FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2149117537> ?p ?o ?g. }

• W2149117537 endingPage "43213" @default.
• W2149117537 startingPage "43202" @default.
• W2149117537 abstract "Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA–/– knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells. Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA–/– knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells. In addition to the genes encoding the structural and enzymatic proteins common to all retroviruses, the human immunodeficiency virus, type 1 (HIV-1) 1The abbreviations used are: HIV-1/2, human immunodeficiency virus type 1/2; CsA, cyclosporin A; CypA, cyclophilin A; CyPs, cyclophilins; PPIase, peptidyl-prolyl cis/trans isomerase; PAA, polyacrylamide; SFA, sanglifehrin A; SIV, simian immunodeficiency virus; sVpr, synthetic full-length Vpr; CA, capsid; CMV, cytomegalovirus; FACS, fluorescence-activated cell sorter; HA, hemagglutinin.1The abbreviations used are: HIV-1/2, human immunodeficiency virus type 1/2; CsA, cyclosporin A; CypA, cyclophilin A; CyPs, cyclophilins; PPIase, peptidyl-prolyl cis/trans isomerase; PAA, polyacrylamide; SFA, sanglifehrin A; SIV, simian immunodeficiency virus; sVpr, synthetic full-length Vpr; CA, capsid; CMV, cytomegalovirus; FACS, fluorescence-activated cell sorter; HA, hemagglutinin. genome contains four accessory genes that serve to accelerate viral replication. One of these gene products, the highly conserved 96-amino acid viral protein R (Vpr) has received considerable attention, and a number of biological functions have been attributed to its presence in various cellular and extracellular compartments. The most intensively investigated biological functions of Vpr are those affecting the translocation of the preintegration complex of the incoming virus from the cytoplasm to the nucleus and the arrest in the G2 phase of the cell cycle (1Sherman M.P. Greene W.C. Microbes Infect. 2002; 4: 67-73Crossref PubMed Scopus (103) Google Scholar, 2Sherman M.P. Schubert U. Williams S.A. de Noronha C.M. Kreisberg J.F. Henklein P. Greene W.C. Virology. 2002; 302: 95-105Crossref PubMed Scopus (75) Google Scholar, 3Bukrinsky M. Adzhubei A. Rev. Med. Virol. 1999; 9: 39-49Crossref PubMed Scopus (78) Google Scholar). The nuclear targeting function of Vpr has been associated with HIV-1 infection of terminally differentiated macrophages. Regarding the second function of Vpr, which leads to G2 cell cycle arrest in HIV-1-infected and/or Vpr-transfected human cells, it was implicated that this activity provides an intracellular milieu conductive for enhanced viral replication by increasing HIV LTR-driven gene expression (4Goh W.C. Rogel M.E. Kinsey C.M. Michael S.F. Fultz P.N. Nowak M.A. Hahn B.H. Emerman M. Nat. Med. 1998; 4: 65-71Crossref PubMed Scopus (437) Google Scholar). This response has been linked recently (5de Noronha C.M. Sherman M.P. Lin H.W. Cavrois M.V. Moir R.D. Goldman R.D. Greene W.C. Science. 2001; 294: 1105-1108Crossref PubMed Scopus (224) Google Scholar) with the capacity of Vpr to alter the structure of the nuclear lamina, leading to transient, DNA-containing herniations of the nuclear envelope that intermittently rupture. Other studies (6Poon B. Grovit-Ferbas K. Stewart S.A. Chen I.S. Science. 1998; 281: 266-269Crossref PubMed Scopus (164) Google Scholar, 7Stewart S.A. Poon B. Song J.Y. Chen I.S. J. Virol. 2000; 74: 3105-3111Crossref PubMed Scopus (141) Google Scholar, 8Yao X.J. Mouland A.J. Subbramanian R.A. Forget J. Rougeau N. Bergeron D. Cohen E.A. J. Virol. 1998; 72: 4686-4693Crossref PubMed Google Scholar) suggest that the prolonged G2 arrest induced by Vpr ultimately leads to apoptosis of the infected cell. Conversely, early anti-apoptotic effects of Vpr have also been described that are superceded later by its pro-apoptotic effects (9Conti L. Matarrese P. Varano B. Gauzzi M.C. Sato A. Malorni W. Belardelli F. Gessani S. J. Immunol. 