Inhibition of HIV-1 replication by eIF3f INAUGURAL ARTICLE Susana T. Valentea,1, Greg M. Gilmartinb, Christina Motta, Brie Falkarda, and Stephen P. Goffa,1

aDepartment of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, College of Physicians and Surgeons, Columbia University, HHSC 1310c, 701 West 168th Street, New York, NY 10032; and bDepartment of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, Vermont Cancer Center, University of Vermont, Burlington, VT 05405

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2006.

Contributed by Stephen P. Goff, January 21, 2009 (sent for review December 19, 2008)

Viruses often use host machinery in unusual ways to execute differ- of at least 13 nonidentical subunits (8, 9). The active core includes ent steps during their replication. To identify host factors critical for 5 subunits (eIF3a, -b, -c, -g, -i), with the remaining subunits serving virus replication, we screened cDNA expression libraries for or to modulate eIF3 activity (10). eIF3f is a member of the Mov34 fragments that could interfere with HIV-1 vector transduction. family, which are involved in regulation of the proteasome, trans- The DNA clone that most potently inhibited HIV-1 expression encoded lational initiation, pre-mRNA splicing, and transcription (8, 11, 12). the N-terminal 91 aa of the eukaryotic 3 subunit f They share a conserved (Ϸ140-aa) domain named MPN (first (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drasti- observed at the N terminus of the yeast Mpr1 and Pad1p, and hence cally restricted HIV-1 replication by reducing nuclear and cytoplasmic termed ‘‘MPN domain’’). Given these diverse functions, it is not viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3؅ long unexpected that eIF3f might have other roles besides its function in terminal repeat (3؅LTR) region in the viral mRNA. We show that the initiation, and because it is also found in the nucleus it .3؅ end cleavage of HIV-1 mRNA precursors is specifically reduced in is plausible that it might have functions in RNA metabolism N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA We found that expression of N91-eIF3f or full-length eIF3f maturation and that it can specifically interfere with the 3؅ end severely restricted the replication of HIV-1, by specifically targeting processing of HIV-1 mRNAs. the 3Ј end of the viral mRNA. Proviruses were formed normally upon infection but viral mRNA levels thereafter were reduced in cDNA genetic screen ͉ HIV-1 restriction ͉ RNA maturation ͉ the nucleus and cytoplasm. We found that cleavage, rather than translation factor polyadenylation, at the 3Ј end of HIV mRNAs is reduced in the presence of N91-eIF3f in vitro. Additionally, in the absence of he integrated proviral DNA of HIV-1 functions as a single endogenous eIF3f we found that the N91-eIF3f fragment is unable expression unit with a transcriptional promoter in the 5Ј long to restrict the virus. Hence, we have identified an unanticipated role T Ј terminal repeat (LTR) and signals for 3Ј end processing in the for eIF3f in modulating HIV RNA 3 end processing. 3ЈLTR. The 3Ј ends of higher eukaryotic mRNAs are produced by Results a tightly coupled endonucleolytic cleavage and polyadenylation reaction. Polyadenylation regulates subsequent steps in RNA pro- Human cDNA Screen for Genes or Gene Fragments Able to Restrict cessing such as stability, nuclear export and translatability (for Genetically Marked Pseudotyped HIV Particles. In ref. 13, we de- review, see ref. 1). In mammalian cells 3 elements define the core scribed a screen for cDNAs expressing or portions of polyadenylation signal: a highly conserved hexanucleotide proteins capable of restricting HIV replication. Briefly, we con- AAUAAA found 10–30 nt upstream of the cleavage site; a less well structed a cDNA expression library from HeLa cells using an conserved U-rich or GU-rich element located downstream of the MLV-based vector, pBabe-HAZ (14). The cDNAs of HeLa cells cleavage site; and the sequence at the cleavage site itself (CA), stimulated by IFN were expressed as N-terminal fusion proteins which becomes the point of poly(A) addition and is thus referred with the zeocin (zeo) resistance marker. The retroviral vector Ј to as the poly(A) addition site (2, 3). The efficiency of RNA contains a LoxP site in the 3 LTR, which after reverse transcrip- processing correlates with the binding of polyadenylation factors to tion, is duplicated onto both LTRs such that the provirus can be these elements (2). excised from the genome by the Cre recombinase. The cDNA HIV-1 polyadenylation at the 3Ј end of its viral RNAs, and 3Ј library was packaged into virus-like particles (VLPs) by transfection processing of both spliced and unspliced RNAs, might be expected of 293T cells with DNAs encoding the cDNA library, the murine to involve a unique manipulation of host cleavage and polyadenyl- leukemia virus Gag-Pol proteins, and the pantropic envelope of the ation machineries. The HIV retroviral genome contains a repeat vesicular stomatitis virus (VSV-G). The recovered virus was used region (R) that forms both the extreme 5Ј and 3Ј ends. These R to infect TE671 cells, a human rhabdomyosarcoma cell line highly regions contain several controlling functional motifs including the susceptible to HIV infection. The transduced TE671 cells were poly(A) site, which is part of a highly conserved hairpin structure selected for zeocin resistance, ensuring expression of at least 1 in various HIV and SIV isolates (4). Several mechanisms have been member of the cDNA library. To isolate rare HIV-resistant clones, proposed to regulate utilization of the HIV 5Ј and 3Ј poly(A) pools of transduced TE671 cells were repeatedly infected at low signals. Studies have shown in vitro and in vivo that an upstream multiplicity with a pseudotyped retrovirus preparation expressing stimulatory element (USE), located in the U3 region present only the herpes simplex virus (HSV) thymidine kinase (TK) gene. The ϩ on the 3Ј terminus of the viral RNA, is essential for efficient virus-susceptible cells, having become HSV-TK , were then elim- polyadenylation (5, 6). We performed a genetic screen of a human cDNA library for Author contributions: S.T.V. and S.P.G. designed research; S.T.V., G.M.G., C.M., and B.F. factors disruptive of fundamental molecular interactions that are performed research; S.T.V., G.M.G., and S.P.G. contributed new reagents/analytic tools; critical for HIV replication. Our most potent clone encoded the S.T.V., G.M.G., and S.P.G. analyzed data; and S.T.V. and S.P.G. wrote the paper. N-terminal 91 residues of the eukaryotic initiation factor 3 subunit The authors declare no conflict of interest. f (N91-eIF3f). eIF3f is a subunit of the multisubunit eIF3 transla- Freely available online through the PNAS open access option. tion factor. Within the cytoplasm, eIF3f functions as a component 1To whom correspondence may be addressed. E-mail: [email protected] or spg1@ of the multisubunit eIF3 translation factor that promotes binding of columbia.edu.

