The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains
© Solbak et al; licensee BioMed Central Ltd. 2011
Received: 1 July 2011
Accepted: 20 December 2011
Published: 20 December 2011
Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.
Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.
For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.
The viral protein R (Vpr) is encoded by the human immunodeficiency viruses types 1 and 2 (HIV-1/HIV-2), the simian immunodeficiency viruses (SIV) and primate lentiviruses [1, 2]. This accessory protein facilitates transport of the pre-integration complex into the nucleus of non-dividing cells  and fulfils multiple functions in the viral life cycle, including increase of viral replication in non-dividing host cells, induction of G2 cell-cycle arrest [4, 5], apoptosis [6, 7] and transduction through cell membranes  (The multiple functions of Vpr are reviewed in [9–11]). Vpr interacts with several cellular factors, including the human peptidyl prolyl isomerase cyclophilin A (CypA) [12–14]. One of the main problems with existing antiretroviral therapy is that the viruses can develop drug resistance, which necessitates identification of new potential drug targets that overcome this problem. One approach that recently has received increased attention, is targeting host factors essential for the pathogen life cycle, rather than pathogen components directly [15–17]. CypA could be such a target as it is dispensable for cell viability [18, 19], and viral replication of HIV-1 is determined to be effectively inhibited by use of selective inhibitors of CypA [20–25].
Recently, we investigated the interactions of CypA with the N-terminus of Vpr at atomic resolution . Prolyl cis/trans isomerization of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. However, of the N-terminal peptides of Vpr investigated, only those containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in surface plasmon resonance (SPR) biosensor experiments. Extensive analysis revealed that the binding region of N-terminal Vpr to CypA consisted of the heptapeptide motif RHFPRIW centered at Pro-35 .
The biological significance of the interaction of Vpr with CypA, including the extensively studied interaction of CypA with HIV-1 capsid (CA), that is crucial for viral replication [26, 27], is still not completely understood (Reviewed in [25, 28]). However, specific inhibitors of the prolyl cis/trans isomerase activity of CypA, such as cyclosporine A and SDZ-NIM811, inhibit HIV-1 replication [20–25] and a possible role of CypA in both entry and postentry events of the viral life cycle of HIV-1 has been indicated . The interaction of HIV-1 Vpr with CypA is known to occur in vitro and in vivo[12, 13, 25], although the biological consequences thereof are disputed. The original reports concluded that CypA had significance for the de novo synthesis of Vpr, as the Vpr-mediated cell cycle arrest in HIV-1 infected T cells appeared to be eliminated in the absence of CypA activity . More recently it has been suggested that the interaction of Vpr with CypA is independent of the ability of Vpr to induce G2 cell cycle arrest .
Independent of these data, previous studies have unambiguously shown the N-terminal residues from Ala-30 to Phe-34, which are adjacent to or incorporated in the N-terminal binding region of Vpr to CypA, comprised of 32RHFPRIW38 centered at Pro-35, are crucial for the ability of Vpr to induce G2 cell cycle arrest [30–32]. Similarly, the ability of Vpr to induce G2 cell cycle arrest is also influenced by residues in the C-terminal region 71-90 [30, 31, 33–37].
All studies are in agreement that mutation of Pro-35 with Ala disrupts the interaction of Vpr with CypA [12, 13], which is consistent with a conformational change in the hydrophobic core . On the other hand, intriguingly, Ardon et al. reported that mutation of the C-terminal residue Arg-80 with Ala also prevented coimmunoprecipitation of Vpr with CypA . However, the molecular explanation for the latter C-terminal mutation of Vpr to interfere with the interaction of the protein with CypA has remained elusive . Mutation of Arg-80 with Ala may cause a change in the folded structure of full-length Vpr or, could in theory, alter the structure of a specific novel C-terminal binding region of Vpr to CypA. The Pro residues of HIV-1NL4-3 Vpr are located in the N-terminal domain of the protein, namely at positions 5, 10, 14 and 35. All previously determined binding domains of various proteins interacting with CypA have hitherto required at least one Pro residue as part of the binding region.
However, a limited number of studies have shown that CypA is able to bind to peptides that do not contain Pro residues through an alternative non-Pro-dependent binding region . According to Demange et al., CypA exhibits two binding sites, of which the S1' site requires a Pro-containing substrate whereas the S2'-S3' site facilitates binding to peptides that do not contain Pro . Saphire et al. reported that a Vpr-CypA fusion protein which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication, which may indicate an important role of CypA associated with Vpr .
These facts, together with the observation of considerable amounts of CypA in virions  prompted us to study the interaction of Vpr with CypA in order to identify potential novel binding sites. This paper presents SPR binding studies of the interactions of CypA with full-length synthetic Vpr (s Vpr) and C-terminal s Vpr peptides. A novel specific non-Pro-containing C-terminal binding region of Vpr facilitating strong binding to CypA has been identified. The contribution of this novel C-terminal binding region together with the previously characterized N-terminal binding region RHFPRIW centered at Pro-35 has been demonstrated through characterization and quantification of the interaction of full-length Vpr with CypA.
To investigate the interaction between full-length Vpr and CypA in more detail, and to find an explanation for the previously undefined role of the C-terminus of Vpr with respect to this interaction, we performed interactions studies by SPR spectroscopy between highly pure recombinant CypA and full-length Vpr and C-terminal Vpr peptides. Additionally, possibly structural changes in the C-terminal region caused by substitution of Arg-80 by Ala were investigated by nuclear magnetic resonance (NMR) spectroscopy.
