HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA*

HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.

HIV-1 depends on host factors to complete its life cycle (1)(2)(3)(4)(5)(6)(7)(8). Hundreds of human proteins involved in the interaction with HIV-1 for modulating viral replication have been identi-fied by genome-wide RNA interference screen or affinitytagged protein purification using mass spectrometry (1,(3)(4)9). A comprehensive understanding of how host machineries are manipulated during HIV-1 infection can facilitate the identification of host targets for antiviral drugs or gene therapy.
The CRM1 (chromosome region maintenance 1, 2 also known as exportin 1) nuclear export pathway is typically used to transport host protein cargoes and small RNAs, which has been found to engage HIV-1 regulatory protein Rev for directing the nuclear export of unspliced and partially spliced HIV-1 RNAs (10 -15). These incompletely spliced HIV-1 RNAs contain a Rev response element (RRE), and the coordinate assembly of multiple Rev molecules on RRE recruits human protein complex containing CRM1 and Ran-GTP (GTP-binding nuclear protein Ran). Rev contains a leucine-rich nuclear export signal (NES) domain located near the carboxyl terminus, through which Rev interacts with CRM1, and the Rev-RRE-CRM1/Ran-GTP complex is then transported to the cytoplasm (6,(11)(12)(13)(14)(15)(16). Similarly, other retroviruses such as mouse mammary tumor virus and human T cell lymphotropic virus encode Rev-like regulatory proteins Rem or Rex, respectively, for hijacking the host CRM1 pathway to accomplish nuclear export of unspliced viral transcripts (17)(18)(19)(20)(21)(22).
Several other cellular factors have also been identified as Rev co-factors that are required for efficient Rev function, such as the RNA binding motif protein 14, the single-stranded nucleic acid-binding protein Pur-alpha, eukaryotic initiation factor-5A, and the DEAD (Asp-Glu-Ala-Asp) box RNA helicase DDX1, DDX3, and DDX5 (23)(24)(25)(26)(27)(28)(29)(30)(31). However, some host factors recruited by Rev can interfere with its function. For example, the nucleoporins Nup98 and Nup214 can be recruited by Rev to the nucleoli, while the nucleoporin (NP repeat) domains of Nup98 and Nup214 are able to competitively inhibit Rev's nuclear export. However, the inhibition induced by the Nup98 NP domain could be counteracted by excess Rev (32). Since the Rev-RRE complex has been proposed to be an attractive therapeutic target (33), the detailed elucidation of Rev/CRM1-associated cellular factors is essential for developing new strategies for antiviral approaches.
In this study, we revealed a novel role of the host protein Naf1 (HIV-1 Nef-associated factor 1) in promoting Rev/CRM1 function during HIV-1 production. Naf1 is also known as virionassociated nuclear shuttling protein and has been identified as a HIV-1 Nef-binding protein that increases the expression of cell surface CD4 (34). Naf1 is also known as A20-binding inhibitors of NF-B activation (ABIN-1) (35)(36), and three ABIN proteins have been identified to bind with the deubiquitinating protein A20 for inhibiting NF-B activation (37)(38)(39)(40).
Here we found that Naf1 is associated with CRM1 for nucleocytoplasmic shuttling and promotes the nuclear export of unspliced mRNA of HIV-1 gag. The novel role of cellular factor Naf1 in regulating HIV-1 production could provide a potential therapeutic target for controlling HIV-1 infection.

Experimental Procedures
Cells and Virus Stock-HEK293T cells were cultured in DMEM medium (Hyclone) containing 10% fetal bovine serum (FBS) (Hyclone) and 100 units/ml penicillin and 100 g/ml streptomycin. Calcium-phosphate-mediated transfection of HEK293T cells was used to generate virus stock as described previously (41). Pseudotyped single-cycle infectious HIV-H131/VSV-G was cotransfected with the plasmid pHIV/H131 and the expression plasmid vesicular stomatitis virus G (VSV-G) protein. Harvested supernatants of transfected cells that contained viral particles were filtered and titrated with p24 gag capture enzyme-linked immunosorbent assay (ELISA) as previously described (42).