eukaryotic initiation factor 2 (eIF2), GTP, and Met-tRNAi with the This article contains supporting information online at www.pnas.org/cgi/content/full/ MICROBIOLOGY 40S to form a 43S preinitiation complex (7). eIF3 consists 0900557106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900557106 PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4071–4078 Downloaded by guest on September 27, 2021 Fig. 1. Characterization of the recovered cDNA. (A) Schematic representation of the H2 cDNA fragment. H2 cDNA inserted into retroviral Mo-MuLV vector, pBabeHAZ. ATG1, at influenza hemaglutinin (HA) tag epitope; ATG2, alternative start codon within HA coding sequence; ATG3 start codon for H2(N91-eIF3f)-Zeo. (B) Homology of H2 cDNA N-terminal portion with eIF3f . MPN domain, Mrp-1Pad1N-terminal sequence (EXnHXHX10D). (C)(Left) Western blot analyses (WB) of nuclear and cytoplasmic extracts, from empty vector control TE671 cells (TE) and H2 cellular clone, with an anti-Sh-BLE (zeocin resistance protein). Anti-actin served as a loading control. (Center) detection of both encoded H2 proteins with anti-Sh-BLE or anti-eIF3f antibodies. GAPDH was loading control. (Right) Determination of expression of N91-eIF3f and endogenous eIF3f, using anti-eIF3f antibody in H2 cells; TE cells were used as negative control. (D) Endogenous expression of eIF3f determined using anti-eIF3f antibody. GAPDH was used as a loading control. (E) TE671 cells expressing empty vector control pBabe-HAZ (TE) or H2 cells expressing N91-eIF3f were infected at the indicated multiplicities of infection with VSV-HIV-Puro. Forty-eight hours later, puromycin was added to the medium, and, 5–8 days later, drug-resistant colonies in the plates were counted after Giemsa staining. Results are representative of 3 independent experiments. Error bars represent standard error. (F) Cells tested for susceptibility to a Mo-MuLV vector, VSV-MLV-Neo virus. Transduced drug resistant colonies were scored after Giemsa staining. (G) Cre recombinase was stably introduced into H2 cells by cotransfection with p-Puro. Six puromycin resistant clones were expanded and deletion of H2-Zeo DNA was monitored by PCR. Clones were tested for resistance to VSV-HIV-Neo at a multiplicity of 1 ϫ 10Ϫ2.(H) The H2-Zeo fragment was recovered from H2 cells, cloned into pBabe-HAZ, used to generate retroviral supernatants by transfection, and reintroduced into naı¨ve TE671 cells. Zeocin resistant clones were tested for resistance to transduction after infection with VSV-HIV-Puro. Relative infectivity was determined by comparison to the TE cell line. Results shown are typical of those obtained in 3 independent experiments.

inated by exposure to the nucleotide analogue ganciclovir (GCV). Characterization of Recovered cDNA and Its Activity. The H2 cDNA The rare surviving HSV-TK-negative clones were recovered as was recovered from the H2 resistant cell clone by PCR, and potentially HIV-resistant. A total of 1 ϫ 105 clones were screened sequence analysis revealed an insert of 330 nt, encoding 91 aa and 3 resistant clones were recovered; one expressing the N- identical to the N-terminal portion of the eukaryotic initiation terminal region of hnRNPU is reported in ref. 13. The other 2 factor 3 subunit p47 or f (eIF3f) (15) fused to the zeocin resistance clones, H2 and H3, were more profoundly resistant and were found gene. The gene product was termed N91-eIF3f (Fig. 1A). An to encode the same cDNA. The 3Јuntranslated region of these 2 alternative start site was located upstream from the cDNA insert, clones was found to be different, demonstrating that 2 independent in frame with the fusion protein, encoding the identical protein but clones had been identified rather than siblings clones. The inde- with an additional 16 aa at the N terminus (Fig. 1B). The 2 proteins pendent recovery of the same cDNA twice strongly demonstrates were expressed at approximately equivalent amounts as determined the importance of this gene fragment as having antiviral activity. by Western blot with antiserum detecting the Zeocin resistance Here, we describe the activity of one of the cDNA clones, dubbed protein (Fig. 1C Left) in H2 cells. TE671 cells expressing empty H2, recovered in 1 of these 2 virus-resistant clones. vector control pBabe-HAZ will for simplicity be termed the TE cell