Characterization of the interaction of C-terminal Vpr75-90 with CypA using surface plasmon resonance spectroscopy
Estimated kinetic constants for binding of full-length HIV-1 Vpr and C-terminal Vpr peptides to CypA.
Amino acid sequence
K D (μM)
ka 1 (1/Ms)
0.28 ± 0.27
8.1 ± 6.1 × 104
6.3 ± 4.6 × 10-3
Vpr75-90 (R76Q, V83I, T84I)
GCQ HSRIGII RQRRAR
4.7 ± 2.6
1.6 ± 0.1 × 104
0.071 ± 0.037
Vpr75-90 (R76Q, V83I, R80A, T84I)
GCQ HSA IGII RQRRAR
2.7 ± 1.9
4.2 ± 0.8 × 104
0.095 ± 0.061
7.5 ± 2.6
3.8 ± 0.6 × 103
0.027 ± 0.005
Vpr1-96 (918 RU CypA)
ka1 = 1.3 ± 1.0 × 104
ka2 = 5.9 ± 1.7 × 10-6
kd1 = 1.8 ± 0.2 × 10-3
kd2 = 6.7 ± 3.8 × 10-4
Vpr1-96 (918 RU CypA)
0.31 ± 0.28
3.2 ± 1.2 × 104
0.007 ± 0.005
Vpr1-96 (180 RU CypA)
ka1 = 5.8 ± 0.8 × 103
ka2 = 1.6 ± 1.6 × 10-4
kd1 = 0.022 ± 0.025
kd2 = 7.3 ± 4.0 × 10-4
Vpr1-96 (180 RU CypA)
0.32 ± 0.14
1.6 ± 0.6 × 104
4.22 ± 0.4 × 10-3
Determination of the C-terminal binding region of Vpr to CypA
Mutation of Arg-80 with Ala significantly reduces the binding affinity
Previous studies show that the C-terminal region is important for most of the identified functions of Vpr, and attention has especially been directed to the arginine residues in the C-terminal region. As Arg-80 has previously been reported to be necessary for maintaining binding between Vpr and CypA , SPR experiments were performed on the mutant peptides Vpr75-90 R80A, Vpr75-90 (R76Q, V83I, T84I) and Vpr75-90 (R76Q, V83I, R80A, T84I). A reduction in association was observed for all mutants compared with the wild-type (wt) peptide (Table 1). Interestingly, the peptides with mutation of Arg-80 (Vpr75-90 R80A and Vpr75-90 (R76Q, V83I, R80A, T84I)) showed the lowest binding response (Figure 2). The dissociation constant (KD) of the three mutants were of the same order of magnitude (10-6M) (Table 1). The highest dissociation constant was found for Vpr75-90 R80A, indicating that this mutant exhibits the weakest binding to CypA. In summary, this implies an important function of Arg-80 in maintaining a strong interaction of the C-terminal binding region of Vpr to CypA, notwithstanding the fact that other residues appear to influence the interaction between Vpr and CypA to some extent. The fact that the decapeptide Vpr75-84 fails to bind to CypA (Figure 2) clearly demonstrates that Arg-80 and its nearby surrounding residues are insufficient for the interaction to occur. Reduced binding affinity was also observed for the mutant peptide Vpr75-90 (R76Q, V83I, T84I) where Arg-80 was conserved (Figure 2), demonstrating the importance of an intact 16 residue C-terminal binding domain of Vpr.
Mutation of Arg-80 with Ala does not influence the weak secondary structure of the C-terminal binding domain of Vpr
C-terminal Vpr binding to CypA confirmed by Isothermal titration calorimetry
Estimated kinetic constants for binding of HIV-1 Vpr75-90 to CypA by ITC
Mean values ± S.E
1.3 ± 0.4
0.09 ± 0.03
-16.9 ± 0.5
7.3 ± 0.3
Full length Vpr binds strongly to CypA as a bivalent analyte
The N- and C-terminal binding regions of Vpr to CypA were accurately determined by SPR biosensor analysis of the interaction of N- and C-terminal peptides with a varying number of residues. These data encouraged characterization of the interaction of full-length Vpr with CypA using SPR spectroscopy.
As a consequence of our discovery that Vpr has one proline containing N-terminal CypA binding region and one non-proline containing C-terminal CypA binding region, these most likely bind to the S1' and S2'-S3' binding sites of CypA, respectively. Hence, CypA could be associated with both binding regions of Vpr simultaneously. Ardon et al. reported that both mutants Vpr P35N and Vpr R80A failed to coprecipitate with CypA , which implies that simultaneous binding of CypA to both binding sites of Vpr is required for CypA to bind to full length Vpr.
The SPR sensorgrams of the interaction of full-length Vpr with immobilized CypA gave an excellent fit to the bivalent analyte model. This strongly supports simultaneous binding of CypA with the N-terminal Pro-containing binding region and the C-terminal non-Pro binding region of Vpr, involving the S1' (Pro-dependent) and S2'-S3' (Pro-independent) binding sites of CypA, respectively (Figure 6). The stoichiometry of the interaction is 1:1 and as an estimate, the interaction was quantified from the simple 1:1 binding model. The sensorgrams of Vpr1-96 were fitted to a 1:1 binding model by the Biacore evaluation software, and kinetic constants were determined (Figure 6, Table 1). The dissociation constant (KD) was determined to be approximately 320 nM.
