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. Author manuscript; available in PMC: 2015 Jun 4.
Published in final edited form as: Virology. 2015 Jan 22;477:10–17. doi: 10.1016/j.virol.2014.12.040

Determinants for degradation of SAMHD1, Mus81 and induction of G2 arrest in HIV-1 Vpr and SIVagm Vpr

Ana Beatriz DePaula-Silva a, Patrick A Cassiday a, Jeffrey Chumley a,b, Alberto Bosque a, Carlos M R Monteiro-Filho a, Cathal S Mahon c, Kelsey R Cone d, Nevan Krogan c, Nels C Elde d, Vicente Planelles a,#
PMCID: PMC4455942  NIHMSID: NIHMS695217  PMID: 25618414

Abstract

Vpr and Vpx are a group of highly related accessory proteins from primate lentiviruses. Despite the high degree of amino acid homology within this group, these proteins can be highly divergent in their functions. In this work, we constructed chimeric and mutant proteins between HIV-1 and SIVagm Vpr in order to better understand the structure-function relationships. We tested these constructs for their abilities to induce G2 arrest in human cells and to degrade agmSAMHD1 and Mus81. We found that the C-terminus of HIV-1 Vpr, when transferred onto SIVagm Vpr, provides the latter with the de novo ability to induce G2 arrest in human cells. We confirmed that HIV-1 Vpr induces degradation of Mus81 although, surprisingly, degradation is independent and genetically separable from Vpr’s ability to induce G2 arrest.

INTRODUCTION

The HIV-1 genome encodes structural proteins (Gag, Pol and Env), regulatory proteins (Tat and Rev), and accessory proteins such as Vif, Vpr, Vpu and Nef. HIV-2, SIVmac and SIVsmm encode Vpr, Vpx, Vif and Nef as accessory proteins. Vpr and Vpx from HIV-2, SIVmac and SIVsmm are highly related to each other and are thought to have arisen through gene duplication (Tristem et al., 1992). SIVagm encodes Vif, Vpr and Nef. A Vpu homolog is not found in the HIV-2/SIVmac/SIVsmm or the SIVagm phylogenetic groups.

From the entry step to the time of release from the host cell, lentiviruses encounter several restriction factors that function to inhibit viral infection and are considered innate immune effector mechanisms. TRIM5α, APOBEC3G, sterile alpha motif (SAM) and HD domain-containing protein 1 (SAMHD1) and tetherin are examples of host restriction factors. These restriction factors are typically overcome by the accessory proteins encoded by lentiviruses (reviewed in (Strebel, 2013)).

Three of the four accessory proteins in each HIV-1 (Vif, Vpr and Vpu) and HIV-2 (Vif, Vpr and Vpx) antagonize innate immunity by a common mechanism, the ubiquitin-proteasome system (UPS). These proteins modify the specificity of cullin-RING ubiquitin ligases (CRUL) such that non-cognate proteins are modified and later degraded.

Vpr has been associated with induction of cell cycle arrest in G2 and apoptosis (He et al., 1995; Jowett et al., 1995; Stewart et al., 1997). Vpr induces these effects via activation of the ATR kinase (Roshal et al., 2003), a result of manipulation of the ubiquitin ligase CRUL4DDB1/DCAF1 (Belzile et al., 2007; DeHart et al., 2007; Hrecka et al., 2007; Le Rouzic et al., 2007; Schrofelbauer et al., 2007; Wen et al., 2007). Recently, the ubiquitination target for the Vpr/CRUL4 complex has been identified (Laguette et al., 2014). Vpr induces premature activation of the SLX4 complex (SLX4com) (Laguette et al., 2014). Vpr increases the binding of DCAF1 to the scaffold protein SLX4 and, together with the polo-like kinase-1 (PLK1), promotes SLX4com remodeling, which results in Mus81 degradation (Laguette et al., 2014). As a consequence of the untimely SLX4com activation, replication forks are processed incorrectly causing cell cycle arrest in G2 (Laguette et al., 2014). The authors showed that by activating SLX4, Vpr prevents the viral DNA from stimulating cellular DNA sensors, which would normally trigger a type-I interferon response (Laguette et al., 2014)

