We examined a series of site-directed point mutants of human immunodeficiency virus type 1 (HIV-1) Vif for their interaction with cellular anti-viral factors APOBEC3G/APOBEC3F. Mutant viruses that display growth-defect in H9 cells did not counteract effectively APOBEC3G and/or APOBEC3F without exception, as monitored by single-cycle infectivity assays. While growth-defective mutants of Vif C-terminal region were unable to suppress APOBEC3G/APOBEC3F, some N-terminal region mutants did neutralize one of APOBEC3G/APOBEC3F. These data have suggested that members of APOBEC3 family other than APOBEC3G/APOBEC3F are not important for anti-HIV-1 activity. Furthermore, APOPEC3G/APOBEC3F were found to differently associate with Vif in virions as analyzed by equilibrium density centrifugation. Taken together, these results indicated that interaction of HIV-1 Vif and APOBEC3G is distinct from that between Vif and APOBEC3F

Adachi, Akio

Miyake, Ariko

Nagao, Tamiko

Nomaguchi, Masako

Uchiyama, Tsuneo

Yamashita, Tomoki

Tokushima University Institutional Repository

INTRODUCTIONThe genome of HIV-1 encodes Vif as one of ac-cessory viral proteins that are unique to primateimmunodeficiency viruses. HIV -1 Vif efficientlyneutralizes the strong anti - retroviral effects ofAPOBEC3G/APOBEC3F in natural target cells bytargeting them to proteasomal degradation (1).Through this activity of Vif, HIV-1 can grow in Tlymphocytes and macrophages, the two major tar-gets cells of the virus. Extensive studies by us andothers have revealed that HIV-1 Vif consists of twomajor functional domains with various motifs (2-4).HIV-1 Vif binds to APOBEC3G/APOBEC3F by itsN - terminal region (2 - 10), and degrades themthrough interaction with its C - terminal region(2, 11-14). While Vif mediates proteasomal degra-dation of both APOBEC3 proteins, it binds to themdifferently via several distinct motifs (2-10). In someCD4+ cells such as H9, primary T lymphocytes, andmacrophages that are non-permissive for HIV-1without Vif, APOBEC3G/APOBEC3F are found toact against HIV-1. However, biological and biochemi-cal basis for interaction of Vif and APOBEC3G/APOBEC3F is not well understood yet.APOBEC3G/APOBEC3F belong to a family ofpolynucleotide cytidine deaminases with diversefunctions (1). Although it has been well estab-lished that APOBEC3G/APOBEC3F are potent anti-retroviral cellular factors, several other APOBECfamily members have been reported to be able toinhibit HIV-1 (1). However, the extent of contribu-tion by these APOBEC proteins to block or impedeHIV-1 replication remains to be determined.ORIGINALDifferent interaction between HIV-1 Vif and its cellulartarget proteins APOBEC3G/APOBEC3FTamiko Nagao*, Tomoki Yamashita*, Ariko Miyake, Tsuneo Uchiyama,Masako Nomaguchi, and Akio AdachiDepartment of Microbiology, Institute of Health Biosciences, the University of Tokushima GraduateSchool, Tokushima, JapanAbstract : We examined a series of site-directed point mutants of human immunodeficiencyvirus type 1 (HIV-1) Vif for their interaction with cellular anti-viral factors APOBEC3G/APOBEC3F. Mutant viruses that display growth-defect in H9 cells did not counteract ef-fectively APOBEC3G and/or APOBEC3F without exception, as monitored by single-cycleinfectivity assays. While growth-defective mutants of Vif C-terminal region were unableto suppress APOBEC3G/APOBEC3F, some N-terminal region mutants did neutralize oneof APOBEC3G/APOBEC3F. These data have suggested that members of APOBEC3 familyother than APOBEC3G/APOBEC3F are not important for anti-HIV-1 activity. Further-more, APOPEC3G/APOBEC3F were found to differently associate with Vif in virions asanalyzed by equilibrium density centrifugation. Taken together, these results indicatedthat interaction of HIV-1 Vif and APOBEC3G is distinct from that between Vif andAPOBEC3F. J. Med. Invest. 57 : 89-94, February, 2010Keywords : HIV-1, Vif, APOBEC3G, APOBEC3FReceived for publication November 20, 2009 ; accepted Decem-ber 21, 2009.