Recently we determined that the N-terminal binding region of Vpr to CypA is comprised of the heptapeptide 32RHFPRIW38 centered at Pro-35 . Mutation of the central Pro residue (P35A) causes a loss of binding through disruption of the Pro-dependent N-terminal heptapeptide binding region. In the context of the full length molecule this mutation also results in merging of helix 1 and 2, which in turn causes a substantial change in the hydrophobic core of the protein as the anti-parallel folded structure of the N-terminal and central helices is no longer possible .
In this paper we have characterized a C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA. This region of Vpr does not contain any Pro residues, but binds much more strongly to CypA than to the N-terminal binding region (Figure 1). The fact that the mutant peptide Vpr75-90 R80A binds considerably weaker to CypA than the wt peptide (Figure 2) confirmed that Arg-80 is a key residue in the C-terminal binding region. Arg-80 is considered to be located in proximity to, but not included in, the well-defined helix 3 (residues 55-77) of the structured protein formed under membranous conditions at physiological pH . The secondary structure NOEs of residues 78-90 of full-length Vpr found by Morellet et al.  are mainly restricted to those between NH-NH(i, i+1), in addition to a few Hα-NH(i, i+2) and Hα-NH(i, i+3), indicating that these residues comprise a region with a relatively weak α-helical structure. Indeed, our NMR data of wt Vpr75-90 and Vpr75-90 R80A confirmed this interpretation and revealed that the propensity for weak helical structure of the C-terminal binding region is essentially unaffected by the mutation of Arg-80 to Ala (Figure 3). Thus, the mutation prevents binding of Vpr to CypA through a local change in the C-terminal binding region rather than any change in the tertiary structure of Vpr.
In aqueous solution Vpr is present as high order aggregates (~decamers) with a lower percentage of higher multimers . According to Fritz et al. oligomerization is mediated by the Vpr hydrophobic core but not by the flexible N- and C-terminal domains . The C-terminal binding domain of Vpr to CypA is located beyond the residues that have been shown experimentally to be involved in oligomerization of Vpr. In agreement with this we used the statistical mechanics algorithm TANGO, which identifies aggregation-prone regions of peptides and denatured proteins using a set of balanced physico-chemical parameters [48, 49]. According to the TANGO algorithm, a score of ≤ 0.02% indicates no aggregation, 0.02-5.0% indicates moderate aggregation, and ≥ 5.0% indicates high aggregation propensities. Application of this program predicted a region of 16 residues (Ala-55 to Ile-70) populating the oligomerization state to more than 5% per residue (5.09 - 35.56% per residue). Four further residues were predicted with much lower score, namely Thr-53 (1.30%), Trp-54 (3.50%), His-71 (0.84%) and Phe-72 (0.80%). The fact that the dissociation constant of full-length Vpr is in the same order of magnitude as the dissociation constant of the C-terminal binding domain Vpr75-90 does not indicate that oligomerization of Vpr influences the interaction with CypA significantly.
Previous studies have shown that the mutants Vpr P35A and Vpr R80A were key residues for coimmunoprecipitation of Vpr and CypA, and when replaced abrogated the coimmunoprecipitation to CypA . Although, these authors concluded that residues beyond Vpr1-40 also are important for binding to CypA, no further explanation was provided. Our data independently confirm the importance of these residues for the interactions and indicate that the C-terminal binding is quantitatively stronger than the N-terminal binding to CypA (Figure 1). However, the fact that the decapeptide Vpr75-84 fails to bind to CypA (Figure 2) clearly demonstrates that Arg-80 and its nearby surrounding residues are insufficient for the interaction to occur. Reduced binding affinity was also observed for a mutant Vpr75-90 peptide where Arg-80 was conserved, demonstrating the importance of an intact 16 residue C-terminal binding domain of Vpr.
A simultaneous interaction of both Vpr sites with CypA was suggested as SPR sensorgrams of full-length Vpr interacting with immobilized CypA gave an optimal fit with a bivalent analyte model (Figure 5). Taken together, these data indicate that a simultaneous binding of the N- and C-terminal domains is required for full-length Vpr to interact with CypA. The dissociation constant (KD) of full-length Vpr was found to be approximately 0.32 μM (Table 1), which implies that the Vpr-CypA interaction may be stronger than the well characterized interaction of HIV-1 CA with CypA, which has been determined previously by SPR spectroscopy (KD 16 ± 4 μM) . This suggests that the CypA-Vpr interaction has a functional role in the relationship between the host and pathogen.
Most residues of the N- and C-terminal binding domains of Vpr are highly conserved among HIV-1 strains (with the exception of Ile-37 of the N-terminal binding domain and Arg-77, Val-83, Thr-84, Arg-85, Gln-86 and Ala-89 of the C-terminal binding ), indicating that maintenance of these structural regions of the protein is important for the viral life cycle.
CypA may represent a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses HIV-1 replication. All previous studies of protein-protein interactions involving human CypA have hitherto been limited to Pro-containing substrates. The fact that a non-proline-containing 16-residue region of a protein is able to bind with high affinity to CypA, presented for the first time in this manuscript, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 are associated with the identified N- and C-terminal binding domains of the protein to CypA.