Transfection and HIV-1 Infection Assays-Transfection was performed by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 6 h post-transfection, the medium containing the mixture of plasmids and transfection reagents was replaced with fresh DMEM supplemented with 10% FBS, and then cells were further cultured for 24 h. For infection, Naf1-stable-knocking-down or off-target HEK293T cells were inoculated with HIV-H131/VSV-G for 4 h (2 ng p24 gag ), and after washing, cells were further cultured for 24 h, then culture supernatants and cell lysates were harvested to quantify viral production by p24 gag capture ELISA.
Cytotoxicity Assay by MTT Method-LMB cytotoxicity was assessed by MTT method as described previously (45). Briefly, HEK293T cells (2 ϫ 10 4 ) seeded on a 96-well plate were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/H131 plasmid for 18 h. Various concentrations of LMB were added for an additional 6 h of cell culture. 20 l of MTT (3-(4,5cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (the stock concentration is 5 mg/ml) was added and incubated at 37°C for 4 h. Then 100 l of 10% SDS (sodium dodecyl sulfate)-50% DMF (N,NЈ-dimethyl formamine) was added and incubated until the formazan was dissolved completely. The plates were read on a Bio-Tek ELx 800 enzyme-linked immunosorbent assay (ELISA) reader at 595/630 nm. The cell viability (% of untreated control) was calculated by measuring absorbance values.
Nucleo-cytoplasmic HIV-1 RNA Fractionation and Quantification-HEK293T cells were co-transfected with pCMV-Myc-Naf1 (0.4 g) or pCMV-tag3B vector with pHIV/H131 (0.4 g) for 24 h. HEK293T cells with stable Naf1 knockdown or the off-target control were inoculated with HIV-H131/VSV-G for 4 h (4 ng p24 gag ), and after washing, cells were further cultured for 24 h. Total RNA from the cells was extracted using TRIzol reagent (Life Technologies). Nuclear and cytoplasmic RNA fractions were purified by using a PARIS Protein and RNA isolation kit (Ambion, Life Technologies) following the manufacturer's protocol. U6 snRNA and 18S rRNA were used as nuclear or cytoplasmic RNA control, respectively.
Confocal Microscopy-HEK293T cells were seeded on the Cell Imaging Dish (35 ϫ 10 mm) (Eppendorf), and transfected with pEGFP-Naf1 (0.2 g) for 24 h as above. Three-dimensional reconstruction was performed using Nikon A1 ϩ laser scanning confocal microscope, and live-cell imaging was performed with Leica TCS SP8 laser scanning confocal microscope. Nuclei were indicated with Hoechst 33258 (Sigma).
Statistical Analysis-Statistical analysis was performed using a paired t test with SigmaStat 2.0 software (Systat Software, San Jose, CA).

Results
Naf1 Expression Is Essential for HIV-1 Production-To better understand the effect of Naf1 on HIV-1 replication, we first investigated the role of Naf1 in HIV-1 production. The expression of endogenous Naf1 in HEK293T cells was successfully knocked-down by using specific shRNA (Fig. 1A), then cells were transfected with the HIV-1-derived expression vector H131, and viral production was monitored by quantifying the p24 gag from both cell culture supernatants and cell lysates. The vector pH131 was derived from the HIV-1-eGFP construct with the depletions of nef, vif, vpr, vpu, and env, but this vector possesses HIV-1 rev and tat gene (43)(44). The interference with Naf1 expression significantly reduced p24 gag production (Fig. 1B).