*equal contributionAddress correspondence and reprint requests to Akio Adachi,Department of Microbiology, Institute of Health Biosciences,the University of Tokushima Graduate School, Kuramoto-cho,Tokushima 770-8503, Japan and Fax : +81-88-633-7080.The Journal of Medical Investigation Vol. 57 201089In this study, we have determined a single-cycleviral infectivity of numerous HIV-1 vif mutants inthe presence of APOBEC3G or APOBEC3F, andcompared the results with growth characteristics ofthe mutants in non-permissive H9 cells. In addition,we have examined status of Vif/APOBEC3 com-plexes in cells and virions by equilibrium densitycentrifugation. We demonstrate here the distinctinteraction of Vif and the two APOBEC3 proteins.MATERIALS AND METHODSCellsHuman monolayer cell lines 293T (15) and MAGI(16) were maintained in Eagles’s minimal essentialmedium containing 10% heat-inactivated fetal bovineserum.TransfectionSub-confluent 293T cells in 90 mm dishes were co-transfected with a proviral clone and a flag-taggedexpression vector of APOBEC3G or APOBEC3F(3, 4) by the calcium-phosphate co-precipitationmethod (17). On day 2 post-transfection, cells andcell-free virus samples were prepared as previouslydescribed (3, 4, 17). Viral amount was determinedby the HIV-1 p24 antigen ELISA kit (ZeptoMetrixCorp.).MAGI assayViral single-cycle infectivity per 0.1 ng of Gag-p24was determined by the MAGI assay as previouslydescribed (16).Analysis of Vif and APOBEC3G/APOBEC3F com-plexes in cells and virionsTransfected 293T cells and virions produced inthe cells were lysed in a lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mMEDTA and 1% protease inhibitor cocktail (Sigma-Aldrich)). For preparation of virions, culture super-natants from transfected 293T cells were filteredthrough 0.45-μm filters, and partially purified byultracentrifugation through 25% sucrose for 2 h at80,000g using SW41 rotor. Cell and virion lysateswere resolved by SDS-PAGE, and electrophoreti-cally transferred to polyvinilidene fluolide mem-branes. Membranes were treated with anti-FLAG(Sigma-Aldrich), anti-Vif (NIH AIDS Research andReferences Reagent Program) or anti-p24 (18) anti-body, and visualized by ECL plus Western blottingdetection system (Amersham Pharmasia BiotechInc.). For analysis by equilibrium density centrifu-gation, cell and virion lysates were loaded on thetop of 5-50% sucrose gradient, and spun for 16 h at100,000g using SW41 rotor. Eight fractions werecollected from each tube, and precipitated by trichlo-roacetic acid. Samples were then analyzed by im-munoblotting as described above.DNA constructsProviral vif mutant clones of HIV-1 NL4-3 havebeen previously described (3, 4, 19).RESULTS AND DISCUSSIONWe have previously generated a series of HIV-1vif mutants from wild-type (WT) NL4-3 clone, andanalyzed them biologically and biochemically (Fig.1) (3, 4, 19). Approximately half of the site-directedpoint mutants were defective for virus growth innon-permissive H9 cells, and were predicted to besusceptible to anti-viral activity of APOBEC3 pro-teins. In fact, mutants designated W21A, S32A,W38A, Y69A and H108A did not overcome the anti-HIV-1 action of APOBEC3G/APOBEC3F (3, 4).Moreover, while mutants Y40A and H43A did notneutralize the activity of APOBEC3G, E76A andW79A did not suppress APOBEC3F (3, 4).In order to do a more systemic genetic analysisof anti-APOBEC activity of HIV-1 Vif, we selectedfifteen clones from our proviral mutants (3, 4, 19)as shown in Fig. 1. These included mutants carry-ing mutations in known functional motifs or outsidethe regions. A mutant without Vif (ΔVif) did notgrow at all in H9 cells (Fig. 1) (3, 19). The othermutants except for H28A were growth-defective inH9 cells to some extent (Fig. 1) (3, 19). The mu-tants and expression vectors of APOBEC3G orAPOBEC3F were co-transfected into 293T cells,and equal amounts of resultant viruses as deter-mined by Gag-p24 ELISA were assayed for thesingle-cycle infectivity in MAGI cells. All the mu-tants were simultaneously examined for infectivityin each experiment to know relative anti-APOBECactivity. Fig. 2 shows the MAGI infectivity of variousviruses derived from proviral clones (Fig. 1). Inthe