The synthesis, purification and molecular characterization of synthetic Vpr1-96 (s Vpr), the C-terminal peptides Vpr75-90 , Vpr69-78, Vpr75-84, Vpr81-90 and Vpr87-96 and the mutants Vpr75-90 (R76Q, V83I, T84I), Vpr75-90 (R76Q, V83I, R80A, T84I), Vpr75-90 (R80A) were performed as described in detail elsewhere [8, 53] and the purities were checked by HPLC, MALDI-MS and positive ion ESI-MS . The mass spectrum of s Vpr exhibited a significant M-18 peak corresponding to loss of a water molecule from the intact protein during synthesis. As tandem mass spectrometry ( MS/MS) analysis revealed that the water molecule was eliminated from residues 6 or 7, belonging to a region that has been experimentally shown not to bind to CypA , the protein was considered suitable for our studies.
Cyclophilin A (CypA)
The production and purification of recombinant human CypA has been described previously .
Nuclear magnetic resonance spectroscopy
2D 1H Total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) NMR experiments were performed at 600.13 MHz on a Bruker Avance 600 MHz instrument equipped with an UltraShield Plus magnet and a triple resonance cryoprobe with gradient unit. Individual samples were dissolved in 600 μl 100 mM aqueous DPC-d38 micelles 10% D2O (v/v) at concentrations between 1-2 mM. The 2D NMR experiments were performed at 300 K without spinning with mixing times of 110 ms for the TOCSY experiments and 250 ms for the NOESY experiments. Efficient suppression of the water signal was achieved by application of excitation sculpting in the 1D 1H and the 2D 1H TOCSY and NOESY NMR experiments . 1H signal assignments of the NMR spectra were achieved by identification of the individual spin systems in the 2D 1H TOCSY spectra, combined with observations of sequence-specific short-distance crosspeaks (Hα-HN i, i+1) in the 2D 1H-1H NOESY spectra [53, 77]. Readily recognizable spin systems were used as starting points for correlation of the individual spin systems observed in the TOCSY and NOESY spectra with individual residues in the peptide sequences. Acquisition of data, processing and spectral analysis were performed with Bruker Topspin 1.3 software.
Surface plasmon resonance spectroscopy
SPR [42–44] measurements were performed at 25°C on a Biacore T100 instrument (Biacore AB, Uppsala, Sweden) equipped with CM5 research-grade sensor chips. CypA was immobilized to 150, 180, 918 and 5003 RU, using standard amine-coupling chemistry. The reference flow cells were treated correspondingly except for CypA immobilization. The synthetic full length Vpr, as well as fragments and mutants thereof, were dissolved at four different concentrations in the running buffer (HBS-EP buffer pH 7.4; 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA and 0.005% surfacant). The samples were injected over the flow cells at a flow rate of 30 μl/min. Data were collected at 2.5 Hz during 60 s association and 180 s or 240 s dissociation phases, and were automatically corrected for bulk buffer effects and non-specific binding of Vpr peptides to the chip matrix. All SPR data was acquired from two individual series of experiments using four different analyte concentrations with a duplicate injection of one of the individual concentrations.
Analysis of biosensor data
Affinity, association and dissociation rate constants were obtained from sensorgrams by the Biacore T100 evaluation software version 2.0.1 in accordance with the global curve fit model. Sensorgram data for the four different concentrations were fitted to several binding models including 1:1 (Langmuir) binding model (A+B ↔ AB), two-state reaction (conformational change) model (A+B ↔ AB ↔ AB*), heterogeneous ligand (HL) model (interaction one: A+B1 ↔ AB1; interaction two: A+B2 ↔ AB2) and bivalent analyte (BA) model (A+B ↔ AB; AB+B↔ABB). Kinetic constants were calculated for the best fitted model.
Isothermal titration calorimetry
ITC was performed in a VP-ITC titration calorimeter (MicroCal Inc.). CypA and Vpr75-90 were prepared in filtered HBS-EP buffer pH 7.4 and the samples were degassed prior to titration. Two individual titrations were performed by adding 14.8 μM and 11 μM CypA, respectively to the sample cell and titrated with a 210 μM stock solution of Vpr75-90. CypA samples were subjected to 30-50 injections (5-10 μL) of the Vpr75-90 peptide with a 360 or 420 s interval between each injection at 25°C. The mean of the heat from the last 5 injections was subtracted from the raw data to correct for experimental heat dilution. The binding isotherms were fitted to a one site binding model using the Origin v7 software (OriginLab). Chi-square minimization was performed iteratively to obtain the best-fit parameters.
Docking analysis of the Vpr-CypA complex
A cartoon of the Vpr-CypA complex was generated based on the detailed experimental characterization of the N- and C-terminal binding domains of Vpr using the ZDOCK algorithm . The X-ray structure of CypA (pdb entry 3K0N)  and the NMR structure of Vpr (pdb entry 1M8L)  were used as rigid units, together with knowledge of the key residues of the Pro-dependent and Pro-independent binding domains of CypA.
The authors are indebted to Dr. David Mitzner for providing recombinant CypA, Professor Aurora Martinez for the access to the isothermal titration calorimeter and Andrea Abrahamik for mass spectral data. We thank the Meltzer foundation for financial support for publishing this paper.