2000; 165: 3293-3300Crossref PubMed Scopus (55) Google Scholar). These pro-apoptotic effects of Vpr may result from either effects on the integrity of the nuclear envelope or direct mitochondrial membrane permeabilization (10Ferri K.F. Jacotot E. Blanco J. Este J.A. Kroemer G. Ann. N. Y. Acad. Sci. 2000; 926: 149-164Crossref PubMed Scopus (67) Google Scholar), perhaps involving Vpr-mediated formation of ion channels in cellular membranes (11Piller S.C. Ewart G.D. Premkumar A. Cox G.B. Gage P.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 111-115Crossref PubMed Scopus (104) Google Scholar).As the biologically relevant activity of the multifunctional Vpr has not yet been clarified, the molecular bases for many of its effects remain elusive. To date structural studies of the full-length molecule have been hampered by the fact that this protein does not crystallize, and the use of NMR techniques is complicated by the strong tendency for Vpr to undergo self-association. In addition, the structure of Vpr depends critically on the solution conditions, and an unresolved apparent heterogeneity in the composition of the polypeptide has been observed (12Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar, 13Henklein P. Bruns K. Sherman M.P. Tessmer U. Licha K. Kopp J. de Noronha C.M. Greene W.C. Wray V. Schubert U. J. Biol. Chem. 2000; 275: 32016-32026Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). However, a somewhat limited model is slowly emerging from structural studies of fragments of Vpr (14Luo Z. Butcher D.J. Murali R. Srinivasan A. Huang Z. Biochem. Biophys. Res. Commun. 1998; 244: 732-736Crossref PubMed Scopus (24) Google Scholar, 15Schuler W. Wecker K. de Rocquigny H. Baudat Y. Sire J. Roques B.P. J. Mol. Biol. 1999; 285: 2105-2117Crossref PubMed Scopus (94) Google Scholar, 16Wecker K. Roques B.P. Eur. J. Biochem. 1999; 266: 359-369Crossref PubMed Scopus (56) Google Scholar, 17Engler A. Stangler T. Willbold D. Eur. J. Biochem. 2001; 268: 389-395Crossref PubMed Scopus (21) Google Scholar, 18Engler A. Stangler T. Willbold D. Eur. J. Biochem. 2002; 269: 3264-3269Crossref PubMed Scopus (18) Google Scholar, 19Bruns K. Fossen T. Wray V. Henklein P. Tessmer U. Schubert U. J. Biol. Chem. 2003; 278: 43156-43169Abstract Full Text Full Text PDF Scopus (51) Google Scholar) and, more recently, of full-length synthetic forms of Vpr (12Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar, 13Henklein P. Bruns K. Sherman M.P. Tessmer U. Licha K. Kopp J. de Noronha C.M. Greene W.C. Wray V. Schubert U. J. Biol. Chem. 2000; 275: 32016-32026Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). These studies were performed in aqueous solution that required the presence of either detergents or micelles to detect the most structured forms of Vpr. The secondary structures in Vpr emerging from these analyses (summarized in the accompanying paper (19Bruns K. Fossen T. Wray V. Henklein P. Tessmer U. Schubert U. J. Biol. Chem. 2003; 278: 43156-43169Abstract Full Text Full Text PDF Scopus (51) Google Scholar)) suggest the presence of an α-helix-turn-α-helix motif between residues 17 and 48 and an amphipathic α-helix between amino acids 53–55 and 78–83 (12Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar, 15Schuler W. Wecker K. de Rocquigny H. Baudat Y. Sire J. Roques B.P. J. Mol. Biol. 1999; 285: 2105-2117Crossref PubMed Scopus (94) Google Scholar). These helices most likely play a key role in self-association and the interaction of Vpr with heterologous proteins (13Henklein P. Bruns K. Sherman M.P. Tessmer U. Licha K. Kopp J. de Noronha C.M. Greene W.C. Wray V. Schubert U. J. Biol. Chem. 2000; 275: 32016-32026Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Zhao L.J. Wang L. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 32131-32137Abstract Full Text PDF PubMed Google Scholar). Both helical domains are highly conserved and contribute to the formation of a novel nuclear import signal (21Sherman M.P. de Noronha C.M. Pearce D. Greene W.C. J. Virol. 2000; 74: 8159-8165Crossref PubMed Scopus (64) Google Scholar). The distal helix also contains a leucine-rich nuclear export signal whose function is inhibited by leptomycin B (22Sherman M.P. de Noronha C.M. Heusch M.I. Greene S. Greene W.C. J. Virol. 2001; 75: 1522-1532Crossref PubMed Scopus (108) Google Scholar). The C terminus of Vpr contains a basic amino acid-rich segment between residues 73 and 96 for which little definite structure has been assigned (15Schuler W. Wecker K. de Rocquigny H. Baudat Y. Sire J. Roques B.P. J. Mol. Biol. 1999; 285: 2105-2117Crossref PubMed Scopus (94) Google Scholar). However, this region is conserved and influences the stability and, potentially, the structure of the entire protein (8Yao X.J. Mouland A.J. Subbramanian R.A. Forget J. Rougeau N. Bergeron D. Cohen E.A. J. Virol. 1998; 72: 4686-4693Crossref PubMed Google Scholar). It contains a bipartite, arginine-rich nuclear import signal that can promote nuclear uptake of heterologous proteins via the nuclear pore complex (22Sherman M.P. de Noronha C.M. Heusch M.I. Greene S. Greene W.C. J. Virol. 2001; 75: 1522-1532Crossref PubMed Scopus (108) Google Scholar, 23Jenkins Y. McEntee M. Weis K. Greene W.C. J. Cell Biol. 1998; 143: 875-885Crossref PubMed Scopus (175) Google Scholar). Various investigations have shown that the C-terminal domain also participates in a number of specific protein-protein and protein-nucleic acid interactions such as with NCp7 (24de Rocquigny H. Petitjean P. Tanchou V. Decimo D. Drouot L. Delaunay T. Darlix J.L. Roques B.P. J. Biol. Chem. 1997; 272: 30753-30759Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), Tat (25Sawaya B.E. Khalili K. Rappaport J. Serio D. Chen W. Srinivasan A. Amini S. Gene Ther. 1999; 6: 947-950Crossref PubMed Scopus (23) Google Scholar), RNA (26de Rocquigny H. Caneparo A. Delaunay T. Bischerour J. Mouscadet J.F. Roques B.P. Eur. J. Biochem. 2000; 267: 3654-3660Crossref PubMed Scopus (34) Google Scholar), and the adenine nucleotide translocator of the mitochondrial pore (27Jacotot E. Ravagnan L. Loeffler M. Ferri K.F. Vieira H.L. Zamzami N. Costantini P. Druillennec S. Hoebeke J. Briand J.P. Irinopoulou T. Daugas E. Susin S.A. Cointe D. Xie Z.H. Reed J.C. Roques B.P. Kroemer G. J. Exp. Med. 2000; 191: 33-46Crossref PubMed Scopus (401) Google Scholar).In contrast to the C-terminal domain, few functional properties have been attributed to the N-terminal domain of Vpr, and its participation as an interaction surface with other molecules has received less attention. Many previous structural characterizations of Vpr (for instance see Refs. 12Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar, 13Henklein P. Bruns K. Sherman M.P. Tessmer U. Licha K. Kopp J. de Noronha C.M. Greene W.C. Wray V. Schubert U. J. Biol. Chem. 2000; 275: 32016-32026Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, and 20Zhao L.J. Wang L. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 32131-32137Abstract Full Text PDF PubMed Google Scholar) have shown the important influence of environmental factors and indicate that in vivo folding of Vpr will probably require the presence of structure-stabilizing interacting factors (for review see Ref. 3Bukrinsky M. Adzhubei A. Rev. Med. Virol. 