4072 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900557106 Valente et al. Downloaded by guest on September 27, 2021 line. The protein fragments were observed both in the cytoplasm imately to the same level as the level of endogenous eIF3f (Fig. 2C and in the nucleus of H2 cells. Endogenous eIF3f is reported to Lower). The same results were obtained when eIF3f-myc was reside both in the nucleus and in the cytoplasm of cells (16). Using overexpressed in 293T cells (Fig. 2D). These results support the INAUGURAL ARTICLE an antibody specific for eIF3f, only the lower molecular weight form notion that the restriction observed is not cell type-dependent. They was recognized (Fig. 1C Center), probably because of a different also show that, remarkably, a 2-fold increase in eIF3f can block virus conformation of the longer fusion protein or masking of the transduction. recognized epitopes. A slower migrating band on these blots is a To determine whether N91-eIF3f-mediated restriction of HIV-1 nonspecific band in all cell lysates. We have generated an antibody expression acts through an involvement with the endogenous eIF3f, directed against eIF3f in rabbits. This polyclonal antibody more we asked whether RNAi-mediated knockdown of eIF3f in N91- specifically recognizes full-length eIF3f compared with the N91- eIF3f-expressing cells would affect viral restriction. We used ret- eIF3f fragment (Fig. 1C Right). It is not possible to correlate the roviral vectors encoding appropriate short hairpin RNAs (shRNAs) amounts of N91-eIF3f expression to endogenous eIF3f expression to knockdown endogenous eIF3f expression in TE or H2 cell lines. by Western blot using this particular antibody. The protein levels of A primer targeted to the noncoding sequence of the human eIF3f full-length eIF3f, the parental TE cell line, or H2 cells were gene was used and clonal cell lines stably expressing the RNAi comparable as judged by Western blot (Fig. 1D), suggesting that the construct, and a scrambled vector control, were tested for both N-terminal fragments did not affect the expression or accumulation eIF3f expression and ability to restrict VSV-HIV-Neo virus in a of the endogenous protein in the H2 cell line. plate assay (Fig. 2 E and F). In both TE and H2 cells, nearly The H2 cell line was tested for sensitivity to HIV-based retroviral complete knockdown of eIF3f was achieved (Fig. 2E). Knockdown vectors by counting drug resistant colonies after infection with a of endogenous eIF3f had no effect on N91-eIF3f expression in H2 reporter virus, VSV-HIV-Puro, and was found to be profoundly clones, as measured using an anti-zeocin antibody (Fig. 2E), nor did resistant to virus transduction. H2 cells yielded Ϸ30- to 100-fold it seem to affect cell viability. The loss of endogenous expression of fewer transductants compared with the TE cell line (Fig. 1E). The eIF3f in H2 cells was accompanied by a loss of restrictive HIV-1 H2 cell line was somewhat less resistant to a Moloney murine activity (Fig. 2F), with a regain of sensitivity to infection to levels leukemia virus (MLV) vector; only a 4- to 5-fold restriction was equivalent to unaltered TE cells. We noted that the sensitivity to observed (Fig. 1F). infection of empty vector control TE cells very modestly increased To confirm that the library derived cDNA was responsible for the as a result of eIF3f knockdown. Whether this modest increase of observed restriction, H2 cells were transfected with a plasmid infectivity in TE cell is specific to HIV-1 or rather because of an encoding the Cre recombinase to mediate excision of the cDNA overall increase in translation efficiency needs to be studied further. insert via the surrounding LoxP sites in the vector LTRs. Clones in Nevertheless, our results indicate that N91-eIF3f alone without WT which Cre-induced recombination had deleted the provirus were levels of eIF3f is unable to mediate restriction. tested for resistance to infection by VSV-HIV-Neomycin virus. We next assessed how N91-eIF3f expressing cells responded to These clones consistently became virus sensitive (Fig. 1G). The selection for breakthrough expression of an HIV reporter con- correlation between the presence of the insert and resistance to struct. After infection of H2 cells with VSV-HIV-Puro, we forced viral infection confirmed that the continuous presence of the cDNA expression of the vector by culturing the cells in increasing con- was necessary for the resistance to HIV-1. The cDNA insert present centrations of puromycin. We observed a loss of N91-eIF3f expres- in the H2 clone was recovered by PCR amplification and recloned sion in the cell population with increasing selective pressure for the into the pBabe-HAZ vector. After packaging, the cDNAs were reporter (Fig. 2G). These results suggest that cells expressing higher introduced into naı¨ve TE671 cells. The newly transduced cellular amounts of N91-eIF3f were negatively selected in competition with clones became highly resistant to infection with VSV-HIV-Puro those cells allowing higher HIV-1 gene expression. (Fig. 1H), whereas control cells transduced with pBabe-HAZ vector (TE) alone did not. Expression of the H2 cDNA was thus both Identification of Step in the HIV-1 Life Cycle Blocked by N91-eIF3f. To necessary and sufficient to confer resistance to HIV-1 infection. determine whether the viral block mediated by N91-eIF3f occurred before or after proviral integration, H2 cells and an empty vector Restriction Ability of N91-eIF3f Not Fused to Zeocin and of Full-Length control line were infected with VSV-HIV-Puro virus at several eIF3f. To determine whether the fusion of the N91-eIF3f ORF to different multiplicities and propagated without puromycin selec- the Zeocin ORF was needed to block HIV-1 transduction, TE671 tion. Seven days after infection, total cellular DNA was extracted or 293T cells were transfected with an expression vector for and the proviral copy number was determined by real time quan- N91-eIF3f carrying only a c-myc tag (N91-eIF3f-myc) and clones titative PCR (qPCR) (Fig. 3A). The proviral copy number in the stably expressing the DNA were selected. The expression of N91- resistant clone was not significantly different from that found in the eIF3f-myc in each clone was assessed by RT-PCR and the suscep- TE cells, under the same conditions under which the number of tibility to HIV-1 was determined by scoring for HIV-Puro trans- transduced colonies was substantially reduced (Fig. 1E). These duction efficiency (Fig. 2A). The clones expressing N91-eIF3f-myc results are consistent with a viral block induced by N91-eIF3f at showed strong resistance to HIV-1 transduction compared with some time after integration of the provirus into the host genome. control clones expressing the empty vector or to clones whose To define the postintegration block, the levels of viral mRNAs expression of the fragment was undetectable. This result indicates present in the cytoplasm of the resistant clones were assessed after that the N91-eIF3f peptide without fusion to Zeocin is sufficient for infection of TE and H2 cells with VSV-HIV-Neo. Assays of viral restriction. Additionally, the phenotype was observed in 2 different mRNA levels were determined by qRT-PCR, using primers di- cell lines, TE671 and 293T, suggesting that the activity is not rected to the neomycin resistance gene (Fig. 3B Left). The number cell-type specific. of neomycin resistant colonies was determined in parallel (Fig. 3B The full-length eIF3f was tested for its ability to block HIV-1. Right). We observed a Ͼ10-fold reduction in the mRNA levels in TE671 cellular clones stably expressing the full-length protein fused the cytoplasm of H2 cells compared with cells expressing the empty to the c-myc epitope were created by transfection. The full-length vector control, corresponding well with the reduction in neomycin protein was expressed both in the nucleus and in the cytoplasm in resistant colonies. We also evaluated the expression of viral mRNA TE671 cells (Fig. 2 B and C). The TE671 cellular clones overex- in the nucleus of H2 and TE cells upon infection with VSV-HIV- pressing eIF3f-myc strongly blocked HIV-Puro infection. These TK. RT-PCR (Fig. 3C Left) and qRT-PCR (Fig. 3C Right) assays results suggest that overexpression of the full length eIF3f restricts of nuclear RNA revealed that the levels of nuclear viral RNA in H2