- Paxton W, Connor RI, Landau NR: Incorporation of Vpr into Human Immunodeficiency Virus Type 1 Virions: Requirement for the p6 Region of gag and Mutational Analysis. J Virol 1993, 67: 7229–7237.PubMed CentralPubMedGoogle Scholar
- Sherman MP, Schubert U, Williams SA, de Noronha CMC, Kreisberg JF, Henklein P, Greene WC: HIV-1 Vpr displays natural protein-transducing properties: Implications for viral pathogenesis. Virology 2002, 302: 95–105. 10.1006/viro.2002.1576View ArticlePubMedGoogle Scholar
- de Noronha CMC, Sherman MP, Lin HW, Cavrois MV, Moir RD, Goldman RD, Greene WC: Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr. Science 2001, 294: 1105–1108. 10.1126/science.1063957View ArticlePubMedGoogle Scholar
- Levy DN, Fernandes LS, Williams WV, Weiner DB: Induction of cell differentiation by human immunodeficiency virus 1 vpr. Cell 1993, 72: 541–550. 10.1016/0092-8674(93)90073-YView ArticlePubMedGoogle Scholar
- Rogel ME, Wu LI, Emerman M: The Human Immunodeficiency Virus Type 1 vpr Gene Prevents Cell Proliferation during Chronic Infection. J Virol 1995, 69: 882–888.PubMed CentralPubMedGoogle Scholar
- Ayyavoo V, Mahalingam S, Rafaeli Y, Kudchodkar S, Chang D, Nagashunmugam T, Williams WV, Weiner DB: HIV-1 viral protein R (Vpr) regulates viral replication and cellular proliferation in T cells and monocytoid cells in vitro. J Leukoc Virol 1997, 62: 93–99.Google Scholar
- Stewart SA, Poon B, Jowett JBM, Chen ISY: Human immunodeficiency virus type 1 vpr induces apoptosis following cell cycle arrest. J Virol 1997, 71: 5579–5592.PubMed CentralPubMedGoogle Scholar
- Henklein P, Bruns K, Sherman MP, Tessmer U, Licha K, Kopp J, de Noronha CMC, Greene WC, Wray V, Schubert U: Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G(2) cell cycle arrest. J Biol Chem 2000, 275: 32016–32026.View ArticlePubMedGoogle Scholar
- Kogan M, Rappaport J: HIV-1 accessory protein Vpr: relevance in the pathogenesis of HIV and potential for therapeutic intervention. Retrovirology 2011, 8: 25. 10.1186/1742-4690-8-25PubMed CentralView ArticlePubMedGoogle Scholar
- de Rocquigny H, Fritz JV, Briant L, Mely Y, Bouaziz S: HIV-1 viral protein r: from structure to function. Future Virology 2010, 5: 607–625. 10.2217/fvl.10.47View ArticleGoogle Scholar
- Zhao RY, Li G, Bukrinsky MI: Vpr-Host Interactions During HIV-1 Viral Life Cycle. J Neuroimmune Pharmacol 2011, 6: 216–229. 10.1007/s11481-011-9261-zView ArticlePubMedGoogle Scholar
- Ardon O, Zimmerman ES, Andersen JL, DeHart JL, Blackett J, Planelles V: Induction of G(2) arrest and binding to cyclophilin A are independent phenotypes of human immunodeficiency virus type 1 Vpr. J Virol 2006, 80: 3694–3700. 10.1128/JVI.80.8.3694-3700.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Zander K, Sherman MP, Tessmer U, Bruns K, Wray V, Prechtel AT, Schubert E, Henklein P, Luban J, Neidleman J, Greene WC, Schubert U: Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J Biol Chem 2003, 278: 43202–43213. 10.1074/jbc.M305414200View ArticlePubMedGoogle Scholar
- Solbak S, Reksten T, Wray V, Bruns K, Horvli O, Raae A, Henklein P, Henklein P, Roder R, Mitzner D, Schubert U, Fossen T: The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding. BMC Struct Biol 2010., 10:Google Scholar
- Moss RB, Davey RT, Steigbigel RT, Fang F: Targeting pandemic influenza: a primer on influenza antivirals and drug resistance. J Antimicrob Chemoth 2010, 65: 1086–1093. 10.1093/jac/dkq100View ArticleGoogle Scholar
- Kellam P: Attacking pathogens through their hosts. Genome Biol 2006., 7:Google Scholar
- Prussia A, Thepchatri P, Snyder JP, Plemper RK: Systematic Approaches towards the Development of Host-Directed Antiviral Therapeutics. Int J Mol Sci 2011, 12: 4027–4052. 10.3390/ijms12064027PubMed CentralView ArticlePubMedGoogle Scholar
- Luban J, Braaten D: Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells. Embo Journal 2001, 20: 1300–1309. 10.1093/emboj/20.6.1300PubMed CentralView ArticlePubMedGoogle Scholar
- Luban J, Colgan J, Asmal M: Isolation, characterization and targeted disruption of mouse Ppia: Cyclophilin A is not essential for mammalian cell viability. Genomics 2000, 68: 167–178. 10.1006/geno.2000.6295View ArticlePubMedGoogle Scholar
- Billich A, Hammerschmid F, Peichl P, Wenger R, Zenke G, Quesniaux V, Rosenwirth B: Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporine A analog with activity against human immunodeficiency virus (HIV) type 1: interference with HIV Protein-cyclophilin A interactions. J Virol 1995, 69: 2451–2461.PubMed CentralPubMedGoogle Scholar
