Induction of G2 Arrest and Binding to Cyclophilin A Are Independent Phenotypes of Human Immunodeficiency Virus Type 1 Vpr

Division of Cellular Biology and Immunology, Department of Pathology, University of Utah School of Medicine, 30 N 1900 East, SOM 5C210, Salt Lake City, Utah 84132, USA.
Journal of Virology (Impact Factor: 4.44). 05/2006; 80(8):3694-700. DOI: 10.1128/JVI.80.8.3694-3700.2006
Source: PubMed


Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl cis-trans isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and function (specifically,
induction of G2 arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R (Vpr). In the present study, we examine the role of
the Vpr-CypA interaction on Vpr-induced G2 arrest. We find that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35
of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA or its binding
to Vpr is dispensable for the ability of Vpr to induce G2 arrest. Vpr expression in CypA−/− cells leads to induction of G2 arrest in a manner that is indistinguishable from that in CypA+ cells. CsA abolished CypA-Vpr binding but had no effect on induction of G2 arrest or Vpr steady-state levels. In view of these results, we propose that the interaction with CypA is independent of
the ability of Vpr to induce cell cycle arrest. The interaction between Vpr and CypA is intriguing, and further studies should
examine its potential effects on other functions of Vpr.

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Available from: Orly Ardon, Oct 08, 2015
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    • "than VprL23F , that mutant harbours an intact sequence of helix a - 1 . However , mutation of Pro - 35 results in an elongation of the helix towards the C - terminus of the molecule and a significant global change of the structure of Vpr ( Votteler et al . , 2007 ) . In contrast to VprL23F , the VprP35A mutant is still able to induce G2 - arrest ( Ardon et al . , 2006 ; Votteler et al . , 2007 ) , and most likely binds to the NE . However , the changed structure , caused by the P35A mutation , somehow renders the VprP35A mutant insen - sitive to caspase 3 - inhibition as it does not accumulate at the NE upon Z - DEVD - fmk treatment ."
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    ABSTRACT: The HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1(NL4-3)-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest.
    Virology 07/2012; 432(2):444-51. DOI:10.1016/j.virol.2012.06.027 · 3.32 Impact Factor
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    • "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 [12]. 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 [12]. 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. "
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    ABSTRACT: 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.
    BMC Structural Biology 12/2011; 11(1):49. DOI:10.1186/1472-6807-11-49 · 1.18 Impact Factor
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    • "The loop region centred at Pro-35 of Vpr comprising the binding motif to CypA maintains the integrity of the N-terminal and middle helices of Vpr and is functionally important for incorporation of Vpr into virus particles and replication of R5-tropic HIV-1 in human lymphoid tissue [28]. CypA acts as a catalyst of prolyl cis/trans interconversions of Pro 5, 10, 14 and 35 of N-terminal HIV-1 Vpr, which may indicate a folding chaperone role of CypA in HIV replication compatible with the fact that inhibitors of CypA, which inhibit CypA-Vpr binding [9,10] and CypA-mediated prolyl cis/trans catalysis, also suppress HIV-1 replication in cell culture. "
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    ABSTRACT: Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation 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. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.
    BMC Structural Biology 10/2010; 10(1):31. DOI:10.1186/1472-6807-10-31 · 1.18 Impact Factor
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