To verify the necessity of Naf1 for HIV-1 production during viral infection, the HIV-derived H131 vector was used to generate VSV-G pseudotyped, single-cycle HIV-H131/VSV-G. HEK293T cells with or without knock-down of Naf1 were infected with HIV-H131/VSV-G and HIV-1 production was examined by quantifying p24 gag . Similar results indicate that knockdown of Naf1 significantly impaired HIV-1 production (Fig. 1C).
In contrast, when Naf1 was overexpressed (Fig. 1D), enhanced HIV-1 p24 gag production from both supernatants and cell lysates was observed (Fig. 1E). Furthermore, when Naf1 expression was complemented in HEK293T cells with stable Naf1 knockdown (Fig. 1F), HIV-1 p24 gag production from both supernatants, and cell lysates was recovered (Fig. 1G). Together, these data demonstrate that Naf1 expression is required for efficient HIV-1 production in virus-producing cells.
Naf1 Enhances the Nuclear Export of Unspliced HIV-1 gag mRNA-To examine the mechanism of Naf1-promoted HIV-1 production, the potential effect of Naf1 on viral transcription was investigated. HIV-1-based expression vector H131 was transfected into Naf1-overexpressing HEK293T cells. Timecourse analysis showed that Naf1 overexpression did not affect the expression of total HIV-1 gag mRNA ( Fig. 2A), suggesting FIGURE 1. Naf1 expression is required for efficient HIV-1 production. A, endogenous Naf1 expression in HEK293T cells was knocked down with specific shRNA and detected by immunoblotting. B and C, knock-down of Naf1 significantly suppresses HIV-1 production in comparison to off-target control in HEK293T cells transfected with HIV-1-based expression vector pH131 (B), or infected with HIV-H131/VSV-G vector (C), and HIV-1 production was quantified by measuring p24 gag amount 24 h later in cell culture supernatants and cell lysates. D and E, Naf1 overexpression enhances HIV-1 production. HEK293T cells were transfected with pCMV-Myc-Naf1, and Naf1 expression was detected by immunoblotting (D). In Naf1-overexpressed cells, viral p24 production was increased (E). F and G, Naf1 complementation in stable Naf1-knocking-down HEK293T cells restores HIV-1 production. Naf1 expression was complemented in stable Naf1-knocking-down HEK293T cells by transfected with pCMV-Myc-Naf1 for 24 h (F), and cells were transfected with the pHIV/H131 plasmid for additional 24 h and viral production was quantified by p24 gag assay (G). GAPDH was used as a loading control (A, D, F). One representative of at least four independent repeats for each result is shown. The results were analyzed by the paired Student's t test; **, p Ͻ 0.01 and ***, p Ͻ 0.001 were considered statistically significant.
Naf1 has been shown to be a nucleo-cytoplasmic shuttling protein (46), which prompted us to investigate the potential effect of Naf1 on nuclear export of transcribed HIV-1 mRNA (Fig. 2B). The pHIV/H131 plasmid was transfected into Naf1overexpressing or vector control HEK293T cells, and the cytoplasmic and nuclear fractions were separated and the levels of HIV-1 mRNAs were quantified (Fig. 2D). U6 snRNA and 18S rRNA was used as nuclear and cytoplasmic RNA control, respectively (Fig. 2C). Naf1 overexpression significantly enhanced the nuclear export of HIV-1 unspliced gag mRNA, but did not significantly alter the nucleo-cytoplasmic distribution of multiple-spliced HIV-1 tat-rev mRNA (Fig. 2D).
