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Journal of Virology logoLink to Journal of Virology
. 2013 Jul;87(13):7726–7736. doi: 10.1128/JVI.00124-13

A Carboxy-Terminally Truncated Human CPSF6 Lacking Residues Encoded by Exon 6 Inhibits HIV-1 cDNA Synthesis and Promotes Capsid Disassembly

Takanori Hori 1, Hiroaki Takeuchi 1,✉, Hideki Saito 1, Ryuta Sakuma 1, Yoshio Inagaki 1, Shoji Yamaoka 1,✉
PMCID: PMC3700264  PMID: 23658440

Abstract

Since HIV-1 replication is modulated at multiple stages by host cell factors, identification and characterization of those host cell factors are expected to contribute to the development of novel anti-HIV therapeutics. Previous studies showed that a C-terminally truncated cytosolic form of cleavage and polyadenylation-specific factor 6 (CPSF6-358) inhibits HIV-1 infection through interference with HIV-1 trafficking to the nucleus. Here we identified and characterized a different configuration of C-terminally truncated human CPSF6 (hCPSF6-375) through cDNA expression cloning coupled with ganciclovir-mediated lethal selection. Notably, hCPSF6-375, but not mouse CPSF6-358 (mCPSF6-358) as previously reported, remarkably interfered with viral cDNA synthesis after HIV-1 infection. Moreover, we found that hCPSF6-375 aberrantly accelerated the disassembly of the viral capsid in target cells, while CPSF6-358 did not. Sequence comparison of CPSF6-375 and CPSF6-358 cDNAs showed a lack of exon 6 and additional coding sequence for 54 amino acid residues in the C terminus of hCPSF6-375. Mutational analyses revealed that the residues encoded by exon 6, but not the C-terminal 54 residues in hCPSF6-375, is responsible for impaired viral cDNA synthesis by hCPSF6-375. This is the first report demonstrating a novel mode of HIV-1 inhibition by truncated forms of CPSF6 that involves rapid capsid disassembly and inhibition of viral cDNA synthesis. These findings could facilitate an increased understanding of viral cDNA synthesis in light of the viral capsid disassembly.

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) replication requires the help of host cell factors, and diverse cellular pathways are hijacked by HIV-1 for efficient completion of the viral replication cycle (1). A wide range of cellular factors and processes are exploited by HIV-1 during various stages of replication, which involves the uncoating steps that form into a reverse transcription complex, intracellular trafficking of the viral preintegration complex (PIC) to the cell nucleus, and integration of the viral DNA into a host cell chromosome for generation of the provirus (2). Several genome-wide small interfering RNA (siRNA) analyses have demonstrated over 250 cellular factors that influence HIV-1 infection (3–5). On the other hand, cDNA expression cloning also proved to be a powerful strategy for discovering functional properties of cellular genes that may potentially contribute to identification of host targets for anti-HIV therapeutics (6–10). By screening with a mouse cDNA expression library, a C-terminally truncated form of mouse cleavage and polyadenylation-specific factor 6 (mCPSF6) was recently identified as a novel functional protein that blocks HIV-1 trafficking to the nucleus (7). The truncated protein, mCPSF6-358, reduced the amount of the 2-long terminal repeat (LTR) circular form of viral DNA that is located in the nucleus, but not the late product of viral cDNA, indicating impairment of the nuclear entry of HIV-1 (7). More recently, it was shown that the C-terminal 58 residues of mCPSF6-358 are sufficient for HIV-1 inhibition and that mCPSF6-358 residues 313 to 327 contribute to antiviral activity (11). A subsequent report showed that a synthesized peptide corresponding to mCPSF6 (residues 313 to 327) binds specifically to the N-terminal domain of HIV-1 capsid (12). However, it has remained unclear how the capsid–mCPSF6-358 interaction interferes with the nuclear import of the viral PIC.

Recent studies suggest that proper uncoating is a key step for reverse transcription. (i) In the case of restriction of HIV-1 by rhesus monkey TRIM5α, productive reverse transcription is abrogated by accelerated disassembly of viral capsids (13). (ii) The Vif, Nef, and integrase proteins are essential for optimal stability of the viral core that leads to efficient viral cDNA synthesis in target cells (14–16). (iii) Capsid mutations that impair HIV-1 infection are unable to achieve proper uncoating and reverse transcription (17–20). Overall, these observations suggest that proper uncoating is functionally linked to reverse transcription of HIV-1.

In the present study, we identified a novel C-terminally truncated form of human CPSF6 (hCPSF6) lacking residues encoded by exon 6 (Ex6), hCPSF6-375, by lethal selection of cells resistant to HIV infection following transduction of a human cDNA expression library and focused on its ability to inhibit viral cDNA synthesis in light of the viral capsid disassembly.

MATERIALS AND METHODS

Cells.

HEK293, HeLa, and Plat-E packaging cells (21) were propagated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) and penicillin-streptomycin. MT-4/CCR5 and MOLT-4 cells were maintained in complete RPMI 1640 medium supplemented with 10% FBS and penicillin-streptomycin.

Preparation of virus stocks.

HEK293T cells cultured in a 10-cm dish were cotransfected with 8 μg of pNL4-3luc (env nef mutant) (22) and 2 μg of pHCMV-G (vesicular stomatitis virus G protein [VSV-G]) using FuGENE 6 (Roche Applied Science, Mannheim Germany) according to the manufacturer's instructions. Virus supernatant was harvested at 48 h posttransfection and filtered through a 0.45-μm-pore syringe filter. HIV-1NL4-3 virus stock was prepared by transfection of HeLa cells as previously reported (23). Titers of the virus stocks were quantitated by HIV-1 CA (p24) enzyme-linked immunosorbent assay (ELISA) (ZeptMetrix Corporation, Buffalo, NY) and by determination of the reverse transcriptase (RT) activity (24). For production of retrovirus vectors, Plat-E cells were cotransfected with retrovirus vector plasmids and VSV-G using FuGENE 6 transfection reagent. Culture supernatant of Plat-E cells was collected 60 h after transfection and filtered through a 0.45-μm-pore syringe filter.

Construction of cDNA library and screening.