- Dorfman T, Weimann A, Borsetti A, Walsh CT, Gottlinger HG: Active-site residues of cyclophilin A are crucial for its incorporation into human immunodeficiency virus type 1 virions. J Virol 1997, 71: 7110–7113.PubMed CentralPubMedGoogle Scholar
- Franke EK, Luban J: Inhibition of HIV-1 replication by cyclosporine A or related compounds correlates with the ability to disrupt the Gag-cyclophilin A interaction. Virology 1996, 222: 279–282. 10.1006/viro.1996.0421View ArticlePubMedGoogle Scholar
- Rosenwirth B, Billich A, Datema R, Donatsch P, Hammerschmid F, Harrison R, Hiestand P, Jaksche H, Mayer P, Peichl P, Quesniaux V, Schatz F, Schuurman HJ, Traber R, Wenger R, Wolff B, Zenke G, Zurini M: Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppresive cyclosporine analog. Antimicrob Agents Chemother 1994, 38: 1763–1772.PubMed CentralView ArticlePubMedGoogle Scholar
- Thali M, Bukovsky A, Kondo E, Rosenwirth B, Walsh CT, Sodroski J, Gottlinger HG: Functional association of cyclophilin A with HIV-1 virions. Nature 1994, 372: 363–365. 10.1038/372363a0View ArticlePubMedGoogle Scholar
- Votteler J, Wray V, Schubert U: Role of cyclophilin a in HIV replication. Future Virol 2007, 2: 65–78. 10.2217/17460722.214.171.124View ArticleGoogle Scholar
- Bosco DA, Eisenmesser EZ, Pochapsky S, Sundquist WI, Kern D: Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. PNAS 2002, 99: 5247–5252. 10.1073/pnas.082100499PubMed CentralView ArticlePubMedGoogle Scholar
- Bosco DA, Kern D: Catalysis and binding of cyclophilin a with different HIV-1 capsid constructs. Biochemistry 2004, 43: 6110–6119. 10.1021/bi049841zView ArticlePubMedGoogle Scholar
- Ivery MTG: Immunophilins: Switched on protein binding domains? Medicinal Research Reviews 2000, 20: 452–484. 10.1002/1098-1128(200011)20:6<452::AID-MED2>3.0.CO;2-6View ArticlePubMedGoogle Scholar
- Saphire ACS, Bobardt MD, Gallay PA: Cyclophilin a plays distinct roles in human immunodeficiency virus type 1 entry and postentry events, as revealed by spinoculation. J Virol 2002, 76: 4671–4677. 10.1128/JVI.76.9.4671-4677.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Dimarzio P, Choe S, Ebright M, Knoblauch R, Landau NR: Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol 1995, 69: 7909–7916.Google Scholar
- Jacquot G, Le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, Benichou S: Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages. Retrovirology 2007., 4:Google Scholar
- Selig L, Benichou S, Rogel ME, Wu LI, Vodicka MA, Sire J, Benarous R, Emerman M: Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest. J Virol 1997, 71: 4842–4846.PubMed CentralPubMedGoogle Scholar
- Berglez JM, Castelli LA, Sankovich SA, Smith SC, Curtain CC, Macreadie IG: Residues within the HFRIGC sequence of HIV-1 Vpr involved in growth arrest activities. Biochem Biophys Res Commun 1999, 264: 287–290. 10.1006/bbrc.1999.1511View ArticlePubMedGoogle Scholar
- Gaynor EM, Chen ISY: Analysis of apoptosis induced by HIV-1 Vpr and examination of the possible role of the hHR23A protein. Exp Cell Res 2001, 267: 243–257. 10.1006/excr.2001.5247View ArticlePubMedGoogle Scholar
- Sawaya BE, Khalili K, Gordon J, Srinivasan A, Richardson M, Rappaport J, Amini S: Transdominant activity of human immunodeficiency virus type 1 Vpr with a mutation at residue R73. J Virol 2000, 74: 4877–4881. 10.1128/JVI.74.10.4877-4881.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Yao XJ, Mouland AJ, Subbramanian RA, Forget J, Rougeau N, Bergeron D, Cohen EA: Vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1-infected dividing Jurkat T cells. J Virol 1998, 72: 4686–4693.PubMed CentralPubMedGoogle Scholar
- Macreadie IG, Castelli LA, Hewish DR, Kirkpatrick A, Ward AC, Azad AA: A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. PNAS 1995, 92: 2770–2774. 10.1073/pnas.92.7.2770PubMed CentralView ArticlePubMedGoogle Scholar
- Votteler J, Studtrucker N, Sorgel S, Munch J, Rucker E, Kirchhoff F, Schick B, Henklein P, Fossen T, Bruns K, Sharma A, Wray V, Schubert U: Proline 35 of human immunodeficiency virus type 1 (HIV-1) Vpr regulates the integrity of the N-terminal helix and the incorporation of Vpr into virus particles and supports the replication of R5-tropic HIV-1 in human lymphoid tissue ex vivo. J Virol 2007, 81: 9572–9576. 10.1128/JVI.02803-06PubMed CentralView ArticlePubMedGoogle Scholar
- Demange L, Moutiez M, Vaudry K, Dugave C: Interaction of human cyclophilin hCyp-18 with short peptides suggests the existence of two functionally independent subsites. Febs Letters 2001, 505: 191–195. 10.1016/S0014-5793(01)02814-9View ArticlePubMedGoogle Scholar