To confirm this finding, a single-cycle infectious HIV-H131/ VSV-G vector was used to infect HEK293T cells with stable knock-down, of Naf1, and the nucleo-cytoplasmic distribution of HIV-1 mRNA was measured. Naf1 knock-down decreased the nuclear export of unspliced mRNA of HIV-1 gag from 33% to 18%, but only slightly altered the nuclear export of the multiple-spliced HIV-1 tat-rev mRNA from 74% to 77% (Fig. 2E). We have observed that these viral unspliced RNAs at 24 h posttransfection or -infection were predominantly located in the nucleus (Fig. 2, D and E), which might suggest the different export dynamic for unspliced viral mRNA compared with multiple-spliced viral mRNA. Together, these data suggest that Naf1 expression facilitates efficient nuclear export of unspliced HIV-1 gag mRNA. HEK293T cells were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/H131 for 24 h. Cells were harvested at the indicated times post-transfection, and total mRNAs were isolated, and the levels of HIV-1 gag mRNA were quantified with qRT-PCR and normalized to ␤-actin mRNA. B, scheme for detection of cytoplasmic/nuclear mRNA. Cells were collected for isolation of cytoplasmic and nuclear mRNA, and the relative ratios of cytoplasmic and nuclear mRNA (unspliced gag mRNA or multi-spliced tat-rev mRNA) were analyzed by qRT-PCR. C, U6 snRNA and 18S rRNA was used as nuclear or cytoplasmic RNA control, respectively. C: cytoplasm, N: nucleus. D, Naf1 overexpression enhances the nuclear export of HIV-1 unspliced gag mRNA. Naf1-overexpressing or parental (pCMV-Myc vector) HEK293T cells were transfected with HIV/H131 for 24 h, then cells were collected and total mRNA were isolated, and HIV-1 gag mRNA were quantified as above, the relative cytoplasmic and nuclear distribution of HIV-1 mRNA were calculated, and 4 repeats were summarized. The horizontal lines in the dot plot (low panels) indicate the mean with S.D. E, Naf1 knock-down decreases the nuclear export of HIV-1 unspliced gag mRNA. The Naf1-stable-knock-down HEK293T or off-target cells were infected with the HIV-H131/VSV-G vector for 24 h, and the cytoplasmic/nuclear distribution of HIV-1 mRNA was monitored and the relative values were calculated. ***, p Ͻ 0.001 was considered significant as determined by the paired Student's t test. FEBRUARY 26, 2016 • VOLUME 291 • NUMBER 9

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Naf1 Association with CRM1 Is Required for Augmenting HIV-1 Production-It has been reported that Naf1 nucleo-cytoplasmic shuttling is CRM1-dependent (46). To confirm the association between Naf1 and CRM1, co-immunoprecipitation assays were performed. The expression plasmids encoding FLAG-tagged Naf1 and HA-tagged Rev were co-transfected into HEK293T cells, then cells were harvested and lysed for immunoprecipitation with anti-Flag beads, and then immunoblotting was performed with specific antibodies against FLAG (Naf1), CRM1, or HA (Rev), respectively (Fig. 3A). Similarly, we Naf1 Regulates HIV-1 Production also co-transfected HEK293T cells with FLAG-tagged CRM1 and Myc-tagged Naf1 or HA-tagged Rev for co-immunoprecipitation (Fig. 3B). Our results confirmed the association between Naf1 and CRM1, and showed that Naf1 did not interact directly with HIV-1 Rev (Fig. 3, A and B).