Construction of the retroviral cDNA library was performed as described previously (25). Briefly, complementary DNAs were synthesized from poly(A) RNA of MT-1 cells with random hexamer primers using the SuperScript Choice system (Invitrogen) according to the manufacturer's instructions. The synthesized cDNAs were inserted between the BstXI sites of pMRX (26) using BstXI adaptors (Invitrogen), generating a retroviral cDNA expression library. HEK293 cells were transduced with VSV-G-pseudotyped retroviruses expressing this cDNA library. The cells were then infected with VSV-G-pseudotyped NL4-3 thymidine kinase (NL4-3-TK) vector (env nef mutant) and subjected to lethal selection with ganciclovir (GCV) (2 μg/ml) to exclude cells infected with the NL4-3-TK vector. After the repeated infection and selection, susceptibility to HIV-1 infection was assessed by infection with NL4-3luc vector and genomic DNA was isolated from surviving cell pools. Inserted cDNAs were amplified by PCR from the genomic DNA, cloned into pCR2.1 (Invitrogen), and sequenced. The cDNA was subcloned into the pMRX-HA-ires-puro vector (27), generating pMRX-HA-hCPSF6-375. Mutated nucleotides were corrected to match the reference sequence (NM_007007.2) by site-directed mutagenesis.

Construction of expression vectors.

An env-defective variant of HIV-1 NL4-3, pNL4-3env(−) carrying a 580-bp deletion between the BglII restriction site in the env gene, was constructed. To generate a herpes simplex virus thymidine kinase (HSV-TK) gene-carrying NL4-3env(−) vector, the last 133 bp of the env gene was amplified from pNL4-3 using Pfu DNA polymerase (Stratagene, La Jolla, CA) with the following primers: 5′-TCTAGAATTCTCGAGTGTTAACTTGCTCAATGCCACAGCCATAGC-3′ and 5′-GATGGCCGGGGTACGAAGCCATCTTATAGCAAAATCCTTTCCA-3′. The HSV-TK gene was amplified from the pIgκ2tkH vector (28) using Pfu DNA polymerase with the following primers: 5′-ATGGCTTCGTACCCCGGCCATCAACACGCGTC-3′ and 5′-CCGCTCGAGTTTCAGTTAGCC-3′. The fragments of the last 133 bp of the env gene and HSV-TK gene were fused by fusion PCR. After confirmation of the nucleotide sequence, fused DNA fragments were extracted after digestion with HpaI and XhoI and inserted into HpaI and XhoI sites of pNL4-3env(−), generating pNL4-3tk. pNL4-3 CA N74D-luc (env and nef negative) was generated by site-directed mutagenesis on pCR2.1 using the NL4-3 gag gene (nucleotides [nt] 689 to 2225) of the HIV-1NL4-3 genome (GenBank accession no. M19921) as the template. Primers NL4-3 CA-N74D-5 (5′-TTAAAAGAGACCATCGATGAGGAAGCTGCAGAA-3′) and NL4-3 CA-N74D-3 (5′-TTCTGCAGCTTCCTCATCGATGGTCTCTTTTAA-3′) were used for N74D substitution. The mutated DNA fragment was inserted to the BssHII and ApaI sites of pNL4-3luc (env nef mutant). The resultant plasmid was referred to as pNL4-3 CA N74D-luc. For construction of murine CPSF6 mutant expression vectors, the cDNAs of murine CPSF6 variants were amplified by PCR using NIH 3T3 cell-derived cDNAs as the template. Primers BglII mCPSF6 F (5′-AAAGATCTGCCACCATGGCGGACGGTGTGGAC-3′) and mCPSF6 321R stop NotI (5′-AATGCGGCCGCCTAGCCTGGAGGTGGAGGTGGTC-3′) were used for amplification of mCPSF6-321 and mCPSF6-358. Primers BglII mCPSF6 F (5′-AAAGATCTGCCACCATGGCGGACGGTGTGGAC-3′) and m6CT 375 R stop NotI (5′-AATGCGGCCGCCTAACTATCTGATGTTGGCATGC-3′) were used for amplification of mCSPF6-375 and mCPSF6-412. The amplified cDNAs were inserted into pCR2.1 and sequenced. The cDNAs were inserted into the BamHI and NotI sites of pMRX-IRES-puro (29). The resultant plasmids were referred to as pMRX-mCPSF6-321, pMRX-mCPSF6-358, pMRX-mCPSF6-375, and pMRX-mCPSF6-412, respectively. For generation of human CPSF6 mutant expression constructs, the cDNAs of hCPSF6-321 and hCPSF6-375 were amplified by PCR using pMRX-HA-hCPSF6-375 as the template. Primers BamHI hCPSF6 F (5′-AAGGATCCGCCACCATGGCGGACGGCGTGGACCAC-3′) and hCPSF6 321R stop NotI (5′-AATGCGGCCGCCTAGCCTGGAGGTGGAGGTGGTC-3′) were used for amplification of hCPSF6-321. Primers BamHI hCPSF6 F (5′-AAGGATCCGCCACCATGGCGGACGGCGTGGACCAC-3′) and hCPSF6 375R stop NotI (5′-AATGCGGCCGCCTAGCTATCTGATGTAGGCATGC-3′) were used for amplification of hCPSF6-375. hCSPF6-358 and hCPSF6-412 were generated by site-directed mutagenesis using mCPSF6-358 and mCPSF6-412 on pCR2.1 as the templates, respectively, because our attempts to amplify the cDNA containing exon 6 in human cells were unsuccessful, and there exist only two amino acid substitutions at residues S153N and G192S in comparison with mCPSF6. Primers mCPSF6-S153N-5 (5′-GAACTTCATGGTCAGAATCCTGTTGTAACTCCA-3′) and mCPSF6-S153N-3 (5′-TGGAGTTACAACAGGATTCTGACCATGAAGTTC-3′) were used for S153N substitution. Primers mCPSF6-G192S-5 (5′-GGTCCTCCAGGAGGCAGTTCACGCGCAGCGTTT-3′) and mCPSF6-G192S-3 (5′-AAACGCTGCGCGTGAACTGCCTCCTGGAGGACC-3′) were used for G192S substitution. Full-length hCPSF6 was amplified by RT-PCR using as the template total RNA isolated from MOLT-4 human T cells by RNeasy minikit (Qiagen, Inc., Valencia, CA). Primers BamHI hCPSF6 F (5′-AAGGATCCGCCACCATGGCGGACGGCGTGGACCAC-3′) and hCPSF6 NotI (5′-AATGCGGCCGCCTAACGATGACGATATTCGCGCTCTC-3′) were used. RT-PCR was performed with the PrimeScript II high-fidelity one-step RT-PCR kit (TaKaRa Bio, Inc., Shiga, Japan). The amplified PCR products were inserted into pCR2.1 and sequenced. The cDNA was inserted into the BamHI and NotI sites of pMRX-ires-puro (29). The resultant plasmids were referred to as pMRX-hCPSF6-321, pMRX-hCPSF6-358, pMRX-hCPSF6-375, pMRX-hCPSF6-412, and pMRX-hCPSF6, respectively.

