Vpr induces cell cycle arrest in G2 independently of the presence or absence of CypA.
Our previous studies have shown that infection with a lentiviral vector (pHR-VPR-IRES-GFP, referred to as pHR-VPR in this study) (29
) that encodes a Vpr-IRES-GFP tandem cistron faithfully recapitulates the effects of Vpr, including induction of cell cycle arrest (37
), apoptosis (5
), and transactivation of the viral long terminal repeat (29
). To test whether the presence of CypA is necessary for induction of cell cycle arrest, we infected CypA−/−
) or unmodified Jurkat cells (herein referred to as CypA+
) with pHR-VPR or a control vector, pHR-GFP (29
). Vector-infected cells were analyzed 24 h postinfection for DNA content and, separately, for GFP expression using flow cytometry (Fig. ). CypA−/−
Jurkat cells exhibited cell cycle arrest (68% cells in G2
/M; 65% GFP+
cells) comparable to that of CypA+
Jurkat cells (77% cells in G2
/M; 70% GFP+
cells). The CypA expression status of the cells was verified by Western blotting (rabbit anti-CypA; EMD Biosciences, San Diego, CA) and confirmed the lack of CypA expression in CypA−/−
cells (Fig. ).
FIG. 1. CypA is not required for Vpr-mediated cell cycle arrest. (A) Cell cycle analysis of CypA+ and CypA−/− Jurkat cells infected with pHR-VPR, encoding Vpr and GFP, or the control vector, pHR-GFP, encoding GFP only. Vector-infected (more ...)
Since lentiviral vectors allow expression of high levels of heterologous proteins, it is possible that ectopic expression of Vpr in the previous experiment may have overcome a potential restriction (such as biochemical instability of Vpr or an inactive conformation) derived from the lack of CypA. To validate the above results in a more relevant expression system, we performed similar experiments in which Vpr was introduced by infection with replication-competent HIV-1NL4-3
). As a negative control, we utilized the isogenic, Vpr-negative mutant HIV-1NL4-3
vprX, which contains a frameshift mutation that completely inactivates Vpr (5
). We performed infections of CypA+
cells at a multiplicity of infection (MOI) of 0.5. Infected cultures were analyzed at 96 h by flow cytometry after combined staining for intracellular p24 and DNA content. The p24-positive and -negative cells from each infection were then electronically gated and analyzed for cell cycle distribution (Fig. ). Infections of CypA−/−
cells were routinely low (between 0.5% and 2.5% at 96 h postinfection) and viruses failed to spread in these cells, consistent with a known requirement for CypA in target cells during the uncoating step (9
). In contrast, infections of CypA+
cells were at a high level (between 25% and 60% at 96 h postinfection) and showed viral spread (data not shown). Despite the low frequency of infected cells in CypA−/−
cultures, it was possible to use electronic gating to analyze the cell cycle of infected and uninfected cells separately. As expected, CypA+
cell infection with HIV-1NL4-3
, but not with HIV-1 NL4-3
VprX, resulted in a dramatic increase of the G2
/M peak after 96 h (Fig. ; 63.2% and 29.2% cells in G2
/M, respectively). The lack of CypA in CypA−/−
cells did not affect the sensitivity of cells to G2
arrest by infection with HIV-1NL4-3
(67.3% cells in G2
/M). After 7 days of infection, the percentages of HIV-1NL4-3
-infected cells exhibiting G2
/M DNA content were 80.1% and 60.8% for CypA+
cells, respectively (Fig. ).
FIG. 2. CypA is not required for Vpr-induced cell cycle arrest in the context of HIV-1 infection. Vpr was introduced by infection with replication-competent HIV-1NL4-3. The isogenic, Vpr-negative mutant, HIV-1NL4-3vprX, was used as a negative control. Infections (more ...)
The above results indicate that Vpr expression is associated with induction of G2
arrest in a manner that is indistinguishable between CypA+
cells, whether Vpr is expressed from a lentiviral vector or from infectious, full-length HIV-1. The apparent discrepancy between our results and those reported by Zander et al. (34
) prompted us to reexamine the Vpr-CypA interaction. Vpr binding to CypA was previously reported (8
), and this interaction was postulated to be required for Vpr stability and for its ability to induce G2
). Bruns et al. indicated that the proline residue at position 35 of Vpr was essential for the interaction with CypA (8
). In order to directly examine the interaction between CypA and Vpr, we performed coimmunoprecipitation studies with wild-type Vpr as well as with three Vpr mutants. Vpr(P35N) (8
) and Vpr(P35A) disrupt proline-35. Vpr(R80A) is unable to induce G2
arrest or apoptosis (5
). 293FT cells were transfected with pHR-VPR, Vpr mutants, or GFP. Twenty-four hours posttransfection, cells were lysed and anti-CypA antibody was used for immunoprecipitation. Immunoprecipitates were then analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis followed by Western blotting, using an anti-HA antibody that recognizes an amino-terminal hemagglutinin epitope present in all Vpr vector constructs.
In agreement with the previous finding, we found that Vpr efficiently coprecipitated with CypA (Fig. , lane 5). In contrast, both Vpr(P35N) and Vpr(P35A) were impaired in their abilities to coprecipitate with CypA, as evidenced by extremely faint Vpr bands (Fig. , lanes 3 and 4). Vpr(R80A) also failed to coprecipitate with CypA, as indicated by the absence of a detectable Vpr band (Fig. , lane 2). The blot shown on the top panel of Fig. was then stripped and reprobed with anti-CypA antibody in order to verify that equal amounts of CypA had been immunoprecipitated in experiments 1 through 6 (Fig. , second blot). Analysis of the steady-state levels of Vpr by Western blotting of input cell lysates (Fig. , third blot from the top, lanes 2′ to 5′) revealed that all Vpr mutants were expressed at comparable or higher levels than wild-type Vpr.