To investigate whether association with CRM1 is required for Naf1 augmentation of HIV-1 production, the specific inhibitor LMB was used to block CRM1-dependent nuclear export pathway. LMB is known to bind to CRM1 to prevent the for-mation of nuclear export complex (47)(48)(49). We found that LMB treatment significantly impaired Naf1-promoted HIV-1 production in both supernatants and cell lysates (Fig. 3, C and  D). LMB treatment did not cause apparent cytotoxicity (Fig.  3E). Similarly, when CRM1 expression in HEK293T cells was knocked-down with specific shRNA, Naf1-augmented HIV-1 production was significantly attenuated (Fig. 3, F and G). The CRM1 knock-down was confirmed by Western blotting (Fig.  3H). Conversely, CRM1 overexpression significantly increased FIGURE 3. Naf1 association with CRM1 is required for augmenting HIV-1 production. A and B, Naf1 associates with CRM1. HEK293T cells were cotransfected with plasmids expressing Naf1-Flag and Rev-HA (A), or CRM1-Flag and Myc-Naf1 or Rev-HA (B), the whole cells lysates were prepared, immunoprecipitations were performed with anti-Flag coated magnetic beads, and immunoblotting was performed with indicated specific antibodies. C and D, LMB treatment impairs Naf1-mediated enhancement of HIV-1 Gag production. HEK293T cells were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/ H131 plasmid for 18 h. LMB (20 nM) or media were added for an additional 6 h in the cells. Viral production was quantified as above and the relative fold enhancement was calculated (C), and the summary of 4 repeat experiments (individual dots) was presented (D). E, LMB treatment does not have cytotoxicity. Transfected HEK293T cells were incubated with various concentrations of LMB for 6 h, and the LMB cytotoxicity was assessed with MTT method. Cell viability (% of untreated control) was calculated. One representative from three independent repeats is shown. F-H, CRM1 knock-down attenuates Naf1mediated enhancement of HIV-1 Gag production. HEK293T cells were transfected with CRM1 specific shRNAs or off-target control for 24 h, then cells were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/H131 plasmid for an additional 24 h. The viral production was quantified as above and the relative fold enhancement was calculated (F), and the summary of 4 repeats (individual dots) was presented (G). The CRM1 knock-down was confirmed by immunoblotting (H). I-K, CRM1 overexpression enhances Naf1 function. HEK293T cells were transfected with pcDNA3.1-CRM1-HA for 24 h, then cells were transfected with the pHIV/H131 plasmid for an additional 24 h (I), and viral productions were quantified as above and the relative fold enhancement was calculated (J), and the summary of 4 repeats (individual dots) was presented (K). *, p Ͻ 0.05 was considered significant as determined by the paired Student's t test. . The shuttling of Naf1 between the nucleus and the cytoplasm is required for promoting HIV-1 production. A, Naf1 distribution observed with confocal microscopy. HEK293T cells were transfected with peGFP-Naf1 expression plasmid for 24 h, and cells were observed with confocal microscopy. Nuclei were indicated with Hoechst 33258 (blue) stain. B, Naf1 distribution investigated by Western blotting. HEK293T cells were transfected with pCMV-Myc-Naf1 for 24 h, and the cytoplasmic (cyto.) and nuclear (nuc.) components were fractionated for immunoblotting. C, amino acids sequence of Naf1 NESs, NLS, and mutations. D, expression of Naf1 and its mutation detected with immunoblotting. E, distribution of Naf1 and its NLS mutant detected with immunoblotting. F, disruption of NESs or NLS of Naf1 attenuates the enhancement for HIV-1 production. HEK293T cells were transfected with wild type pCMV-Myc-Naf1 or different mutants for 24 h, then cells were further transfected with HIV/H131 plasmid for an additional 24 h, and HIV-1 production was measured by detecting p24 gag amounts in culture medium and cell lysates. *, p Ͻ 0.05 and **, p Ͻ 0.01 are considered significant difference as determined by paired Student's t test. FEBRUARY 26, 2016 • VOLUME 291 • NUMBER 9

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Naf1-mediated augmentation of HIV-1 production (Fig. 3, I, J, and K). Together, these data demonstrate the requirement of CRM1 for Naf1-promoted HIV-1 production.
Disruption of NESs or NLS of Naf1 Attenuates the Enhancement for HIV-1 Production-Naf1 is a nucleo-cytoplasmic shuttling protein with characteristic intracellular trafficking (46). To confirm the intracellular trafficking of Naf1, the Naf1-GFP-expressing plasmid pEGFP-Naf1 was transfected into HEK293T cells, and Naf1 cellular localization was monitored. Naf1-GFP was distributed both in the cytoplasm and the nucleus as observed by confocal microscopy (Fig. 4A). When the transfected HEK293T cells were fractionated into the cytoplasmic and nuclear components, the distribution of Naf1 in both cytoplasm and nucleus was confirmed by Western blotting (Fig. 4B). The nucleo-cytoplasmic shuttling of Naf1 was also observed by time-lapse imaging (supplemental Videos A and B).