Single-round infection assay.

For infection of HEK293 cells, 3 × 105 cells were infected with 10 ng (p24) of VSV-G-pseudotyped NL4-3luc (VSV-G/NL4-3luc) (22) in 12-well plates, harvested, and lysed at 20 h postinfection. For infection of MT-4/CCR5 or MOLT-4 cells, 5 × 105 cells were infected with 10 ng (p24) of VSV-G/NL4-3luc in 24-well plates and harvested and lysed at 24 h postinfection. To compare the infectivities between VSV-G/NL4-3luc and VSV-G/NL4-3 CA N74D-luc, we used virus normalized with the RT count equivalent for 10 ng (p24) of VSV-G/NL4-3luc. To determine the luciferase activity, cell lysates were mixed with luciferase substrate and light emission was measured in a GloMAX multidetection system (Promega Corp, Madison, WI).

Fate-of-capsid assay.

The fate-of-capsid assay was performed as previously described (13) with minor modifications. Approximately 5 × 106 HEK293 cells carrying the empty vector or those stably expressing hCPSF6-375, hCPSF6-412, or hemagglutinin-tagged mCPSF6-358 (mCPSF6-358-HA) were preplated in a 10-cm dish 1 day before the assay. Cells were inoculated with 5 × 106 RT counts of VSV-G/NL4-3luc at 4°C for 30 min, incubated at 37°C for 4 or 8 h, and then washed twice with ice-cold phosphate-buffered saline (PBS) containing 0.005% trypsin-EDTA to detach viruses from the cellular surface and once with ice-cold PBS to remove trypsin. Washed cells were resuspended in 1 ml of hypotonic lysis buffer (10 mM Tris-HCl [pH 8.0], 10 mM KCl, 1 mM EDTA, protease inhibitor cocktail [Nacalai Tesque, Inc., Kyoto, Japan]) and incubated on ice for 15 min. Swollen cells were lysed in a 7-ml Dounce homogenizer with a “tight” pestle (15 gentle strokes making a half-turn of pestle per each stroke), and cell lysates were cleared by centrifugation at 2,000 × g for 3 min at 4°C. Fifty microliters of the cleared cell extract was collected as input. Cleared extracts (0.8 ml) were layered over 20% to 60% sucrose cushions prepared in PBS and centrifuged at 4°C at 115,000 × g for 70 min in a Beckman SW50.1 rotor. Three fractions (1.1 ml each) were collected from the top of the gradient. Aliquots of each fraction and input were subsequently subjected to immunoblotting.

Western blotting.

Whole-cell lysates were prepared as follows. Cells were washed twice with PBS, suspended in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate) (250 μl per 5 × 106 cells). The protein concentration was determined by Bradford assay, and the concentration was adjusted to 15 μg/15 μl with RIPA buffer. The lysate was mixed with an equal volume of 2× sample buffer (4% sodium dodecyl sulfate, 125 mM Tris-HCl [pH 6.8], 10% 2-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue). Samples were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and reacted with a rabbit polyclonal antibody to CPSF6 (Abcam, Inc., Cambridge, MA), a mouse monoclonal antibody to HIV-1 p24 (Abcam, Inc., Cambridge, MA), and a rabbit polyclonal antibody to β-actin (Abcam, Inc., Cambridge, MA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ), and proteins were visualized by Western Lightning Plus-ECL (PerkinElmer, Waltham, MA) or enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL).

Quantitation of viral cDNA.

Prior to infection, virus solution was treated with 100 U of RNase-free DNase I (Roche Applied Science, Indianapolis, IN) in the presence of 10 mM MgCl2 for 30 min at 37°C. For infection of HEK293 cells, 7 × 105 target cells were incubated for 6, 12, or 24 h with VSV-G/NL4-3luc virus supernatant containing 23 ng of p24. For infection of MT-4/CCR5 or MOLT-4 cells, 5 × 105 cells were incubated for 4, 8, or 24 h with an NL4-3 virus supernatant containing 50 ng of p24. Total cellular DNA was extracted using the DNeasy tissue kit (Qiagen, Inc., Valencia, CA). HIV-1 inactivated by incubation at 65°C for 30 min was used as a negative control. In real-time PCR, the primers U5-gag/F2 (5′-GTAGTGTGTGCCCGTCTGTTG-3′) (nt 553 to 573) and U5-gag/R2 (5′-CAAGCCGAGTCCTGCGT-3′) (nt 689 to 705) and probe U5-gag/probe2 (5′-FAM-TGGCGCCCGAACAGGGACTT-TAMRA-3′ [where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine]) (nt 636 to 655) were used for amplification and quantitation of the U5-gag region of the HIV-1 late reverse transcription product (30). For standardization, the β-globin mRNA was quantitated as previously described (30). Real-time PCR was carried out with the StepOnePlus real-time PCR system (Applied Biosystems, Carlsbad, CA). The ratios of viral cDNA level to β-globin cDNA level are shown.

Analysis of HIV-1 replication in human T cells.

MT-4/CCR5 cells (1 × 105) were exposed to HIV-1 supernatant containing 100 pg of p24. Virus production was monitored for 14 days postinfection by RT activity in the culture supernatants.