presence of APOBEC3G and APOBEC3F, ΔVifmutant displayed less than 20% and 30% infectivityin a single-replication cycle, respectively, relativeto that of WT virus. As expected, L24A and H28A,mutants with almost normal growth phenotype inT. Nagao, et al. HIV-1 Vif and APOBEC3G/F90H9 cells (Fig. 1) (3), behaved similarly with WT vi-rus in the presence of APOBEC3G/APOBEC3F.However, L24A was somewhat sensitive to A3F ac-tivity in agreement with its slightly poor growth inH9 cells (Fig. 1) (3). As can be easily seen in Fig. 2,mutants other than L24A and H28A showed con-siderable defects against APOBEC3G, APOBEC3F,or both. Following mutants are typical examples :Figure 2 : Single-cycle infectivity of various vif mutants in the presence of APOBEC3G/APOBEC3F. Virus samples were preparedfrom 293T cells co-transfected with a proviral clone (15 μg) and an expression vector of flag tagged APOBEC3G/APOBEC3F (1 μg).Single-cycle infectivity was determined by MAGI assay (16), and viral infectivity relative to that of WT NL4-3 clone is shown. A3G,APOBEC3G ; A3F, APOBEC3F ; WT, wild- type virus NL4-3 ; ΔVif, Vif -minus virus NL-Nd (22).Figure 1 : Major functional domains of HIV-1 Vif protein and its mutants used in this study. Functionally important domains or motifsin Vif (2-4), and positions and designations of the mutants are indicated at the upper part. All mutants are derived from HIV-1 NL4-3clone (17) (GenBank accession no. AF324493). At the lower part, growth property of mutant viruses in non-permissive H9 cells (3,19) are summarized as follows. Wild type, virus growth similar to that of wild type virus ; No, no detectable virus growth ; Slightlypoor, virus growth kinetics are similar to those of wild type virus but virion production at the peak is around 50% of that by wild typevirus ; Poor, virus growth kinetics are significantly delayed relative to those of wild type virus (at least 3 days). Abbreviations : WT,wild- type virus NL4-3 ; ΔVif, Vif -minus virus NL-Nd (22).The Journal of Medical Investigation Vol. 57 February 2010 91Y40A/APOBEC3G, W79A/APOBEC3F, H108A/APOBEC3G and APOBEC3F. The experiment wasrepeated with similar results. In summary, Vif mu-tants with a growth-defect in H9 cells were alwayssusceptible to anti-viral activity of APOBEC3G and/or APOBEC3F to a various extent as shown in Table1. Of note, viral ability to neutralize APOBEC3G/APOBEC3F paralleled perfectly with viral growthproperty in H9 cells (Fig. 1).It has been reported that APOBEC3G/APOBEC3Fare present in high molecular mass (HMM) com-plexes in some cells such as immortalized or acti-vated primary T cells and that they reside in lowmolecular mass (LMM) complexes in the restingprimary T cells (20, 21). It has also been shownthat only LMM complexes exhibit the cytidinedeaminase activity in vitro (20, 21). These findingsprompted us to examine whether HIV-1 Vif is asso-ciated with the protein complexes of APOBEC3G/APOBEC3F. We employed equilibrium density cen-trifugation to segregate HIV-1 Vif and APOBEC3G/APOBEC3F complexes. As a control Vif mutant,H108A, which can bind to both of APOBEC3G/APOBEC3F (3, 4) and are unable to neutralizeAPOBEC3G/APOBEC3F (Table 1), was chosenand used in this experiment. Cell and virion lysateswere prepared from 293T cells co-transfected withan expression vector for APOBEC3G/APOBEC3Fand WT or H108A mutant proviral clone. Lysateswere then centrifuged onto 5-50% sucrose gradient,and samples collected from centrifugal tubes weresubjected to immunoblot analysis. As shown inFig. 3, in the absence of Vif, while Gag-p24 wasmostly observed in low density fractions in virus-producer cells and virions, APOBEC3G/APOBEC3Fwere mainly present in high density fractions inTable 1. Ability of various vif mutants to neutralize APOBEC3G/APOBEC3FMutant A3G A3FW21A - -L24A ++ +H28A ++ ++S32A - -W38A - -Y40A - ++H43A + ++Y69A - -E76A ++ -W79A ++ -D104A + +H108A - -C114S - -C133S - -H139A - -A3G, APOBEC3G ; A3F, APOBEC3F.