1999; 9: 39-49Crossref PubMed Scopus (78) Google Scholar). Furthermore, we have recently focused our attention on the N-terminal domain of Vpr (19Bruns K. Fossen T. Wray V. Henklein P. Tessmer U. Schubert U. J. Biol. Chem. 2003; 278: 43156-43169Abstract Full Text Full Text PDF Scopus (51) Google Scholar) and found that two of the four conserved prolines at residues 14 and 35 in this domain exhibit an unusually high content of cis-conformations of the imidic bond. Based on the cis/trans phenomenon that could be important for the folding of Vpr, it was projected that a potential interaction between Vpr and a host peptidyl-prolyl isomerase (PPIase) might regulate the interconversion of the imidic bond of these N-terminal proline residues of Vpr in vivo.As for most other intracellular parasites, the replication of HIV depends on the interaction with host cell factors, and some of these are incorporated specifically into progeny virions. Among the most abundant cellular proteins and the first ever found in HIV-1 virions is cyclophilin A (CypA). This protein is specifically incorporated into HIV-1 virions, but not into virions of other lentiviruses, through an interaction with a proline-rich region of the HIV-1 capsid (CA) protein. In particular, Pro-222, which is conserved in the CA region of all HIV-1 Gag polyproteins, appears to be important for the interaction of HIV-1 Gag with CypA (Refs. 28Franke E.K. Yuan H.E. Luban J. Nature. 1994; 372: 359-362Crossref PubMed Scopus (645) Google Scholar and 29Thali M. Bukovsky A. Kondo E. Rosenwirth B. Walsh C.T. Sodroski J. Gottlinger H.G. Nature. 1994; 372: 363-365Crossref PubMed Scopus (559) Google Scholar; for review see Refs. 30Luban J. Cell. 1996; 87: 1157-1159Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and 31Ott D.E. Rev. Med. Virol. 2002; 12: 359-374Crossref PubMed Scopus (95) Google Scholar). Earlier studies using two-hybrid screens found that HIV-1 Gag binds to most of the known members of the family of CyPs (32Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (700) Google Scholar). CyPs were originally described as binding partners of cyclosporin A (CsA), an immunosuppressive cyclic undecapeptide used clinically to prevent allocraft rejection (33Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1442) Google Scholar). CsA binds with nanomolar affinity to CypA, and this complex inhibits calcineurin, a calcium-dependent phosphatase that regulates the expression of various cytokine genes in activated T cells (for review see Ref. 34Ivery M.T. Med. Res. Rev. 2000; 20: 452-484Crossref PubMed Scopus (116) Google Scholar). CyPs belong to the PPIases, a group of enzymes found in organisms ranging from prokaryotes to humans, that catalyze the otherwise relatively slow cis/trans isomerization of peptidyl-prolyl bonds in vitro. As CyPs have been proposed to regulate protein folding in vivo (34Ivery M.T. Med. Res. Rev. 2000; 20: 452-484Crossref PubMed Scopus (116) Google Scholar) it was conceivable that the family of CyPs, and CypA in particular, are possible candidates for providing a PPIase activity that regulates cis/trans interconversion in Vpr.A large body of evidence supports a function of CypA in formation of infectious HIV-1 viruses (for review see Refs. 30Luban J. Cell. 1996; 87: 1157-1159Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and 31Ott D.E. Rev. Med. Virol. 2002; 12: 359-374Crossref PubMed Scopus (95) Google Scholar). These studies were based either on mutation within gag or the use of competitive CypA inhibitors such as CsA that interfere with the CypA-Gag interaction (35Steinkasserer A. Harrison R. Billich A. Hammerschmid F. Werner G. Wolff B. Peichl P. Palfi G. Schnitzel W. Mlynar E. et al.J. Virol. 1995; 69: 814-824Crossref PubMed Google Scholar, 36Billich A. Hammerschmid F. Peichl P. Wenger R. Zenke G. Quesniaux V. Rosenwirth B. J. Virol. 1995; 69: 2451-2461Crossref PubMed Google Scholar, 37Braaten D. Aberham C. Franke E.K. Yin L. Phares W. Luban J. J. Virol. 1996; 70: 5170-5176Crossref PubMed Google Scholar, 38Braaten D. Ansari H. Luban J. J. Virol. 1997; 71: 2107-2113Crossref PubMed Google Scholar, 39Dorfman T. Weimann A. Borsetti A. Walsh C.T. Gottlinger H.G. J. Virol. 