HIV-1 to the same extents as its N-terminal fragment. The over- cells were drastically reduced: The levels were reduced Ͼ85% in the MICROBIOLOGY expression of eIF3f-myc in the 4 independent clones was approx- H2 cell line compared with control. These results suggest that H2

Valente et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4073 Downloaded by guest on September 27, 2021 Fig. 2. Expression of N91-eIF3f and eIF3f and resistance to HIV infection. (A)(Left) Susceptibility of TE671 clones expressing N91-eIF3f-myc (H2-myc) to infection as scored by HIV-Puro colony counting. Clones 1, 2, 3, 4, 5 expressed H2-myc; clones 6 and 7 did not. Clone TEpcDNAcl.1 stably expresses empty vector control. The TE and H2 cell lines were included as negative and positive controls, respectively. Cells were infected at an MOI of 1.5 ϫ 10Ϫ3 with VSV-HIV-Puro. Forty-eight hours later, puromycin was added to the medium, and, 5–8 days later, resistant colonies in the plates were counted after Giemsa staining. (Right) N91-eIF3f-myc (H2-myc) was transfected into 293T cells, and clones were obtained and tested as for TE671. One empty vector control clone and 2 N91-eIF3f-myc expressing clones were tested for resistance, using the indicated MOIs of VSV-HIV-Puro. (B) Susceptibility of TE671 cells expressing full-length eIF3f-myc. Cellular clones were infected at an MOI of 2.5 ϫ 10Ϫ3 with VSV-HIV-Puro, and infectivity assayed as in A.(C)(Upper) Expression of transgene protein was assessed in the nucleus and cytoplasm by WB, using an anti c-myc antibody and anti-actin antibody as a loading control. (Lower) Expression of the transgene was determined in total cellular lysates for comparison with endogenous levels of eIF3f, using anti-eIF3f antibody. (D) Full-length eIF3f-myc was stably expressed in 293T cells; eIF3f-myc expression assayed by WB. Cellular clones were tested for resistance at an MOI of 1 ϫ10Ϫ3 with VSV-HIV-Puro. A,B, and Dare representative of as least 3 independent experiments. (E) RNAi-mediated knockdown of eIF3f and restriction to HIV mediated by N91-eIF3f. TE or H2 cells were transduced with pGIPZ-RNAi construct targeting eIF3f or a scrambled negative control. Lysates of individual clones were analyzed by WB for eIF3f, H2-zeo, or GAPDH as a loading control. TE and H2 lysates were loaded as control. (F) Clones knocked down for eIF3f or scrambled control were tested for susceptibility to infection with HIV-Neo at an MOI of 3 ϫ 10Ϫ2. The average number of puromycin resistance colonies obtained for each set of 3 clones for 3 independent experiments is presented. Error bars represent standard error. (G) TE and H2 cells were infected with VSV-HIV-Puro and selected for viral expression, using increasing amounts of selective drug (puromycin 1 and 5 ␮g/mL). Detection of H2 cDNA expression by WB as selective pressure for HIV expression increases, using anti-She-BLE and actin as loading control.

