-tagged shRNA-resistant full-length or mutant TNPO3 expression vectors were imaged by confocal microscopy. GFP fusion proteins are shown in green. The nucleus was counterstained with Hoechst (blue) and the plasma membrane with wheat germ agglutinin (red).

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to the nucleus. HIV-1 CA does appear to stay associated with the reverse-transcribing viral particle as it is transported from the cell periphery to the nucleus (28). A link also appears to exist between viral entry and TNPO3 dependence, suggesting that differences in trafficking of the viral particle to the nucleus could influence the nuclear import of the PIC (37). It is also possible that CA could change TNPO3 dependence by affect-ing the timaffect-ing of uncoataffect-ing or the dockaffect-ing of the viral particle to the nuclear pore. This appears to be possible, as scanning electron microscopy imaging of newly infected cells shows that uncoating occurs at the nuclear pore following the completion of reverse transcription (2).

Our A76V mutation in SIVmac239 CA matched the pheno-type of the previously described N74D, E45A, and Q63A/ Q67A HIV-1 CA mutants, rendering the virus TNPO3 inde-pendent and resistant to mCPSF6-358 (10, 24, 43). However, the A76V SIVmac239 CA mutant and the N74D HIV-1 CA mutant differ from the other two HIV-1 CA mutants in that they are still capable of infecting nondividing cells. Therefore, these two mutations are unlikely to cause a defect in uncoating as the E45A and Q63A/Q67A HIV-1 CA mutants do. Altera-tion of TNPO3 dependence also appears to affect downstream integration steps (31). In agreement with this finding, we found that knockdown of TNPO3 had a more pronounced effect on the number of 2-LTR circles than on the number of infected cells. This may indicate that viruses that are imported through a TNPO3-independent pathway are more efficient at integra-tion while those that are imported through a TNPO3-depen-dent pathway frequently fail to integrate and instead form nonproductive 2-LTR circles.

The relative sensitivities to TNPO3 knockdown may be caused by differences in the affinities of different viral capsids for binding to CPSF6. This mechanism predicts that the HIV-1 N74D and SIVmac239 A76V CA mutations reduce the affinity of CA for CPSF6, and the differences in lentivirus sensitivities to TNPO3 knockdown reflect differences in the affinities of the lentivirus CA for CPSF6. HIV-1 and SIVmac239 were equally sensitive to Nup153 knockdown, suggesting that the protein plays a role in nuclear import that is different from that of TNPO3.

The picture that has emerged for the role of TNPO3 in lentiviral nuclear import illustrates the caution that should be exercised in the interpretation of viral dependency factors identified in genome-wide screens. A large number of such cellular genes have been identified, and while some of these clearly encode products that play a direct role in infection, others may play a much less direct role. TNPO3, for example, may not serve as a viral nuclear import protein itself but rather may prevent another cellular protein from interfering with viral nuclear import. Other dependency factors that have been identified may also play indirect roles in infection. The TNPO3 cargo-binding domain mutants and the A76V CA SIVmac239 mutant reported here will provide useful reagents for the fur-ther analysis of the mechanism by which TNPO3 regulates lentiviral nuclear import. Of particular importance will be the determination of whether CPSF6 or other cellular proteins are affected by TNPO3 knockdown to establish the block to lenti-virus nuclear import.

ACKNOWLEDGMENTS

We thank Paul Bieniasz for the HIV SCA A76V packaging struct, Heinrich Göttlinger for the SIVmac239 CA-p2 gag-pol con-structs, Joseph Sodroski for the HIV SCA packaging concon-structs, Eric Poeschle for the FIV reporter virus constructs, and Vineet Kewal-Ramani for the EIAV reporter virus constructs and mCPSF6-358-expressing HeLa cells. We thank Yan Deng for assistance with confocal imaging at the NYULMC Imaging Core (NCRR S10 RR023704-01A1).

The work was supported by NIH funding (R21AI073237 and 5R01 AI067059-03) and by a postdoctoral fellowship to E.C.L. from the Cancer Research Institute. N.R.L. is a scholar of the Elizabeth Glaser Pediatric AIDS Research Foundation.

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Figure

FIG. 1. TNPO3 knockdown inhibits HIV-1 and SIVmac239 nuclear import. (A) HeLa cells were transfected with nontarget control (NTC),TNPO3, Nup153, or mock siRNA and then infected with HIV-1 and SIVmac239 reporter virus
FIG. 2. TNPO3 knockdown in dividing and nondividing cells blocks infection with multiple lentiviruses
FIG. 3. The carboxy-terminal cargo-binding domain of TNPO3 is required for SIVmac239 infection
FIG. 4. DrosophilaTNPO3 using ClustalW. Identical amino acids are shaded dark gray, and conservative changes are shaded light gray
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