- Saphire ACS, Bobardt MD, Gallay PA: trans-complementation rescue of cyclophilin A-deficient viruses reveals that the requirement for cyclophilin A in human immunodeficiency virus type 1 replication is independent of its isomerase activity. J Virol 2002, 76: 2255–2262. 10.1128/jvi.76.5.2255-2262.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Franke EK, Yuan HEH, Luban J: Specific incorporation of cyclophilin A into HIV-1 virions. Nature 1994, 372: 359–362. 10.1038/372359a0View ArticlePubMedGoogle Scholar
- Jonsson U, Fagerstam L, Ivarsson B, Johnsson B, Karlsson R, Lundh K, Lofas S, Persson B, Roos H, Ronnberg I, Sjolander S, Stenberg E, Stahlberg R, Urbaniczky C, Ostlin H, Malmqvist M: Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 1991, 11: 620-&.PubMedGoogle Scholar
- Karlsson R: Real-Time Competitive Kinetic Analysis of Interactions between Low-Molecular-Weight Ligands in Solution and Surface-Immobilized Receptors. Anal Biochem 1994, 221: 142–151. 10.1006/abio.1994.1390View ArticlePubMedGoogle Scholar
- Stenberg E, Persson B, Roos H, Urbaniczky C: Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J Colloid Interface Sci 1991, 143: 513–526. 10.1016/0021-9797(91)90284-FView ArticleGoogle Scholar
- Morellet N, Bouaziz S, Petitjean P, Roques BP: NMR structure of the HIV-1 regulatory protein VPR. J Mol Biol 2003, 327: 215–227. 10.1016/S0022-2836(03)00060-3View ArticlePubMedGoogle Scholar
- Wishart DS, Sykes BD, Richards FM: The Chemical-Shift Index: A Fast and Simple Method for the Assignment of Protein Secondary Structure through NMR Spectroscopy. Biochemistry 1992, 31: 1647–1651. 10.1021/bi00121a010View ArticlePubMedGoogle Scholar
- Fritz JV, Dujardin D, Godet J, Didier P, De Mey J, Darlix JL, Mely Y, de Rocquigny H: HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag. Journal of Virology 2010, 84: 1585–1596. 10.1128/JVI.01691-09PubMed CentralView ArticlePubMedGoogle Scholar
- Fernandez-Escamilla AM, Rousseau F, Schymkowitz J, Serrano L: Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nature Biotechnology 2004, 22: 1302–1306. 10.1038/nbt1012View ArticlePubMedGoogle Scholar
- Pande VS: A universal TANGO? Nature Biotechnology 2004, 22: 1240–1241. 10.1038/nbt1004-1240View ArticlePubMedGoogle Scholar
- Yoo SH, Myszka DG, Yeh CY, McMurray M, Hill CP, Sundquist WI: Molecular recognition in the HIV-1 capsid/cyclophilin a complex. J Mol Biol 1997, 269: 780–795. 10.1006/jmbi.1997.1051View ArticlePubMedGoogle Scholar
- Ke HM: Similarities and differences between human cyclophilin A and other β-barrel structures: Structural refinement at 1.63 Å resolution. J Mol Biol 1992, 228: 539–550. 10.1016/0022-2836(92)90841-7View ArticlePubMedGoogle Scholar
- Chen R, Li L, Weng ZP: ZDOCK: An initial-stage protein-docking algorithm. Proteins 2003, 52: 80–87. 10.1002/prot.10389View ArticlePubMedGoogle Scholar
- Bruns K, Fossen T, Wray V, Henklein P, Tessmer U, Schubert U: Structural characterization of the HIV-1 Vpr N terminus - Evidence of cis/trans-proline isomerism. J Biol Chem 2003, 278: 43188–43201. 10.1074/jbc.M305413200View ArticlePubMedGoogle Scholar
- Srinivasan A, Ayyavoo V, Mahalingam S, Kannan A, Boyd A, Datta D, Kalyanaraman VS, Cristillo A, Collman RG, Morellet N, Sawaya BE, Murali R: A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: Potential impact on CTL epitopes. Virology Journal 2008., 5:Google Scholar
- Arunagiri C, Macreadie I, Hewish D, Azad A: A C-terminal domain of HIV-1-accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4(+) lymphocytes. Apoptosis 1997, 2: 69–76.View ArticlePubMedGoogle Scholar
- Godet AN, Guergnon J, Croset A, Cayla X, Falanga PB, Colle JH, Garcia A: PP2A(1) Binding, Cell Transducing and Apoptotic Properties of Vpr(77–92): A New Functional Domain of HIV-1 Vpr Proteins. Plos One 2010., 5:Google Scholar
- Jacotot E, Ferri KF, El Hamel C, Brenner C, Druillennec S, Hoebeke J, Rustin P, Metivier D, Lenoir C, Geuskens M, Vieira HLA, Loeffler M, Belzacq AS, Briand JP, Zamzami N, Edelman L, Xie ZH, Reed JC, Roques BP, Kroemer G: Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein R and Bcl-2. J Exp Med 2001, 193: 509–519. 10.1084/jem.193.4.509PubMed CentralView ArticlePubMedGoogle Scholar
- Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HLA, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G: The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med 2000, 191: 33–45. 10.1084/jem.191.1.33PubMed CentralView ArticlePubMedGoogle Scholar