cDNA expression prevents the accumulation of viral mRNAs in the DNAs, or the control expression constructs, and then scored for nucleus and in the cytoplasm. The reduction of viral mRNAs was their ability to produce puromycin resistant colonies (Fig. 4B). An likely to be posttranscriptional, because different vectors with Ϸ14-fold specific restriction of the HIV-puro reporter was ob- different promoters all exhibited the same reduction of cytoplasmic served in H2 compared with TE cells. In contrast, parallel con- and nuclear viral RNA. structs carrying SV40 or BGH poly(A) sequences showed no restriction, and comparable reporter gene expression in both cell Target of N91-eIF3f Restriction. Inspection of the different retroviral lines. This result shows that the HIV-1 3ЈLTR is the target for vectors used as reporters of susceptibility to transduction of H2 cells N91-eIF3f mediated restriction. identified the 3ЈLTR as the only common feature of the transcripts The drastic reduction of viral nuclear RNA in H2 cells and the subject to negative selection (Fig. 4A). This region contains many involvement of the 3ЈLTR in the restriction hinted at the possibility regulatory sequences including the putative AAUAAA poly(A) that RNA precursor cleavage and/or polyadenylation might be signal, a GϩU rich region, and the USE element, all required for affected by N91-eIF3f. To test whether the HIV-1 polyadenylation efficient cleavage and polyadenylation of nascent HIV-1 transcripts signal could be properly recognized in the presence of N91-eIF3f, (17). To test the role of the 3ЈLTR as a target of N91-eIF3f a 32P-labeled fragment of the HIV-1 RNA precursor was synthe- restriction, we replaced the 3ЈLTR region of the HIV-Puro vector sized with the correct 3Јend for polyadenylation (Fig. S1). Nuclear with the simian virus 40 (SV40) or the bovine growth hormone extracts from HeLa cells expressing the H2 cDNA or pBabe-HAZ (BGH) 3Ј regions. TE and H2 cells were transfected with these or control HeLa cells were prepared and incubated with the

4074 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900557106 Valente et al. Downloaded by guest on September 27, 2021 INAUGURAL ARTICLE

Fig. 3. Analysis of viral block in TE and H2 cell lines. (A) Clones were infected at different multiplicities of VSV-HIV-Puro virus. Total DNA was extracted 7 days after infection and copy number of integrated provirus determined by qPCR. Proviral copy number was normalized per 100 ng of total DNA. Data are representative of 3 independent experiments; error bars represent the standard error. (B)(Left) Analysis of viral mRNA expression. Cytoplasmic mRNA was extracted 7 days after VSV-HIV-Neo infection at the indicated multiplicities. First strand cDNA synthesis and amplification of the target DNA was performed by qPCR, using primers recognizing the neomycin reporter gene. Results were normalized to copies of viral mRNA per copy of GAPDH. (Right) Resistance to infection by HIV-Neo was assessed in parallel. Neomycin was added to the medium 48 h after infection, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results shown are typical of those obtained in 3 independent experiments. (C)(Left) Nuclear and cytoplasmic RNA was extracted 7 days after infection with 10-fold decreasing dilutions of VSV-HIV-TK. After first strand cDNA synthesis, TK and GAPDH cDNA was amplified by PCR. (Right) TE and H2 cells were infected at an MOI of 3 ϫ 10Ϫ2 with HIV-Puro. Seven days after infection; nuclear RNA was extracted and quantified by qRT-PCR, using primers recognizing the puromycin reporter gene. Results are relative to values of GAPDH and the ratio found in TE cells was taken as reference. All data are representative of 3 independent experiments. Errors are standard error of the mean. HI, heat-inactivated.

32P-labeled precleaved RNA and its polyadenylation was monitored precursor HIV 3ЈLTR(HIVwt)orHIV3ЈLTR with a deletion in by gel electrophoresis (Fig. 4C). We did not observe any significant the core poly(A) hexamer (HIV⌬Hex) were incubated with HeLa decrease in polyadenylated RNA species when nuclear extracts HAZ extracts and included as a 5Ј cleavage control. These results from cells expressing N91-eIF3f were used compared with empty clearly show that the expression of N91-eIF3f specifically reduces vector control, suggesting that polyadenylation of a precleaved the cleavage at the 3ЈLTR of HIV-1 preventing maturation of viral RNA from the HIV 3ЈLTR is not affected by N91-eIF3f. RNAs, which consequently renders them more prone to degrada- We next assessed the specific activity of nuclear extracts from tion, explaining the drastic reduction of HIV-1 nuclear mRNA in TE671 or HeLa cells expressing empty vector control or N91-eIF3f N91-eIF3f expressing cells. for cleavage of RNA at the site of polyadenylation. A longer (338-nt) uncleaved precursor 32P-labeled RNA was incubated with Discussion extracts in vitro, and cleavage was assessed by gel electrophoresis The results presented here show that a fragment of eukaryotic (Fig. 4D). When proper cleavage occurs, a 237-nt 5Ј cleaved product initiation factor 3 subunit f (eIF3f), or the inappropriate over- is detected. Extracts from TE671 or HeLa cells expressing N91- expression of the WT protein, can modulate the host cleavage

eIF3f consistently produced significantly less cleavage product and polyadenylation machinery to specifically decrease 3Јend MICROBIOLOGY compared with extracts of empty vector control cells. The RNA processing of HIV-1 mRNA transcripts. A cDNA encoding the