1997; 71: 7110-7113Crossref PubMed Google Scholar). A more conclusive proof that among the other 14 known members of mammalian CyPs only CypA plays a functional role in supporting HIV-1 replication was provided by selective genetic inactivation of the gene encoding CypA in human CD4+ T cells (40Braaten D. Luban J. EMBO J. 2001; 20: 1300-1309Crossref PubMed Scopus (234) Google Scholar). In addition, the competitive inhibitor CsA exhibits high affinity for the hydrophobic pocket of almost all CyPs and thus competitively inhibits the interaction of CypA with HIV-1 Gag (28Franke E.K. Yuan H.E. Luban J. Nature. 1994; 372: 359-362Crossref PubMed Scopus (645) Google Scholar, 29Thali M. Bukovsky A. Kondo E. Rosenwirth B. Walsh C.T. Sodroski J. Gottlinger H.G. Nature. 1994; 372: 363-365Crossref PubMed Scopus (559) Google Scholar, 32Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 35Steinkasserer A. Harrison R. Billich A. Hammerschmid F. Werner G. Wolff B. Peichl P. Palfi G. Schnitzel W. Mlynar E. et al.J. Virol. 1995; 69: 814-824Crossref PubMed Google Scholar, 37Braaten D. Aberham C. Franke E.K. Yin L. Phares W. Luban J. J. Virol. 1996; 70: 5170-5176Crossref PubMed Google Scholar, 41Rosenwirth B. Billich A. Datema R. Donatsch P. Hammerschmid F. Harrison R. Hiestand P. Jaksche H. Mayer P. Peichl P. et al.Antimicrob. Agents Chemother. 1994; 38: 1763-1772Crossref PubMed Scopus (161) Google Scholar, 42Franke E.K. Luban J. Virology. 1996; 222: 279-282Crossref PubMed Scopus (132) Google Scholar). The immunosuppressive activity of CsA is not correlated with anti-HIV activity, since the nonimmunosuppressive derivative NIM811 ([methyl-Ile4]-cyclosporin) is an even more potent inhibitor of CypA-mediated HIV-1 replication than the parental CsA (29Thali M. Bukovsky A. Kondo E. Rosenwirth B. Walsh C.T. Sodroski J. Gottlinger H.G. Nature. 1994; 372: 363-365Crossref PubMed Scopus (559) Google Scholar, 36Billich A. Hammerschmid F. Peichl P. Wenger R. Zenke G. Quesniaux V. Rosenwirth B. J. Virol. 1995; 69: 2451-2461Crossref PubMed Google Scholar, 39Dorfman T. Weimann A. Borsetti A. Walsh C.T. Gottlinger H.G. J. Virol. 1997; 71: 7110-7113Crossref PubMed Google Scholar, 41Rosenwirth B. Billich A. Datema R. Donatsch P. Hammerschmid F. Harrison R. Hiestand P. Jaksche H. Mayer P. Peichl P. et al.Antimicrob. Agents Chemother. 1994; 38: 1763-1772Crossref PubMed Scopus (161) Google Scholar, 42Franke E.K. Luban J. Virology. 1996; 222: 279-282Crossref PubMed Scopus (132) Google Scholar). Thus, CsA and related nonimmunosuppressive derivatives form an interesting class of drugs that can modulate the interaction of CypA with other proteins.In the present work, we demonstrate an unexpected interplay of Vpr with CypA that depends of the N-terminal domain of Vpr containing conserved proline residues. Most importantly, we report that CypA regulates the expression of Vpr and is required for the Vpr-mediated G2 cell cycle arrest in HIV-1-infected T cells.MATERIALS AND METHODSBIAcore Spectroscopy—Surface plasmon resonance measurements were performed at 25 °C using a BIACORE 2000 (BIAcore AB, Uppsala, Sweden) equipped with a CM5 research-grade sensor chip. Recombinant CypA (Sigma) was immobilized at a concentration ranging from 4200 to 11200 response units using standard amine-coupling chemistry in three flow cells, and a further flow cell without CypA was used as a control. The peptides Vpr1–40, Vpr1–20, Vpr21–40, and respective proline mutants were dissolved at concentrations ranging from 1 to 250 μm in a running buffer (10 mm Hepes, 150 mm NaCl, 50 μm EDTA, 0.005% Tween 20, pH 7.4) and were injected over the flow cells at a flow rate of 5 μl/min. Data were collected at a rate of 2.5 Hz during the 120-s association and dissociation phase. Results were corrected for