Establishment of HEK293, MOLT-4, and MT-4/CCR5 cells stably expressing the hCPSF6 mutant.

HEK293, MOLT-4, and MT-4/CCR5 cells were transduced with retrovirus vector that confer puromycin resistance and express the hCPSF6 mutant. HEK293, MOLT-4, and MT-4/CCR5 cells stably expressing the hCPSF6 mutant were obtained after selection with 4 μg/ml puromycin for HEK293 and 2 μg/ml puromycin for MOLT-4 and MT-4/CCR5, respectively.

Depletion of hCPSF6 in HEK293 cells.

HEK293 cells were transduced with HIV-1-based vectors that confer puromycin resistance and express either nontargeting short hairpin RNA (shRNA) or those targeting hCPSF6 (Sigma-Aldrich, Co., St. Louis, MO). Pools of HEK293 cells expressing shRNAs were established after selection with puromycin (4 μg/ml).

RESULTS

C-terminally truncated hCPSF6 identified in a functional screen.

To identify proteins that interfere with HIV-1 infection by a functional screen using a cDNA expression library, we employed lethal selection with the herpes simplex virus thymidine kinase (HSV-TK) gene to exclude HIV-1-infected cells (Fig. 1A). HSV-TK is a typical suicide protein (31) that, in concert with cellular kinases, specifically converts the prodrug ganciclovir (GCV) into highly toxic GCV triphosphate, which causes DNA polymerase chain termination and eventual cell death. Previous reports showed that HSV-TK gene-carrying HIV-1 replication is inhibited by selective suicide of HIV-1-infected cells in the presence of GCV and is highly efficient in controlling spreading infection of HIV-1 in tissue culture (32). Thus, we generated HSV-TK gene-carrying HIV-1 by the insertion of the HSV-TK gene in pHIV-1NL4-3-env(−) in place of the nef gene. Recombinant HIV-1 expressing HSV-TK and pseudotyped with the vesicular stomatitis virus G glycoprotein (denoted by HIV-1-TK) can efficiently infect cells of many mammalian species, including humans. We used a murine leukemia virus vector to express a human cDNA library prepared from human T-cell line MT-1 in HEK293 cells, which are highly susceptible to HIV-1-TK infection. After several rounds of HIV-1-TK infection at a high multiplicity of infection and subsequent GCV selection, we obtained and characterized surviving cell clones in terms of susceptibility to HIV infection, among which one clone was found to express a carboxy-terminally truncated fragment of human cleavage and polyadenylation factor 6 (hCPSF6-375).

Fig 1.

Fig 1

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C-terminally truncated hCPSF6 identified in a functional screen inhibits virus cDNA synthesis. (A) HEK293 cells were transduced with VSV-G-pseudotyped retrovirus vector expressing a human T-cell-derived cDNA library. After a few passages, the cells were infected with high-titer HIV-1 capable of expressing HSV-TK (VSV-G/NL4-3TK) and subjected to lethal selection with ganciclovir (GCV) to eliminate infected cells. After repeated infection and selection, inserted cDNAs isolated from ganciclovir-resistant cells were amplified by PCR. The cDNAs were subcloned again in a retrovirus vector and expressed in HEK293 cells to verify the property as anti-HIV factor. (B) Immunoblot analysis of CPSF6 mutants. Lysates of HEK293 cells (293), control vector-infected HEK293 cells (EV control), and cells stably expressing hCPSF6-375 or mCPSF6-358-HA were subjected to immunoblot analysis with anti-CPSF6 or anti-β-actin antibodies. (C) Effect of CPSF6 mutants on viral infectivity. HEK293 cells stably expressing CPSF6 mutants were infected with VSV-G/NL4-3luc. Luciferase activity was measured 20 h after infection. The mean luciferase values in three independent experiments are shown. (D) Amount of viral cDNA synthesized after VSV-G/NL4-3luc infection. Total DNA was isolated from a portion of the cells 6 h after infection. Viral cDNA synthesis was quantified by real-time PCR as described in Materials and Methods. The mean value obtained from EV control cells was arbitrarily set as 100%. Mean values and standard deviations from three independent experiments are shown. (E) Localization of CPSF6 mutants in HEK293 cells. Cell lysates were prepared and separated by the Qproteome cell compartment kit (Qiagen, Inc., Valencia, CA). Fractions were subjected to immunoblot analysis with anti-CPSF6 antibody, anti-GAPDH antibody for the cytoplasmic fraction, antivimentin antibody for the cytoskeletal fraction, and anti-lamin A/C antibody for the nuclear fraction. (F) Effects of CPSF6 mutants on infection with HIV-1 carrying the N74D capsid mutation. HEK293 cells stably expressing CPSF6 mutants were infected with VSV-G/NL4-3 CA N74D-luc. Luciferase activity was measured 20 h after infection. The mean luciferase value from EV control cells was arbitrarily set as 100%.

We investigated if hCPSF6-375 affects HIV-1 infection in a manner similar to mCPSF6-358, which interferes with nuclear import of HIV. We established HEK293 cell pools stably expressing hCPSF6-375 or C-terminally hemagglutinin (HA)-tagged mCPSF6-358 (mCPSF6-358-HA), the latter of which was studied in the previous study (7). hCPSF6-375 was expressed at a level comparable to that of mCPSF6-358-HA, as verified by immunoblotting with an antibody to CPSF6 (Fig. 1B). As shown previously (7), mCPSF6-358-HA strongly inhibited infection with the single-round VSV-G-pseudotyped luciferase-carrying HIV-1 vector (VSV-G/NL4-3luc) (Fig. 1C). hCPSF6-375 also inhibited infection with VSV-G/NL4-3luc to a similar extent (Fig. 1C). Inhibition of HIV-1 infection was evident in HEK293 cells expressing hCPSF6-375 or mCPSF6-358-HA over a wide range of virus amounts (1 to 100 ng) in the HIV-1 p24 antigen (Fig. 1C).