Valente et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4075 Downloaded by guest on September 27, 2021 Fig. 4. Viral target for H2 mediated resistance. (A) Comparison between different WT HIV and retroviral vectors gene products (arrow) restricted by H2 cells. Amplified dashed box represents the common features: the U3 deleted 3ЈLTR (dU3LTR), containing the poly(A) signal and a GT rich region, both defining the transcript end. RRE, Rev-responsive element; SFFV, spleen focus forming virus; WPRE, Woodchuck posttranscriptional regulatory element. (B) HIV-puro and a modified HIV-puro with SV40 poly(A) or a BGH poly(A) sequence replacing dU3LTR were transiently transfected into TE or H2 cell lines. Two days after transfection, puromycin was added to the medium, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results are shown as the ratio between the numbers of TE versus H2 resistant colonies and are representative of 3 independent experiments. Error bars represent standard error. (C) In vitro polyadenylation reaction. Poly(A) addition. The 32P-labeled precleaved RNA substrate was incubated in nuclear extracts from HeLa-CD4-pBabe-HAZ, HeLa-CD4-H2, or HeLa cells and the recombinant eIF3f-GST, N91-eIF3f-GST, and GST proteins, with ATP for 30 min at 30 °C. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Lanes 10 and 11 are controls of polyadenylation. Precleaved HIV-1 input and HeLa nuclear extracts with 3ЈdATP or ATP respectively. (D) Poly(A) site cleavage. The 32P-labeled uncleaved 3ЈLTR HIV RNA substrate was incubated with nuclear extracts of TE671 or HeLa cells expressing empty vector control (HAZ) or N91-eIF3f (H2) for 30 min at 30 °C. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. The 3ЈLTR RNA substrate (HIVwt) or a substrate with a core poly(A) hexamer deletion (HIV⌬Hex) were incubated with HeLa HAZ extracts for 5Јcleavage product control.

N-terminal portion of eIF3f, N91-eIF3f, was recovered from a HIV replication. Less efficient was the restriction observed to library screen in 2 independent virus-resistant clones, and was MLV-VLPs (4- to 5-fold). Overexpressed full-length eIF3f was found to be necessary and sufficient to confer a severe block to also able to mediate the inhibition of HIV expression. Expres-