the response unit values of the reference cell (without CypA) to exclude unspecific binding of Vpr to the chip matrix. Experiments were repeated at least three times, each with two different charges of Vpr peptides and CypA, and afforded reproducible data.Northern Blot Analysis—HeLa cells were transfected with pCMV-FLAG-Vpr, and 5 h post-transfection cells were treated with CsA (50 μg/ml), NIM811 (10 μg/ml), SFA (10 μg/ml), or no inhibitor (aliquot of solvent Me2SO). Following 0, 2, or 8 h of treatment, cells were harvested, washed once in ice-cold phosphate-buffered saline, and frozen immediately at –80 °C. Total cell RNA was isolated using Trizol (Invitrogen), and 10 μg of each RNA sample was separated in 1.2% denaturing agarose gel containing 17.8% formaldehyde. Following blotting and UV cross-linking to the nylon membrane (Hybond-N™; Amersham Biosciences) the membrane was pre-hybridized for 5 h at 65 °C in Denhardt's solutions containing salmon sperm DNA and poly(A) RNA according to modification of a standard procedure described previously (43Valent P. Bevec D. Maurer D. Besemer J. Di Padova F. Butterfield J.H. Speiser W. Majdic O. Lechner K. Bettelheim P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3339-3342Crossref PubMed Scopus (97) Google Scholar). For hybridization oligonucleotides (FLAG probe 5′-cttgtcgtcatcgtctttgtatgccat-3′, Vpr probe 5′-agtaacgcctattctgctatgtcgacacccaattctgaaatg-3′) were radioactively labeled with [α-32P]dCTP using terminal transferase (220 582; Roche Applied Science) and added to the hybridization solution for 12 h at 65 °C. After washing, blots were analyzed by autoradiography. For internal control blots were stripped for 30 min in 0.1% SDS at 70 °C and re-hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe (5′-ccatggtggtgaagacgccagtggactcc-3).Transfection, Infections, Immunostaining, Flow Cytometry, and DNA Content Analysis—Cells were transfected with LipofectAMINE 2000™ or calcium phosphate for precipitation of DNA. T cell cultures were infected with isogenic strains of HIV-1NL4–3, differing only by the presence or absence of vpr (44Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar, 45Eckstein D.A. Sherman M.P. Penn M.L. Chin P.S. De Noronha C.M. Greene W.C. Goldsmith M.A. J. Exp. Med. 2001; 194: 1407-1419Crossref PubMed Scopus (104) Google Scholar). Generally, 107 cells were incubated with virus stocks (200 ng of p24Gag) in 1 ml of medium for 2 h prior to washing and subsequent culturing at 1 × 106 cells per ml for the indicated times. HIV-infected cells were fixed and permeabilized with a solution of 1% paraformaldehyde, 1 mg/ml human IgG (Gemini Bio-Products, Inc.), and 0.1% Tween 20 in FACS buffer for at least 1 h. 106 cells were immunostained in 50 μl of FACS buffer with 1:50 dilution of the monoclonal anti-p24 antibody KC57 (Lot 13; Coulter), conjugated to fluorescein isothiocyanate. Cellular DNA content was assessed by additional staining with 0.01 mm To-Pro-3 iodide (Molecular Probes) in the presence of 1 mg/ml RNaseA followed by analysis using a FACS-can™ flow cytometer acquiring linear fluorescence in the FL4 channel. DNA profiles were analyzed with FlowJo software (Treestar).Cell Culture, Pulse-Chase, and Western Blot Analyses—CD4+ human T lymphoma Jurkat T cell lines and the knock-out line PPIA – / – were cultured in RPMI 1640, and HeLa cells (ATCC CCL2) were propagated in Dulbecco's modified Eagle's medium. Cells were metabolically labeled with [35S]methionine (2 mCi/ml), and pulse-chase experiments and immunoprecipitation were performed as described (46Schubert U. Clouse K.A. Strebel K. J. Virol. 