To investigate the mechanism of inhibition, we measured the amount of viral cDNA synthesized after infection of target cells stably expressing either hCPSF6-375 or mCPSF6-358-HA (Fig. 1D). As reported previously (7), quantitative PCR revealed that the amount of the late reverse transcription product was not reduced in mCPSF6-358-HA-expressing cells (Fig. 1D). Interestingly, stable expression of hCPSF6-375 remarkably suppressed viral cDNA synthesis after HIV-1 infection (Fig. 1D). These results suggest that hCPSF6-375 and mCPSF6-358-HA have an inhibitory effect on HIV-1 infection, but have different modes of action at an early stage of HIV-1 infection. To further investigate whether the difference between hCPSF6-375 and mCPSF6-358-HA is due to their cellular localization or not, we fractionated cellular proteins into different compartments. As shown in Fig. 1E, hCPSF6-375 and mCPSF6-358-HA were predominantly localized in the cytoplasm, suggesting that the different actions of hCPSF6-375 and mCPSF6-358-HA on HIV-1 depend on their functional properties.

A previous report showed that the N74D HIV-1 capsid mutant is resistant to inhibition by mCPSF6-358-HA (7). We therefore examined if hCPSF6-375 inhibits N74D HIV-1 infection and found that hCPSF6-375 did not significantly affect infection with N74D HIV-1 (Fig. 1F). Collectively, hCPSF6-375 and mCPSF6-358-HA appear to work against HIV-1 infection in the early phase of infection, though hCPSF6-375 inhibits viral cDNA synthesis.

To investigate if endogenous hCPSF6 is involved in HIV-1 infection, we depleted hCPSF6 in HEK293 cells (hCPSF6-KD). Lentivirus-mediated expression of an hCPSF6-specific shRNA, shRNA 2, successfully suppressed hCPSF6 expression (Fig. 2A). Depletion of hCPSF6 did not significantly alter the infectivity of VSV-G-pseudotyped NL4-3luc, suggesting that hCPSF6 is not a cofactor required for HIV-1 infection (Fig. 2B, compare the lanes containing nontarget shRNA and shRNA 2). We next investigated if elevated expression of full-length human CPSF6 impairs HIV-1 infection. As shown in Fig. 2C and D, stable expression of ectopic human CPSF6 had little effect on HIV-1 infection, suggesting that full-length CPSF6 by itself does not work as a restriction factor on HIV-1.

Fig 2.

Fig 2

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Involvement of full-length hCPSF6 in HIV-1 infection. (A) Immunoblot analysis of CPSF6 expression. Approximately 30 μg of whole-cell lysates from HEK293 cells stably expressing the indicated shRNAs was subjected to immunoblot analyses with anti-CPSF6 or anti-β-actin antibodies. (B) Effect of CPSF6 depletion on viral infectivity. HEK293 cells stably expressing the indicated shRNAs were infected with VSV-G/NL4-3luc. Luciferase activity was measured 20 h after infection. The mean luciferase value from control cells (nontarget shRNA) was arbitrarily set as 100%. (C) Immunoblot analysis of CPSF6 expression. Approximately 30 μg of whole-cell lysates from HEK293 control cells and those stably expressing full-length CPSF6 was subjected to immunoblot analyses with anti-CPSF6 or anti-β-actin antibodies. (D) Effect of full-length CPSF6 on viral infectivity. Experiments were done as described in panel B. The mean luciferase value from EV control cells was arbitrarily set as 100%.

Lack of exon 6-encoded polypeptide in hCPSF6-375 is responsible for impaired viral cDNA synthesis.

To explore the determinant responsible for viral cDNA synthesis inhibition, we compared amino acid sequences of hCPSF6-375 and mCPSF6-358 (Fig. 3A). We found in hCPSF6-375 two amino acid substitutions at residues N153S and S192G, a deletion of amino acids encoded by exon 6 (residues 232 to 268 in mouse CPSF6 and denoted by “Ex6”), and addition of the C-terminal 54 residues (Fig. 3A). We therefore generated two mCPSF6-based mutants with humanized amino acid substitutions S153N and G192S in mCPSF6-358 (hCPSF6-358) and in mCPSF6-412 (hCPSF6-412) (Fig. 3B). We also generated hCPSF6-321 based on hCPSF6-375 (Fig. 3B). Each mutant was assayed for stable expression in HEK293 cells after retrovirus vector transduction and antibiotic selection.

Fig 3.

Fig 3

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Effects of Ex6 deletion on HIV-1 infection. (A) Amino acid alignments of hCPSF6-375 and mCPSF6-358. Dots indicate sequence identity, and dashes indicate gap introduction to preserve alignment. Amino acids in the reticulate area indicate the region of exon 6. (B) Schematic presentation of hCPSF6 mutants. “Ex6” in the reticulate area indicates the domain encoded by exon 6, and “PRD” represents the proline-rich domain. (C) Immunoblot analysis of HEK293 cells expressing truncated forms of hCPSF6. Approximately 30 μg of whole-cell lysates was subjected to immunoblot analyses using anti-CPSF6 or anti-β-actin antibodies. (D) Effect of hCPSF6 mutants on viral infectivity. HEK293 cells stably expressing hCPSF6 mutants were infected with VSV-G/NL4-3luc. Luciferase activity was measured 20 h after infection. The mean luciferase value from EV control cells was arbitrarily set as 100%. (E) Kinetics of viral cDNA synthesis in vivo. Total DNA was isolated from a portion of the cells at the indicated times after infection. Viral cDNA synthesis was quantified by real-time PCR as described in Materials and Methods. Mean values in three independent experiments are shown. (F) Immunoblot analysis of mouse CPSF6 mutants expressed in HEK293 cells. Experiments were done as described in panel C. (G) Effect of mCPSF6 mutants on viral infectivity. Experiments were done as described in panel D. (H) Kinetics of viral cDNA synthesis in vivo. Experiments were done as described in panel E.