- Janoo A, Morrow PW, Tung HYL: Activation of protein phosphatase-2A(1) by HIV-1 Vpr cell death causing peptide in intact CD4+ T cells and in vitro. J Cell Biochem 2005, 94: 816–825. 10.1002/jcb.20347View ArticlePubMedGoogle Scholar
- Lum JJ, Cohen OJ, Nie ZL, Weaver JG, Gomez TS, Yao XJ, Lynch D, Pilon AA, Hawley N, Kim JE, Chen ZX, Montpetit M, Sanchez-Dardon J, Cohen EA, Badley AD: Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis. J Clin Invest 2003, 111: 1547–1554.PubMed CentralView ArticlePubMedGoogle Scholar
- Mologni D, Citterio P, Menzaghi B, Poma BZ, Riva C, Broggini V, Sinicco A, Milazzo L, Adorni F, Rusconi S, Galli M, Riva A, rHoPe SG: Vpr and HIV-1 disease progression: R77Q mutation is associated with long-term control of HIV-1 infection in different groups of patients. Aids 2006, 20: 567–574. 10.1097/01.aids.0000210611.60459.0eView ArticlePubMedGoogle Scholar
- Sabbah EN, Druillennec S, Morellet N, Bouaziz S, Kroemer G, Roques BP: Interaction between the HIV-1 protein Vpr and the adenine nucleotide translocator. Chem Biol Drug Des 2006, 67: 145–154. 10.1111/j.1747-0285.2006.00340.xView ArticlePubMedGoogle Scholar
- Somasundaran M, Sharkey M, Brichacek B, Luzuriaga K, Emerman M, Sullivan JL, Stevenson M: Evidence for a cytopathogenicity determinant in HIV-1 Vpr. PNAS 2002, 99: 9503–9508. 10.1073/pnas.142313699PubMed CentralView ArticlePubMedGoogle Scholar
- Bischerour J, Tauc P, Leh H, de Rocquigny H, Roques B, Mouscadet JF: The (52–96) C-terminal domain of Vpr stimulates HIV-1 IN-mediated homologous strand transfer of mini-viral DNA. Nucleic Acids Res 2003, 31: 2694–2702. 10.1093/nar/gkg364PubMed CentralView ArticlePubMedGoogle Scholar
- Connor RI, Chen BK, Choe S, Landau NR: Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995, 206: 935–944. 10.1006/viro.1995.1016View ArticlePubMedGoogle Scholar
- Gleenberg IO, Herschhorn A, Hizi A: Inhibition of the activities of reverse transcriptase and integrase of human immunodeficiency virus type-1 by peptides derived from the homologous viral protein R (Vpr). J Mol Biol 2007, 369: 1230–1243. 10.1016/j.jmb.2007.03.073View ArticlePubMedGoogle Scholar
- Rajan D, Wildum S, Rucker E, Schindler M, Kirchhoff F: Effect of R77Q, R77A and R80A changes in Vpr on HIV-1 replication and CD4 T cell depletion in human lymphoid tissue ex vivo. Aids 2006, 20: 831–836. 10.1097/01.aids.0000218546.31716.7fView ArticlePubMedGoogle Scholar
- Lu YL, Spearman P, Ratner L: Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J Virol 1993, 67: 6542–6550.PubMed CentralPubMedGoogle Scholar
- Mahalingam S, Collman RG, Patel M, Monken CE, Srinivasan A: Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization. Virology 1995, 212: 331–339. 10.1006/viro.1995.1490View ArticlePubMedGoogle Scholar
- Zhou Y, Lu YL, Ratner L: Arginine residues in the C-terminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest. Virology 1998, 242: 414–424. 10.1006/viro.1998.9028View ArticlePubMedGoogle Scholar
- Yao XJ, Subbramanian RA, Rougeau N, Boisvert F, Bergeron D, Cohen EA: Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J Virol 1995, 69: 7032–7044.PubMed CentralPubMedGoogle Scholar
- Lai MY, Zimmerman ES, Planelles V, Chen JJ: Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo. J Virol 2005, 79: 15443–15451. 10.1128/JVI.79.24.15443-15451.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang SG, Pointer D, Singer G, Feng YF, Park K, Zhao LJ: Direct binding to nucleic acids by Vpr of human immunodeficiency virus type 1. Gene 1998, 212: 157–166. 10.1016/S0378-1119(98)00178-4View ArticlePubMedGoogle Scholar
- de Rocquigny H, Caneparo A, Delaunay T, Bischerour J, Mouscadet JFO, Roques BP: Interactions of the C-terminus of viral protein R with nucleic acids are modulated by its N-terminus. Eur J Biochem 2000, 267: 3654–3660. 10.1046/j.1432-1327.2000.01397.xView ArticlePubMedGoogle Scholar
- Kichler A, Pages JC, Leborgne C, Druillennec S, Lenoir C, Coulaud D, Delain E, Le Cam E, Roques BP, Danos O: Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R. J Virol 2000, 74: 5424–5431. 10.1128/JVI.74.12.5424-5431.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Hwang TL, Shaka AJ: Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients. J Magn Reson A 1995, 112: 275–279. 10.1006/jmra.1995.1047View ArticleGoogle Scholar
- Wüthrich K: NMR of Proteins and Nucleic Acids. John Wiley and Sons, Inc. New York; 1986.Google Scholar
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