1995; 69: 7699-7711Crossref PubMed Google Scholar). For Vpr detection in virions cell, culture supernatants were tested with p24 enzyme-linked immunosorbent assay to assess peak virus replication during a 7- to 10-day period. Virus pellets were normalized for CA content by Western blot and enzyme-linked immunosorbent assay, and aliquots were separated by SDS-PAGE and probed by Western blot with rabbit antibodies specific for CA and Vpr followed by enhanced chemiluminescence staining. For Western blot kinetic analyses, HeLa cells were transfected with pCMV-FLAG-Vpr, pCMV-3HA-Vpr wild type, pCMV-3HA-VprP5,10,14A, or pCMV-3HA-VprP35N, and after the optimum time post-transfection necessary to initiate homologous gene expression that was determined for each construct, cells were harvested by scraping, aliquoted in RPMI, and treated with CsA (50 μg/ml), NIM811 (10 μg/ml), SFA (10 μg/ml), or no inhibitors (aliquot of solvent Me2SO). Samples were taken 0, 1, 2, 4, and 8 h after beginning of drug treatment, and cell lysates were separated in 14% SDS-PAA gels. Proteins were transferred onto nitrocellulose membranes and probed with specific antibodies followed by enhanced chemiluminescence detection. For internal controls blots were stripped for 20 min in 2% mercaptoethanol and 1% SDS at 65 °C and a re-incubated with antibodies specific for actin and CypA.Plasmids and Antibodies—The pCMV-FLAG-Vpr DNA plasmid directs the expression of HIV-1NL4–3 Vpr with the FLAG epitope at the N terminus (47Kino T. Gragerov A. Kopp J.B. Stauber R.H. Pavlakis G.N. Chrousos G.P. J. Exp. Med. 1999; 189: 51-62Crossref PubMed Scopus (187) Google Scholar). The pCMV-3HA-Vpr vector was used to express Vpr with an N-terminal hemagglutin (HA) tag of HIV-1NL4–3 (21Sherman M.P. de Noronha C.M. Pearce D. Greene W.C. J. Virol. 2000; 74: 8159-8165Crossref PubMed Scopus (64) Google Scholar). Mutations at proline residues 5, 10, 14, and 35 were introduced by designing PCR primers containing the indicated changes and cloning into the plasmid directly. The plasmid pNLenv1 represents an env-deleted version of HIV-1NL4–3 (46Schubert U. Clouse K.A. Strebel K. J. Virol. 1995; 69: 7699-7711Crossref PubMed Google Scholar). Antibodies specific for FLAG, actin, and HA were obtained from Sigma, the CypA antibody was from Calbiochem, and the Vpr antibody was as described (13Henklein P. Bruns K. Sherman M.P. Tessmer U. Licha K. Kopp J. de Noronha C.M. Greene W.C. Wray V. Schubert U. J. Biol. Chem. 2000; 275: 32016-32026Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The peroxidase-coupled anti-mouse and anti-rabbit antibodies were obtained from Dianova.In Vitro and in Vivo Complex Formation of Vpr and CypA—1 μg of each, recombinant CypA (Calbiochem) and sVpr, were incubated in 1 ml of phosphate-buffered saline, pH 7.2, at 22 °C for 12 h. Aliquots of the mixture were denatured in SDS-PAGE sample buffer (without reducing agents), incubated at room temperature for 30 min, separated in a 12.5% PAA gel, and analyzed by Western blotting using rabbit anti-CypA (Calbiochem) and anti-Vpr (R-96) antibodies. For co-immunoprecipitation, 3 × 107 HeLa cells transfected with pCMV-FLAG-Vpr were lysed in lysis buffer (140 mm NaCl, 10 mm Tris/HCl, pH 7.4, 1 mm EDTA, protease inhibitor mixture (Roche Applied Science), 1% (w/v) digitonin (Wako)), and the cytosolic fraction was isolated by centrifugation (4 °C, 15 min, 14,000 × g) and incubated with anti-FLAG antibodies for 12 h at 4 °C. Immune complexes were recovered on protein G-Sepharose, washed" @default.
• W2149117537 created "2016-06-24" @default.
• W2149117537 creator A5023784884 @default.
• W2149117537 creator A5030856723 @default.
• W2149117537 creator A5036857062 @default.
• W2149117537 creator A5038060915 @default.
• W2149117537 creator A5052639237 @default.
• W2149117537 creator A5058961803 @default.
• W2149117537 creator A5070067793 @default.
• W2149117537 creator A5075012306 @default.
• W2149117537 creator A5075675860 @default.
• W2149117537 creator A5080348355 @default.
• W2149117537 creator A5081790590 @default.