We first investigated a possible effect of two amino acid substitutions between hCPSF6-358 and mCPSF6-358 on the inhibition of HIV-1 infection. hCPSF6-358 was expressed at a level comparable to that of mCPSF6-358 (Fig. 3C, compare hCPSF6-358 and mCPSF6-358). Infection efficiency was determined by either a standard luciferase assay (Fig. 3D) or by quantitative PCR (Fig. 3E). Untagged mCPSF6-358 as well as mCPSF6-358-HA strongly inhibited infection with VSV-G/NL4-3luc despite lower expression of untagged mCPSF6-358 (Fig. 3C and D, compare mCPSF6-358 and mCPSF6-358-HA). hCPSF6-358 also inhibited infection by VSV-G/NL4-3luc (Fig. 3D, hCPSF6-358). Like mCPSF6-358-HA, the amount of viral cDNA synthesized after HIV-1 infection of hCPSF6-358-expressing cells was not reduced (Fig. 3E, hCPSF6-358). Moreover, untagged and C-terminally HA-tagged mCPSF6-358 had little effect on viral cDNA synthesis after HIV-1 infection (Fig. 3E, compare mCPSF6-358 and mCPSF6-358-HA). These results suggest that the C-terminal HA tag or two-amino-acid difference between hCPSF6-358 and mCPSF6-358 does not apparently alter the inhibitory property of mCPSF6-358-HA. Furthermore, we also generated mouse CPSF6-375 to investigate possible effects of the two amino acid substitutions between hCPSF6-375 and mCPSF6-375 on the inhibition of HIV-1 infection. As shown in Fig. 3F to H, the effects of mCPSF6-375 on HIV-1 inhibition were similar to those of hCPSF6-375, indicating that the two amino acid substitutions do not significantly alter the inhibitory properties, even in the context of CPSF6-375 (Fig. 3G and H, compare hCPSF6-375 and mCPSF6-375).

To assess the contribution of Ex6 in hCPSF6-358 to the inhibition of HIV-1 infection, we used Ex6-deficient hCPSF6-321. hCPSF6-321 was expressed at a level comparable to that of hCPSF6-358, and the HIV-1 infectivity in hCPSF6-321-expressing cells determined by luciferase assay was comparable to that hCPSF6-358-expressing cells (Fig. 3C and D, compare hCPSF6-321 and hCPSF6-358). Importantly, viral cDNA synthesis after HIV-1 infection in hCPSF6-321-expressing cells was dramatically suppressed in comparison with that in hCPSF6-358-expressing cells (Fig. 3E, compare hCPSF6-321 and hCPSF6-358). On the other hand, addition of Ex6 to hCPSF6-375 (hCPSF6-412) substantially attenuated the inhibition of viral DNA synthesis (Fig. 3E, compare hCPSF6-375 and hCPSF6-412). These results suggest that CPSF6 mutants with Ex6 do not inhibit viral cDNA synthesis.

We also assessed the contribution of the C-terminal 54 residues of hCPSF6-375, which are absent from hCPSF6-321, to the inhibition of HIV-1 infection. hCPSF6-321, when expressed at a level comparable to that of hCPSF6-375, achieved similar inhibition of VSV-G/NL4-3luc infection (Fig. 3C and D, compare hCPSF6-321 and hCPSF6-375). The fact that hCPSF6-321 potently suppresses viral cDNA synthesis also suggests that the C-terminal 54 residues in hCPSF6-375 are not essentially required for viral cDNA synthesis inhibition (Fig. 3E, compare hCPSF6-321 and hCPSF6-375). Collectively, these results indicate that the absence of Ex6, but not the C-terminal 54 residues in hCPSF6-375, is responsible for viral cDNA synthesis inhibition.

CPSF6-375 alters the kinetics of HIV-1 capsid disassembly.

Previous reports showed that optimal dissociation of capsid from the HIV-1 core is required for efficient viral cDNA synthesis in target cells (17–19). To investigate the effect of hCPSF6-375 on the stability of the HIV-1 core after HIV-1 entry, we performed the fate-of-the-capsid assay as described previously (13). Previous studies showed that step gradient analyses could distinguish soluble from core-associated HIV-1 proteins (33–36). To clearly separate HIV-1 cores from the soluble capsid protein after HIV-1 entry into target cells, we employed 20% and 60% sucrose cushions for ultracentrifugation, where the HIV-1 core is expected to be enriched at the border of 20% and 60% cushions. Our modified assay is summarized in Fig. 4A. HIV-1 soluble capsid partitions to fraction a, while the integrated HIV-1 core accumulates in fraction c. A buffer fraction (Fig. 4A, fraction b) was set to separate the fractions a and c (Fig. 4, fractions a and c). This assay was applied to examine the fate of the HIV-1 capsid after infection of HEK293 cells expressing hCPSF6-375 or mCPSF6-375-HA and control cells transduced with the empty retrovirus vector (EV). We also used NL4-3 virions without any envelope protein (Env−) as the HIV-1 entry control (13). The virions were allowed to attach to the target cells for 30 min at 4°C, and then the temperature was shifted to 37°C to initiate infection. At 4 or 8 h after temperature shifting, the cells were lysed and the cell lysates analyzed by step gradient centrifugation. The amounts of p24 capsid protein in the cell lysate were similar in hCPSF6-375, mCPSF6-358-HA, and EV control cells (Fig. 4B, input), and p24 capsid protein was not detected in cell lysate derived from EV control cells incubated with the HIV-1 (Env−) virions, indicating that this assay can correctly measure HIV-1 capsid protein in the cytosol of target cells after HIV-1 infection (Fig. 4B, compare EV-Env− and EV-Env+).

Fig 4.

Fig 4

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Deletion of Ex6 facilitates HIV-1 capsid disassembly. (A) Schematic diagram of the fate-of-capsid assay. HEK293 cells expressing CPSF6 mutants or the empty control vector (EV) were incubated with VSV-G/NL4-3luc or NL4-3luc (Env−) at 4°C for 30 min and then at 37°C for the indicated times. Cell lysates were prepared and analyzed on the step gradient (20%/60%) of sucrose as described in Materials and Methods. (B) Aliquots of each fraction were processed for immunoblotting with anti-HIV-1 p24 antibodies. Representative results of a single experiment are shown. The accumulation of viral protein in each fraction was quantified by HIV-1 CA (p24) enzyme-linked immunosorbent assay, and mean values and standard deviations from three independent experiments are shown. (C) Graphic presentation of p24 capsid protein levels measured by p24 ELISA in the whole-cell lysate (left panel, input) or the viral core fraction (right panel, fraction c) at the indicated times shown in panel B. Mean values from three independent experiments are shown.