++, Wild type ability ; +, Partially defective ; -, No ability.Figure 3 : Profile of Vif and APOBEC3G/APOBEC3F complexes in producer cells and progeny virions as revealed by equilibriumdensity centrifugation. Cell and virion lysates were prepared from 293T cells co-transfected with a proviral cloned and an expressionvector of APOBEC3G/APOBEC3F as described in the legend to Fig. 2, and were ultracentrifuged onto 5-50% sucrose gradient. Eightfractions collected from each tube were then analyzed by immunoblotting. WT, wild- type virus NL4-3 ; ΔVif, Vif -minus virus NL-Nd(22) ; Low, low density ; High, high density.T. Nagao, et al. HIV-1 Vif and APOBEC3G/F92the cells. In contrast, in virions, APOBEC3G andAPOBEC3F were found to predominantly exist inLMM and HMM fractions, respectively. We alsoexamined APOBEC3G/APOBEC3F protein com-plexes in the presence of WT and H108A Vif. WTand H108A Vif did not affect the APOBEC3G/APOBEC3F complexes in cells and virions signifi-cantly (panels WT-Flag and H108A-Flag in Fig. 3).However, in producer cells, the mutation of His108partially shifted the Vif complexes from high to lowdensity (panels WT-Vif and H108A-Vif in Fig. 3).Whether this small mutational effect is directly re-lated to the inability of H108A-Vif to neutralizeAPOBEC3G/APOBEC3F remains to be determined.If related, the molecular basis for the phenotypeshould be studied in detail. A systematic analysis ofthe mutants other than H108A would give a more de-finitive answer. In virions, WT and H108A Vif simi-larly formed HMM protein complexes. In summary,the results described above suggested that HIV-1Vif interacts APOBEC3G/APOBEC3F in cells, andthat Vif may be contained in the APOBEC3F HMMcomplex in virions.In this study, we have demonstrated by using a va-riety of HIV-1 vif mutant viruses that APOBEC3G/APOBEC3F are essential to restrict viral replicationin target cells. All growth-defective mutants arealways sensitive to the strong anti-viral effect me-diated by APOBEC3G and/or APOBEC3F (Fig. 1,Fig. 2 and Table 1). This study has clearly indi-cated that HIV-1 Vif interacts with APOBEC3G/APOBEC3F differently. As illustrated in Fig. 1, thereare several motifs in HIV-1 Vif for binding toAPOBEC3G, APOBEC3F, or both. We confirmedprevious results and extended the observations re-ported. Importantly, there may be another domainaround the site of W21-L24-S32-W38 for binding toboth APOBEC proteins (FG-box), not reported yet.Our analysis of Vif-APOBEC protein complexeshere has also suggested that there may be molecu-lar difference between interactions of Vif and the twoAPOBEC proteins in virions. In virions, while Vifand APOBEC3F was present in HMM fractions,(particularly in the case of H108A Vif), APOBEC3Gresided in LMM fractions. In this regard, our find-ing that H108A Vif does not bind to APOBEC3G invirions but does in cells (4) is interesting. In con-trast, WT Vif was shown to bind to APOBEC3G invirions (4). More detailed biochemical studies wouldreveal molecular structural basis for different in-teraction of Vif and APOBEC3G/APOBEC3F.ACKNOWLEDGMENTSWe thank Ms. Kazuko Yoshida for editorial assis-tance. This work is supported in part by a Grant-in-Aid for Scientific Research (B)(21390141) from theJapan Society for the Promotion of Science, and bya Grant-in Aid for Scientific Research on PriorityAreas (21022033) from the Ministry of Education,Culture, Sports, Science and Technology of Japan.REFERENCES1. Holmes RK, Malim MH, Bishop KN : APOBEC-mediated viral restriction : not simply editing?Trends Biochem Sci 32 : 118-128, 2007.2. He Z, Zhang W, Chen G., Xu R, Yu XF : Char-acterization of conserved motifs in HIV-1 Vif re-quired for APOBEC3G and APOBEC3F inter-action. J Mol Biol 381 : 1000-1011, 2008.3. Yamashita T, Kamada K, Hatcho K, Adachi A,Nomaguchi M : Identification of amino acidresidues in HIV-1 Vif critical for binding andexclusion of APOBEC3G/F. Microbes Infect10 : 1142-1149, 2008.4. Yamashita T, Nomaguchi M, Miyake A,Uchiyama T, Adachi A : Status of APOBEC3G/F in cells and progeny virions modulated by Vifdetermines HIV-1 infectivity. Microbes Infect(in press)5. Tian C, Yu X, Zhang W, Wang T, Xu R, Yu XF :Differential requirement for conserved trypto-phans in human