• W2149117537 creator A5086209382 @default.
• W2149117537 date "2003-10-01" @default.
• W2149117537 modified "2023-10-12" @default.
• W2149117537 title "Cyclophilin A Interacts with HIV-1 Vpr and Is Required for Its Functional Expression" @default.
• W2149117537 cites W1514433785 @default.
• W2149117537 cites W1517050036 @default.
• W2149117537 cites W1538467835 @default.
• W2149117537 cites W1569415412 @default.
• W2149117537 cites W1583620363 @default.
• W2149117537 cites W1595218557 @default.
• W2149117537 cites W1659719610 @default.
• W2149117537 cites W1661847997 @default.
• W2149117537 cites W1745607124 @default.
• W2149117537 cites W1944888779 @default.
• W2149117537 cites W1963587681 @default.
• W2149117537 cites W1966116190 @default.
• W2149117537 cites W1968391451 @default.
• W2149117537 cites W1969819429 @default.
• W2149117537 cites W1972558420 @default.
• W2149117537 cites W1973208298 @default.
• W2149117537 cites W1975534778 @default.
• W2149117537 cites W1977513191 @default.
• W2149117537 cites W1979635267 @default.
• W2149117537 cites W1981498914 @default.
• W2149117537 cites W1992095999 @default.
• W2149117537 cites W1999605002 @default.
• W2149117537 cites W2001849808 @default.
• W2149117537 cites W2003603481 @default.
• W2149117537 cites W2010533729 @default.
• W2149117537 cites W2011537253 @default.
• W2149117537 cites W2011952922 @default.
• W2149117537 cites W2012124354 @default.
• W2149117537 cites W2012564733 @default.
• W2149117537 cites W2020597127 @default.
• W2149117537 cites W2024249136 @default.
• W2149117537 cites W2024569169 @default.
• W2149117537 cites W2028400386 @default.
• W2149117537 cites W2035218720 @default.
• W2149117537 cites W2045562714 @default.
• W2149117537 cites W2048731959 @default.
• W2149117537 cites W2052659154 @default.
• W2149117537 cites W2062639699 @default.
• W2149117537 cites W2071910567 @default.
• W2149117537 cites W2076811542 @default.
• W2149117537 cites W2078248506 @default.
• W2149117537 cites W2085570807 @default.
• W2149117537 cites W2087480936 @default.
• W2149117537 cites W2089135460 @default.
• W2149117537 cites W2089850443 @default.
• W2149117537 cites W2091660627 @default.
• W2149117537 cites W2095633554 @default.
• W2149117537 cites W2097744772 @default.
• W2149117537 cites W2099998840 @default.
• W2149117537 cites W2102644356 @default.
• W2149117537 cites W2103368826 @default.
• W2149117537 cites W2105944752 @default.
• W2149117537 cites W2109324904 @default.
• W2149117537 cites W2117034018 @default.
• W2149117537 cites W2121753315 @default.
• W2149117537 cites W2127582557 @default.
• W2149117537 cites W2146734555 @default.
• W2149117537 cites W2148198051 @default.
• W2149117537 cites W2149227294 @default.
• W2149117537 cites W2149401677 @default.
• W2149117537 cites W2153689167 @default.
• W2149117537 cites W2158685964 @default.
• W2149117537 cites W2162682093 @default.
• W2149117537 cites W2165284441 @default.
• W2149117537 cites W2166676812 @default.
• W2149117537 cites W2166987747 @default.
• W2149117537 cites W2169601134 @default.
• W2149117537 cites W2171662092 @default.
• W2149117537 cites W2314491483 @default.
• W2149117537 cites W4235464062 @default.
• W2149117537 doi "https://doi.org/10.1074/jbc.m305414200" @default.
• W2149117537 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12881522" @default.
• W2149117537 hasPublicationYear "2003" @default.
• W2149117537 type Work @default.
• W2149117537 sameAs 2149117537 @default.
• W2149117537 citedByCount "81" @default.
• W2149117537 countsByYear W21491175372012 @default.
• W2149117537 countsByYear W21491175372013 @default.
• W2149117537 countsByYear W21491175372014 @default.
• W2149117537 countsByYear W21491175372015 @default.
• W2149117537 countsByYear W21491175372016 @default.

• next