Immunoblot analyses revealed that the amount of HIV-1 cores in HIV-1-infected cells expressing hCPSF6-375 was smaller than those detected in mCPSF6-358-HA or control EV cells at 4 (Fig. 4B, upper panel) and 8 h (Fig. 4B, lower panel) post-temperature shifting (Fig. 4B, fraction c, compare hCPSF6-375 and mCPSF6-358-HA). This was further confirmed quantitatively by p24 ELISA of each fraction (Fig. 4B). The levels of p24 capsid protein in the cell lysates from control cells or those expressing CPSF6 mutants were comparable (Fig. 4C, input). In contrast, the amount of particulate HIV-1 capsid in cells expressing hCPSF6-375 was obviously decreased at all time points examined (Fig. 4C, fraction c). No p24 capsid protein was detected in fractions lower than fraction c or pellets prepared from cells exposed to VSV-G/NL4-3luc or NL4-3luc (Env−) virions, indicating that the particulate HIV-1 capsid accumulates only in fraction c (data not shown). These data indicate that the presence of hCPSF6-375 in target cells accelerates HIV-1 capsid disassembly in newly infected cells.

CPSF6-375 inhibits HIV-1 replication in human T cells.

To see if the inhibitory effects of hCPSF6-375 on HIV-1 infection are cell type specific or not limited to HEK293 cells, similar experiments were performed with MOLT-4 human T-cell lines. We made use of MOLT-4 cells expressing hCPSF6-321, -358, -375, or -412 or mCPSF6-358-HA and control cells transduced with EV. The presence of comparable amounts of hCPSF6 mutants except hCPSF6-358 was verified by immunoblotting using a CPSF6-specific antibody (Fig. 5A). mCPSF6-358-HA was expressed at a level comparable to that of hCPSF6-375 (Fig. 5A, compare hCPSF6-375 and mCPSF6-358-HA). The effect of hCPSF6 mutants on the infectivity of MOLT-4 cells was analyzed in a single-round infectivity assay with VSV-G/NL4-3luc. As expected, hCPSF6-375 strongly inhibited HIV-1 infection (Fig. 5B, compare EV and hCPSF6-375). Similarly, mCPSF6-358-HA as well as hCPSF6-358 also inhibited infection with VSV-G/NL4-3luc (Fig. 5B, compare hCPSF6-358 and mCPSF6-358-HA).

Fig 5.

Fig 5

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Effects of Ex6 deletion on HIV-1 replication in human T cells. (A) Immunoblot analysis of CPSF6 mutants expressed in MOLT-4 human T cells. Approximately 30 μg of whole-cell lysates was subjected to immunoblot analyses with anti-CPSF6 or anti-β-actin antibodies. (B) Effect of CPSF6 mutants on viral infectivity. MOLT-4 cells stably expressing CPSF6 mutants were infected with VSV-G/NL4-3luc. Luciferase activity was measured 24 h after infection. The mean luciferase value from EV control cells was arbitrarily set as 100%. (C) Kinetics of viral cDNA synthesis in vivo. Total DNA was isolated from a portion of the cells at the indicated times after infection. Viral cDNA synthesis was quantified by real-time PCR as described in Materials and Methods. Mean values in three independent experiments are shown. (D) Immunoblot analysis of hCPSF6-375 and mCPSF6-358-HA expressed in MT-4/CCR5 human T cells. Experiments were done as described in panel A. (E) Effect of CPSF6 mutants on viral infectivity in MT-4/CCR5 cells. Experiments were done as described in panel B. (F) CPSF6 mutants suppress spreading infection of HIV-1. Virus production in parental, EV control, hCPSF6-375-expressing, and mCPSF6-358-HA-expressing MT-4/CCR5 cells was monitored for 14 days by measuring the virus-associated reverse transcriptase activity in the culture supernatants. Mean values and standard deviations from three independent experiments are shown.

How each mutant affects viral cDNA synthesis was determined by measuring the amount of the late reverse transcription product in target cells stably expressing hCPSF6-375 mutants or mCPSF6-358-HA after HIV-1 infection (Fig. 5C). Similar results shown in Fig. 3E were obtained by quantitative PCR indicating that hCPSF6-375 but not mCPSF6-358-HA inhibited viral cDNA synthesis after HIV-1 infection, suggesting that the inhibitory effect of hCPSF6-375 on HIV-1 DNA synthesis is not cell type specific (Fig. 5C, compare hCPSF6-375 and mCPSF6-358-HA).

A recent study showed that mCPSF6-358 inhibits spreading infection with HIV-1 in a human T-lymphoid cell line (7). To further investigate the effect of hCPSF6-375 on spreading infection with HIV-1 in human T cells, we established MT-4/CCR5 human T-cell lines stably expressing hCPSF6-375 and mCPSF6-358-HA (Fig. 5D). The effect of hCPSF6-375 on the infectivity of MT-4/CCR5 cells was first analyzed in a single-round infectivity assay. Consistent with the results shown in Fig. 3D and 5B, both hCPSF6-375 and mCPSF6-358-HA strongly inhibited infection with VSV-G/NL4-3luc (Fig. 5E). These results provide additional evidence that the inhibitory effect of hCPSF6-375 on HIV-1 infection is not cell type specific. We next investigated the effect of hCPSF6-375 on HIV-1 replication in MT-4/CCR5 T cells with the replication-competent NL4-3 virus. Control EV cells well supported HIV-1 replication, with a peak of RT activity on day 6, while HIV-1 replication was inhibited by mCPSF6-358-HA, as reported previously (7). hCPSF6-375 suppressed HIV-1 replication as potently as mCPSF6-358-HA in MT-4/CCR5 T cells (Fig. 5F).