4076 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900557106 Valente et al. Downloaded by guest on September 27, 2021 sion of N91-eIF3f did not alter the expression of endogenous 3ЈLTR region of the viral mRNA is a uniquely sensitive target for eIF3f. However, if expression of endogenous eIF3f was knocked interference by host proteins. HIV-1 replication requires the 3Ј down by RNAi, the overexpression of N91-eIF3f was no longer processing and export of multiple alternatively spliced transcripts, INAUGURAL ARTICLE sufficient to mediate restriction to the virus. This result strongly and unspliced mRNA. The use of a single poly(A) site to produce suggests that the activity of the fragment involves the contribu- both spliced and unspliced mRNAs might involve a unique manip- tion of the WT protein. The knockdown of eIF3f in WT cells was ulation of the host 3Ј processing and export machinery. Ϸ associated with a slight increase ( 30%) in HIV-1 expression, Our data are the first indication that the cleavage at the 3Ј end although it is not clear whether this is specific for HIV or rather of HIV-1 mRNA is sensitive to specific manipulation by host factors is due to an overall increase in translation efficiency, on the concept with a large impact on viral production. We offer the suggestion that that eIF3f might be a negative regulator of translation (18). eIF3f is a new player in 3Јend cleavage in some cellular mRNAs, These results provide evidence for an effect of eIF3f on HIV and provide evidence that a protein fragment can interfere with this gene expression. Overexpression of the N91-eIF3f fragment or the WT protein has a dominant effect to reduce HIV-1 3Ј end unanticipated function to block HIV-1 mRNA processing. Further analysis of these interactions might result in a clearer understanding processing. This effect seems to require the presence of endogenous Ј amounts of eIF3f. Whether the endogenous levels of eIF3f have of the regulation and function of eIF3f in the 3 end processing of already a potential restrictive activity needs to be further studied. HIV-1 pre mRNA, and manipulating these factors and their The fragment N91-eIF3f is not a natural occurring proteolytic interactions may ultimately offer novel means to suppress viral product of eIF3f, and it does not occur naturally in vivo. Its activity replication. was identified from a screen of an overexpression cDNA library that includes many gene fragments. However, its independent recovery Experimental Procedures twice as a restrictive element of HIV gene highlights the impor- HeLa IFN cDNA Library in pBabe-HAZ. HeLa cells were stimulated by IFN-␥ (1 tance of eIF3f in 3Јend processing and raises the expectation that ␮g/mL) overnight and with IFN-␣ (1,000 units/mL) for 3 h. Construction of the additional components of these pathways will also be important in library was described in ref. 13. viral mRNA formation. Even though we used a cDNA library of cells stimulated by the Production of VSV-HIV-1 Reporter Viruses. To generate VSV HIV-HSV-TK virus, 2 IFN to increase our chance of finding an antiviral gene, it is worth ␮g of the retrovirus vector TRIP-TK [a generous gift from G. Gao (Chinese noting here that eIF3f expression is not stimulated by IFN expres- Academy of Sciences, Beijing)] was transiently transfected with fugene (Roche) sion as shown by WB. Thus, both constitutively expressed and into 293T cells, along with 1 ␮g of HIV-Gag-pol DNA (p8.91) and 1 ␮g of pMDG. induced genes can be recovered from these cell-based screens. The culture supernatant was collected at 48, 72, and 96 h, filtered, aliquoted and Experiments to probe the step of viral replication blocked by titrated on TE671 cells. To generate VSV-HIV-Neomycin and VSV-HIV-Puromycin N91-eIF3f revealed a drastic reduction of retroviral transcripts in viruses, the retroviral vectors pSCNW and pSCPW, a kind gift from G. Towers the nucleus and cytoplasm of infected cells. The loss of viral nuclear (University College of London, London), were used, respectively. mRNA was not apparently caused by reduced efficiency of tran- Titrations Using VSV-HIV-1 Reporter Viruses. A total of 105 cells to be tested were scription initiation, because different vectors with different pro- seeded in 10-cm plates and the next day cells were infected with serial dilutions moters driving the reporter gene were all affected. Moreover, of VSV-pseudotyped viruses in 8 mL (total volume) of complete medium. Forty- expression of N91-eIF3f did not cause loss of nuclear mRNA eight hours after infection, the selective drug was added to the culture media. expression of a nonretroviral construct carrying the same promot- TE671 cells were cultured with 5 ␮g/mL Puromycin, 1.5 mg/mL Neomycin or 50 ers and reporter genes. We determined that the target for N91- ␮g/mL Zeocin according to the viral vector used. Culture media was changed eIF3f-mediated restriction was the 3ЈLTR of the HIV pre-mRNA: every 2 days until visible colonies were detected. Colonies were stained with replacement of this region with the BGH or SV40 poly(A) sites Giemsa and counted. abolished the restriction. This result also supports the notion that a reduced level of viral mRNA is not due to reduced transcription Isolation of H2 cDNA from an H2 Cell Line. The recovery of the cDNA insert was initiation. as described in ref. 14. We studied both cleavage and polyadenylation activities of nuclear extracts from empty vector or N91-eIF3f-expressing cells. Fractionation of Nuclear and Cytoplasmic Protein Extracts. This was performed The specific cleavage activity of the HIV-1 poly(A) site by the latter according to Dignam protocol (21). extracts was significantly reduced compared with empty vector control extract. The presence of N91-eIF3f resulted in the reduction Western Blot Analyses. Immunodetection of H2-zeo protein was performed by of premRNA 3Ј processing. A potential insight into the mechanism resolving proteins on a 15% SDS-polyacrylamide gel and electroblotting onto a of N91-eIF3f inhibition of HIV mRNA 3Ј processing is provided by 0.2-␮m nitrocellulose membrane. A 1:3,000 dilution of an anti-Sh ble (Zeocin the observation that eIF3f is known to interact directly, via its MPN resistance protein) rabbit antiserum (Cayla) was incubated with the membrane domain, with the cyclin-dependent kinase CDK11 (16); CDK11 followed by a secondary incubation with a 1:10,000 dilution of sheep anti-rabbit interacts with the SR protein 9G8, and in turn, 9G8 interacts with antibody conjugated to horseradish peroxidase (HRP) (Amersham). The detection of H2-myc or eIF3f-myc was performed using mouse anti-c-myc antibody (Santa the mammalian poly(A) site recognition factor CFIm (19). We suggest that CDK11 might serve to link eIF3f and mRNA 3Ј Cruz Biotechnologies) at a 1:10,000 dilution followed by a secondary incubation with sheep anti-mouse HRP. Detection of endogenous eIF3f was made using an processing. The binding of 9G8 to sequences upstream of the antiserum raised in rabbit, immunized with recombinant N91-eIF3f-GST. Loading poly(A) site of the avian retrovirus RSV has been shown to promote controls were performed using antibodies against actin or GAPDH (Sigma). mRNA 3Ј processing both in vivo and in vitro (20). Intriguingly, multiple sequences related to the consensus 9G8 binding sites are Ј Preparation of RNA for Analysis of Viral Block. Cells of each cell line were infected present in the HIV-1 3 LTR. The interrelated functions of these with HIV-Puromycin or HIV-Neomycin for at least 4 h. Five to 7 days after infection, various gene products in poly(A) site cleavage remain to be cells were trypsinized and collected for RNA preparation. The cytoplasmic and explored. nuclear RNA fractions were separated using the RNA extraction kit (Qiagen) It is of note that the 2 cDNAs discovered as a result of this screen, following the manufacturer’s instruction. The nuclear pellet was washed twice with N86-hnRNPU (13) and N91-eIF3f, both target the 3ЈLTR region of the cytoplasmic lysis buffer and the nuclear RNA was extracted as for total RNA. the HIV-1 mRNA. We can hypothesize why this happened: One can argue that the pressure exerted during the selection process for Real-Time PCR. PCRs were as described in ref. 13.

an antiviral gene was harsh and that we have abrogated all of the MICROBIOLOGY preintegration blocks. Alternatively, we can postulate that the Virology. Titration of WT HIV was done as described in ref. 13.