The leucine-rich NES in bearing proteins, like HIV-1 Rev, mediates CRM1 recruitment for nuclear export (14,47,50). Based on the analysis of amino acid sequence, Naf1 was predicted to contain four putative NESs that interact with CRM1 (51). When point mutations were introduced into NESs (Fig. 4, C and D), the mutation at NES1, 2, or 3, but not NES4, significantly attenuated Naf1-mediated enhancement of HIV-1 p24 gag production (Fig. 4F), suggesting that NES4 might be inadequately required for modulating Naf1 nuclear export. These data confirm that the interaction between Naf1 and CRM1 is required for Naf1-promoted HIV-1 production.
Naf1 has also been predicted to contain a C terminus-located, lysine-enriched, classical NLS (51) (Fig. 4C), and the mutation of NLS retained Naf1 in the cytoplasm (Fig. 4E). Additionally, the NLS mutant lost its capacity for promoting HIV-1 production (Fig. 4F), suggesting that the intact NLS is required for Naf1-mediated enhancement for HIV-1 production. These data indicate that the nucleus and cytoplasm shuttling property of Naf1 is required for promoting HIV-1 production.

Discussion
Our results reveal a novel role of cellular factor Naf1 in enhancing HIV-1 production. Naf1 associates with cellular CRM1 for promoting nuclear export of unspliced HIV-1 gag mRNA. The association of Naf1 and CRM1 is required for Naf1-promoted HIV-1 production, and the inhibition of CRM1 activity or knockdown of its expression hence attenuated Naf1Јs function in enhancing HIV-1 production. Naf1 was predicted to contain four putative NESs that mediate CRM1 recruitment for nuclear export (51). Naf1 also contains a C terminus-located, lysine-enriched, classical NLS (51). Our mutagenesis of NES1-3 and NLS indicates that the shuttling property of Naf1 is essential for promoting viral production, as the disruption of NESs or NLS of Naf1 attenuates the enhancement of HIV-1 production. The mutation at NES4 did not significantly diminish viral production, suggesting a dispensable role of the predicted NES4 in regulating Naf1 trafficking.
It has been reported that Naf1 expression inhibited NF-B activation and HIV-1 infection (37,46,51,53). Naf1 associates with A20, a zinc finger protein with deubiquitinase activity, and facilitates A20-mediated de-ubiquitination of NEMO/IKK␥ and subsequent NF-B inhibition in response of TNF-␣ (40,54,56). HIV-1-LTR contains the NF-B binding sites, and the suppression of Naf1 on NF-B activation may account for the inhibition of HIV-1 promoter LTR-driven gene expression and viral infection. Naf1 can be incorporated into HIV-1 virions and interacts with the matrix protein of HIV-1 (46). HIV-1 matrix is a 17-kDa myristoylated protein derived from Gag precursor polyprotein and identified as a key component of HIV-1 preintegration complex to contribute to its nuclear localization (57)(58)(59). Notably, HIV-1 matrix is also involved in the viral assembly by directing Gag and Gag-Pol polyproteins to the plasma membrane during viral production (55). These data may suggest that Naf1 possesses multifaceted roles in regulating HIV-1 infection.
Naf1 is ubiquitously expressed in human tissues and cell types, particularly in peripheral blood lymphocytes (46). Naf1 can regulate multiple receptor-mediated signal pathways for modulating inflammation (52), which may interact with HIV-1 replication. Further study the potential relationship of Naf1modulating cellular signaling with enhanced HIV-1 production would be informative to better understand Naf1 functions and mechanisms in viral infection. Overall, our data demonstrate the novel role of cellular factor Naf1 in regulating HIV-1 production, and this find might facilitate the identification of host targets for antiviral drugs or gene therapy.