FIG. 3. Coimmunoprecipitation of Vpr with CypA. (A) 293FT cells were transfected with the indicated lentiviral vectors, and a transfection efficiency of 95% was recorded. Twenty-four hours posttransfection, cells were lysed and anti-CypA antibody was added to (more ...)
The above data indicate that the inability of Vpr mutants to coprecipitate with CypA did not stem from a decrease in the mutant protein steady-state levels, which would have suggested a loss of protein stability. In addition, the fact that Vpr(R80A) failed to coprecipitate with CypA indicates that residues outside the Vpr (residues 1 to 40) domain (8
) are also important for binding to CypA and that the presence of proline-35 is not sufficient for such binding.
To further explore the specificity of Vpr-CypA interaction, we performed parallel coimmunoprecipitation experiments in which cells were cultured in the presence of 2.5 μM CsA. CsA binds to CypA and competitively inhibits the interaction between p24 Gag and CypA (9
). CsA incubation did not impair the steady-state levels of Vpr in the cells, as evidenced by Western blotting of input lysate (Fig. , third blot from the top, compare lanes 5′ and 6′). CsA incubation, however, abolished the interaction between CypA and Vpr (Fig. , top blot, compare lanes 5 and 6).
Expression of wild-type or Vpr mutants in CypA−/− Jurkat cells further demonstrated that CypA is dispensable for the steady-state levels of Vpr expression (Fig. , lanes 2, 4, and 5). CypA−/− Jurkat cells presumably contain all cyclophilins other than CypA. Thus, it is formally possible that Vpr may interact with other cyclophilins, and this, in turn, may compensate for the absence of CypA. Since CsA inhibits all known cyclophilins, we asked whether incubation of CypA−/− cells with CsA would affect the levels of Vpr expression. As shown in Fig. (compare lanes 2 and 3), CsA did not appreciably affect the steady-state level of Vpr. The Vpr expression level in CypA−/− cells was similar to that obtained in CypA+ cells (Fig. , compare lanes 2 and 6) when tested in parallel. Thus, we conclude that the presence of CypA is not required for efficient expression of Vpr. We also conclude that even though CsA potently inhibits the Vpr-CypA interaction, this interaction is dispensable for efficient expression of Vpr.
The ultimate goal of the present study was to establish the requirement of CypA toward Vpr-induced G2 arrest. The availability of Vpr mutants defective for CypA binding, as well as the pharmacological inhibitor CsA, allowed us to ask whether binding to CypA could be dissociated from induction of G2 arrest. Expression of wild-type Vpr and Vpr(P35N) induced dramatic G2 arrest in CypA+ cells as well as in CypA−/− Jurkat cells (Fig. ; 85.8% and 60.1%, respectively). Addition of CsA to the cultures did not significantly change the G2 arrest levels (70.3% in CypA+ and 56.7% in CypA−/− cells, respectively). Vpr(P35N) also induced G2 arrest (52.7%) in CsA, although with slightly decreased efficiency compared with wild-type Vpr. In CypA−/−, Vpr(P35N) induced levels of G2 arrest (67.1%) comparable to those of wild-type Vpr. Incubation of cultures with CsA did not significantly affect G2 arrest by Vpr(P35N) (45% in CypA+ and 65% in CypA−/− cells). Therefore, binding to CypA is not required for the induction of G2 arrest by Vpr.
FIG. 4. Induction of G2 arrest by Vpr and Vpr(P35N) and failure of CsA to inhibit Vpr function. CypA+ (A) or CypA−/− (B) Jurkat cells were mock infected or infected with pHR-GFP, pHR-VPR, or pHR-VPR(P35N) and then subdivided into two cultures, (more ...)
The sharp discrepancies between our results and those reported by Zander and colleagues could be reconciled, in part, if CsA treatment induced the loss of stability of Vpr (and, therefore, loss of function as well) in a CypA-dependent manner. However, our findings demonstrate that although CsA inhibits the interaction between Vpr and CypA, the steady-state level of Vpr in the cells remains the same in CsA-treated and untreated cells, and incubation with CsA does not affect Vpr function.
The concentration of CsA used in our experiments was 2.5 μM. We find that this concentration of CsA effectively inhibits CypA-Vpr binding. This concentration is well below the 50-μg/ml (equivalent to 41.6 μM) CsA concentration used by Zander and colleagues (34
) when they observed loss of Vpr stability in cells treated with this CypA inhibitor. We reason that, since genetic removal of CypA failed to inhibit Vpr function or stability, the effects observed with 50 μg/ml CsA may be due to other effects of CsA which are CypA independent. For example, high concentrations of CypA may be cytotoxic. A precedent for the existence of an additional, ill-understood effect of CsA stems from the observation that CsA inhibits the infectivity of progeny HIV-1 virions in producer cells by a mechanism that is independent of CypA inhibition (18
Since CsA is thought to inhibit all known cyclophilins, our observations combining the presence of CsA and the genetic elimination of CypA suggest that cyclophilins other than CypA appear to also be dispensable for Vpr expression and induction of G2 arrest.
We conclude that while in vivo binding between Vpr and CypA is clearly detectable, this binding is not necessary for the stability of Vpr or its ability to induce G2 arrest. The interaction between Vpr and CypA is highly intriguing and should be further investigated as a potential modulator of virus-host interactions.