Valente et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4077 Downloaded by guest on September 27, 2021 Protein Expression. Full-length eIF3f or N91-eIF3f were cloned into pGEX-5X-1 ACKNOWLEDGMENTS. We thank Guangxia Gao (Chinese Academy of Sci- (Amersham), expressed in Escherichia coli as a C-terminal fusion with GST (GST) ence, Beijing), Greg Towers (University College of London, London), and and purified from bacterial lysates, using glutathione sepharose. Uriel Hazan (Institut Cochin de Ge´ne´ tique Mole´culaire, Paris), for reagents and helpful discussion. This work was supported by the Howard Hughes Cleavage and Polyadenylation Assays. Preparation of RNA extracts, nuclear Medical Institute (to S.T.V.), the American Foundation for AIDS Research, protein extractions, and 3Ј processing reactions were performed as described in the Portuguese Ministry of Education, Praxis XXI. S.P.G. is a Howard Hughes refs. 22 and 23. Medical Institute Investigator.

1. Wahle E, Keller W (1992) The biochemistry of 3Ј-end cleavage and polyadenylation of 13. Valente S, Goff SP (2006) Inhibition of HIV-1 gene expression by a fragment of hnRNP messenger RNA precursors. Annu Rev Biochem 61:419–440. U. Mol Cell 23:597–605. 2. Weiss EA, Gilmartin GM, Nevins JR (1991) Poly(A) site efficiency reflects the stability of 14. Gao G, Guo X, Goff SP (2002) Inhibition of retroviral RNA production by ZAP, a complex formation involving the downstream element. EMBO J 10:215–219. CCCH-type zinc finger protein. Science 297:1703–1706. 3. Gil A, Proudfoot NJ (1987) Position-dependent sequence elements downstream of 15. Asano K, et al. (1997) Structure of cDNAs encoding human eukaryotic initiation factor AAUAAA are required for efficient rabbit beta-globin mRNA 3Ј end formation. Cell 3 subunits. Possible roles in RNA binding and macromolecular assembly. J Biol Chem 49:399–406. 272:27042–27052. 4. Berkhout B, Klaver B, Das AT (1995) A conserved hairpin structure predicted for the 16. Shi J, et al. (2003) The p34cdc2-related cyclin-dependent kinase 11 interacts with the poly(A) signal of human and simian immunodeficiency viruses. Virology 207:276– p47 subunit of eukaryotic initiation factor 3 during apoptosis. J Biol Chem 278:5062– 281. 5071. 5. Gilmartin GM, Fleming ES, Oetjen J (1992) Activation of HIV-1 pre-mRNA 3Ј processing 17. Proudfoot N (1991) Poly(A) signals. Cell 64:671–674. in vitro requires both an upstream element and TAR. EMBO J 11:4419–4428. 18. Doldan A, et al. (2008) Loss of the eukaryotic initiation factor 3f in pancreatic cancer. 6. Valsamakis A, Schek N, Alwine JC (1992) Elements upstream of the AAUAAA within the Molecular carcinogenesis 47:235–244. human immunodeficiency virus polyadenylation signal are required for efficient poly- 19. Dettwiler S, Aringhieri C, Cardinale S, Keller W, Barabino SM (2004) Distinct sequence adenylation in vitro. Mol Cell Biol 12:3699–3705. motifs within the 68-kDa subunit of cleavage factor Im mediate RNA binding, protein– 7. Hinnebusch AG (2006) eIF3: A versatile scaffold for translation initiation complexes. protein interactions, and subcellular localization. J Biol Chem 279:35788–35797. Trends Biochem Sci 31:553–562. 20. Maciolek NL, McNally MT (2007) Serine/arginine-rich proteins contribute to negative 8. Hershey JW, et al. (1996) Conservation and diversity in the structure of translation regulator of splicing element-stimulated polyadenylation in rous sarcoma virus. J Virol initiation factor EIF3 from humans and yeast. Biochimie 78:903–907. 81:11208–11217. 9. Browning KS, et al. (2001) Unified nomenclature for the subunits of eukaryotic 21. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA initiation factor 3. Trends Biochem Sci 26:284. polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 10. Asano K, Kinzy TG, Merrick WC, Hershey JW (1997) Conservation and diversity of 11:1475–1489. initiation factor eIF3. J Biol Chem 272:1101–1109. 22. Gilmartin, G. M (1997) in mRNA formation and function, ed Richter JD (Academic, New 11. Aravind L, Ponting CP (1998) Homologues of 26S proteasome subunits are regulators York.), pp 79–98. of transcription and translation. Protein Sci 7:1250–1254. 23. Gilmartin GM, Fleming ES, Oetjen J, Graveley BR (1995) CPSF recognition of an HIV-1 12. Grainger RJ, Beggs JD (2005) Prp8 protein: At the heart of the spliceosome. Rna mRNA 3Ј-processing enhancer: Multiple sequence contacts involved in poly(A) site 11:533–557. definition. Genes Dev 9:72–83.

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