DISCUSSION

In this study, we established an expression cloning system coupled with selective killing of HIV-1-infected cells to isolate anti-HIV-1 factors and obtained a C-terminally truncated CPSF6 protein, hCPSF6-375, which inhibited HIV-1 at an early stage of infection. Lee et al. previously screened a mouse cDNA library and identified mCPSF6-358, showing that it did not affect reverse transcription but inhibited HIV-1 trafficking to the nucleus (7). A notable difference from mCPSF6-358 is the absence of Ex6 in hCPSF6-375.

Our results revealed that both hCPSF6-375 and mCPSF6-358-HA inhibited infection by the single-round VSV-G/NL4-3luc infection (Fig. 1C). One remarkable finding of the present study is that hCPSF6-375 inhibited HIV-1 infection at the reverse transcription stage, suggesting a different mode of action in the early phase of HIV-1 infection in comparison with mCPSF6-358-HA (Fig. 1D). The localization of hCPSF6-375 as well as mCPSF6-358-HA was predominantly cytoplasmic (Fig. 1E), while endogenous hCPSF6 is normally enriched in the nucleus (37), and the different actions of hCPSF6-375 and mCPSF6-358-HA on HIV-1 cannot be explained by the protein expression levels (Fig. 1B, compare hCPSF6-375 and mCPSF6-358-HA). Consistent with the previous report (7), elevated expression of full-length human CPSF6 and depletion of endogenous hCPSF6 had little effect on HIV-1 infection in our present study (Fig. 2), suggesting unique anti-HIV-1 properties of these truncated proteins. We initially attempted to compare hCPSF6-375 and untagged mCPSF6-358 in terms of HIV-1 restriction, but the stable expression level of mCPSF6-358 was much lower than that of hCPSF6-375 (Fig. 3C, compare hCPSF6-375 and mCPSF6-358). We therefore investigated the effect of mCPSF6-358 on HIV-1 infection in the presence or absence of HA tag (Fig. 3D and E). Both untagged and HA-tagged mCPSF6-358 inhibited HIV-1 infection, as determined with VSV-G/NL4-3luc, despite lower expression of untagged mCPSF6-358 (Fig. 3C and D, compare mCPSF6-358 and mCPSF6-358-HA). Untagged and C-terminally HA-tagged mCPSF6-358 had little effect on viral cDNA synthesis after HIV-1 infection (Fig. 3E, compare mCPSF6-358 and mCPSF6-358-HA). On the basis of the results shown in Fig. 3C to E, we decided to use C-terminally HA-tagged mCPSF6 (mCPSF6-358-HA) to compare the anti-HIV-1 effects with those of hCPSF6-375. A previous study showed that N74D capsid mutation in HIV-1 led to HIV-1 resistance to mCPSF6-358 (7). We found that the N74D mutant is also resistant to hCPSF6-375, suggesting that hCPSF6-375 inhibition of HIV-1 also relies on the capsid sequence.

We have provided evidence that lack of Ex6 in C-terminally truncated forms of hCPSF6 is responsible for the inhibition of viral cDNA synthesis after HIV-1 infection (Fig. 1D, 3E, and 5C). Lee et al. showed that mCPSF6-358 residues 301 to 358 are sufficient for inhibition of HIV-1 and minimal residues 313 to 327 contribute to anti-HIV-1 activity (11). The residues 313 to 327 are located downstream of Ex6 in CPSF6-358 and conserved in all hCPSF6 mutants used in this study. hCPSF6-321, the Ex6-deficient form of hCPSF6-358 inhibited HIV-1 infection at a level comparable to that of hCPSF6-358 in a single-round infectivity assay with VSV-G/NL4-3luc (Fig. 3D and 5B, compare hCPSF6-321 and hCPSF6-358). Similar results were obtained when hCPSF6-375 was compared with hCPSF6-412 (Fig. 3D and 5B, compare hCPSF6-375 and hCPSF6-412). These results indicate that Ex6 of CPSF6 is not essentially involved in HIV-1 inhibition.

We tried the fate-of-capsid assay with minor modifications, and our observation that the amount of HIV-1 cores in hCPSF6-375-expressing cells was lower than those detected in control, mCPSF6-358-HA, and hCPSF6-412 cells implies that the presence of hCPSF6-375 in target cells accelerated disassembly of HIV-1 capsid (Fig. 4B and C). A previous report showed that mCPSF6-358 could bind to tubular complexes composed of the HIV-1 capsid protein, suggesting physical interaction between particulate capsid and mCPSF6-358 (7). It is therefore conceivable that mCPSF6-358 binds to particulate capsid after HIV-1 infection, but does not alter the disassembly kinetics of capsid, having no detectable effect on viral cDNA synthesis. Our findings that hCPSF6-375 blocks HIV-1 infection by impairing both the viral cDNA synthesis and optimal stability of the HIV-1 core in target cells are reminiscent of the activity of restriction factors such as TRIM5α (13, 38). A previous study showed that a peptide corresponding to CPSF6 residues 313 to 327 was sufficient for direct binding to HIV-1 capsid (12). Since the CPSF6 residues 313 to 327 are conserved in all hCPSF6-375 mutants mentioned above, it is plausible that the polypeptide encoded by exon 6 of human CPSF6 influences the mode of interaction between HIV-1 capsid and hCPSF6, eventually giving a different outcome. The precise mechanism of the rapid disassembly of HIV-1 capsid in the presence of hCPSF6-375 in target cells remains to be further investigated.

In conclusion, the present study has clearly demonstrated that C-terminally truncated forms of CPSF6, when deleted with exon 6, promote capsid disassembly and interfere with viral reverse transcription, although the mechanism of actions remain to be determined. Elucidation of the mechanism(s) of inhibition of HIV-1 by hCPSF6 mutants could provide new insights for understanding viral cDNA synthesis in light of viral capsid disassembly.

ACKNOWLEDGMENTS

We thank I. S. Chen for providing pNL4-3luc (env nef mutant).

This work was supported by grant 21390136 from the Ministry of Education, Culture, Sports, Science, and Technology to S.Y. and by grant H22-AIDS-007 for Young Scientists of HIV/AIDS Research from the Ministry of Health, Labor, and Welfare of Japan to H.T.

Footnotes

Published ahead of print 8 May 2013

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