immunodeficiency virus type 1Vif for the selective suppression of APOBEC3Gand APOBEC3F. J Virol 80 : 3112-3115, 2006.6. Russel RA, Pathak VK : Identification of two dis-tinct human immunodeficiency virus type 1 Vifdeterminants critical for interactions with hu-man APOBEC3G and APOBEC3F. J Virol 81 :8201-8210, 2007.7. Mehle A, Wilson H, Zhang C, Brazier AJ,McPike M, Pery E, Gabuzda D : Identificationof an APOBEC3G binding site in human immu-nodeficiency virus type 1 Vif and inhibitors ofVif-APOBEC3G binding. J Virol 81 : 13235-13241, 2007.8. Pery E, Rajendran K, Brazier AJ, Gabuzda D :Regulation of APOBEC3 proteins by a novelYXXL motif in human immunodeficiency virustype 1 Vif and simian immunodeficiency virusSIVagm Vif. J Virol 83 : 2374-2381, 2009.9. Dang Y, Wang X, Zhou T, York IA, Zheng YH :The Journal of Medical Investigation Vol. 57 February 2010 93Identification of a novel WxSLVK motif in theN terminus of human immunodeficiency virusand simian immunodeficiency virus Vif that iscritical for APOBEC3G and APOBEC3F neu-tralization. J Virol 83 : 8544-8552, 2009.10. Chen G, He Z, Wang T, Xu R, Yu XF : A patchof positively charged amino acids surroundingthe human immunodeficiency virus type 1 VifSLVx4Yx9Y motif influences its interaction withAPOBEC3G. J Virol 83 : 8674-8682, 2009.11. Yu Y, Xiao Z, Ehrlich ES, Yu X, Yu F : Selec-tive assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex througha novel SOCS box and upstream cysteinesGenes Dev 18 : 2867-2872, 2004.12. Luo K, Xiao Z, Ehrlich E, Yu Y, Liu B, ZhengS, Yu XF : Primate lentiviral virion infectivityfactors are substrate receptors that assemblewith cullin 5-E3 ligase through a HCCH motifto suppress APOBEC3G. Proc Natl Acad SciUSA 102 : 11444-11449, 2005.13. Mehle A, Thomas ER, Rajendran KS, GabuzdaD : A zinc-binding region in Vif binds Cul5 anddetermines cullin selection. J Biol Chem 281 :17259-65, 2006.14. Xiao Z, Ehrlich E, Yu Y, Luo K, Wang T, TianC, Yu XF : Assembly of HIV-1 Vif-Cul5 E3ubiquitin ligase through a novel zinc-bindingdomain-stabilized hydrophobic interface in Vif.Virology 349 : 290-299, 2006.15. Lebkowski JS, Clancy S, Calos MP : Simian vi-rus 40 replication in adenovirus-transformedhuman cells antagonizes gene expression. Na-ture 12 : 169-171, 1985.16. Kimpton J, Emerman M : Detection of replica-tion-competent and pseudotyped human immu-nodeficiency virus with a sensitive cell line onthe basis of activation of an integrated β-galac-tosidase gene. J Virol 66 : 2232-2239, 1992.17. Adachi A, Gendelman HE, Koenig S, Folks T,Willey R, Rabson A, Martin MA : Production ofacquired immunodeficiency syndrome-associ-ated retrovirus in human and nonhuman cellstransfected with an infectious molecular clone.J Virol 59 : 284-291, 1986.18. Akari H, Fukumori T, Adachi A : Cell-depend-ent requirement of human immunodeficiencyvirus type 1 gp41 cytoplasmic tail for Env in-corporation into virions J Virol 74 : 4891-4893,2000.19. Fujita M, Sakurai A, Yoshida A, Matsumoto S,Miyaura M, Adachi A : Subtle mutations in thecysteine region of HIV-1 Vif drastically alter theviral replication phenotype. Microbes Infect 4 :621-624, 2002.20. Chiu YL, Soros VB, Kreisberg JF, Stopak K,Yonemoto W, Greene WC :Cellular APOBEC3Grestricts HIV-1 infection in resting CD4+ Tcells. Nature 435 : 108-114, 2005.21. Wang X, Dolan PT, Dang Y, Zheng YH : Bio-chemical differentiation of APOBEC3F andAPOBEC3G proteins associated with HIV-1 lifecycle. J Biol Chem 282 : 1585-1594, 2007.22. Adachi A, Ono N, Sakai H, Ogawa K, ShibataR, Kiyomasu T, Masuike H, Ueda S : Genera-tion and characterization of the human immu-nodeficiency virus type 1 mutants. Arch Virol117 : 45-58, 1991.T. Nagao, et al. HIV-1 Vif and APOBEC3G/F94

Different interaction between HIV-1 Vif and its cellular target proteins APOBEC3G/APOBEC3F

http://repo.lib.tokushima-u.ac.jp/files/public/7/72821/20170929140659549630/LID201112262010.pdf

Different interaction between HIV-1 Vif and its cellular target proteins APOBEC3G/APOBEC3F

Abstract

Similar works

Full text

Available Versions

Tokushima University Institutional Repository