Nuclear magnetic resonance (NMR) studies indicate that HIV-1 Vpr is comprised of three bundled alpha helices connected by short flexible loops and flanked by flexible amino-and carboxy-terminal unstructured regions (Morellet et al., 2003). The region on Vpr that binds to DCAF1 was mapped within the third α-helix, involving a leucine-rich motif (DeHart et al., 2007; Le Rouzic et al., 2007). The Vpr Q65R amino acid substitution within this region disrupts the interaction between Vpr and DCAF1 resulting in inability to induce G2 arrest (DeHart et al., 2007; Le Rouzic et al., 2007). The C-terminal unstructured region of Vpr is predicted to be required for interaction with the target, since the mutant R80A within that region is capable of interacting with DCAF1 but is unable to cause G2 arrest (DeHart et al., 2007). Furthermore, Vpr R80A acts as a dominant-negative protein because it binds to DCAF1 and blocks the Vpr-binding site (DeHart et al., 2007; Le Rouzic et al., 2007).

Vpx is encapsidated in HIV-2 and SIVmac virions and antagonizes SAMHD1 (Hrecka et al., 2011; Laguette et al., 2011a). SAMHD1 interferes with the ability of the virus to efficiently synthesize viral cDNA during reverse transcription because it reduces the available cellular pools of dNTPs (Goldstone et al., 2011). Consequently, SAMHD1 diminishes the capacity of the virus to infect macrophages, dendritic cells and quiescent T-cells wherein dNTP levels are normally very low (Hrecka et al., 2011; Laguette et al., 2011b). Vpx triggers degradation of SAMHD1 by associating with DCAF1 and recruiting SAMHD1 to the CRUL4DDB1/DCAF1 E3 ligase. SAMHD1 is subsequently ubiquitinated and degraded by the proteasome. Inhibition of the interaction between Vpx and DCAF1 by the mutant Vpx(Q76A) results in failure to degrade SAMHD1. HIV-1 Vpr and SIVmac Vpx appear to bind to similar or overlapping regions on DCAF1, although the amino acid residues in DCAF1 involved in interaction with either accessory protein are not identical (Cassiday et al., 2014).

African green monkeys (AGM) are endemically infected with several strains of simian immunodeficiency viruses collectively known as SIVagm (Jin et al., 1994). This group of primate lentiviruses encode three accessory genes: Vpr, Vif and Nef. SIVagm Vpr is bifunctional because it can induce G2 arrest and also antagonize SAMHD1. Unlike HIV-1 Vpr, SIVagm Vpr is highly species-specific, as it induces G2 arrest in AGM cells but not in human ones (Fletcher et al., 1996; Planelles et al., 1996), and is able to degrade agmSAMHD1 but not human SAMHD1 (hSAMHD1) (Lim and al., 2012).

In order to understand the structure-function relationships for HIV-1 Vpr and SIVagm Vpr we constructed chimeric proteins by exchanging homologous domains of HIV-1NL4-3 Vpr and SIVagm.gri Vpr. We then investigated the ability of the different chimeras to cause cell cycle arrest in G2 and to induce degradation of SAMHD1 and Mus81. We also investigated whether the structural requirements toward degradation of Mus81 are the same as those required for induction of G2 arrest by HIV-1 Vpr.

RESULTS

To study the structure-function relationships in HIV-1 Vpr and SIVagm.gri (henceforth, SIVagm) Vpr, a set of 4 chimeras and 2 truncations were constructed as shown in Figure 1A. The exchange points for the chimeras were designed based on the published nuclear magnetic resonance (NMR) structure of HIV-1 Vpr and the high degree of amino acid homology with SIVagm Vpr (Figure 1B).

Figure 1.

Figure 1

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Construction of chimeras between HIV-1 Vpr and SIVagm.gri Vpr. A. Schematic representation of the chimeras between HIV-1 Vpr and SIVagm Vpr. HIV-1 Vpr is comprised of three alpha helices, symbolized as barrels, flanked by amino-and carboxy-terminal unstructured regions that are connected by short flexible loops. R represents HIV-1Vpr and AGM represents SIVagm.gri Vpr. The subscript numbers denote the amino acid residues. B. Alignment between HIV-1Vpr and SIVagm.gri Vpr, illustrating the amino acid conservation between these two strains. An asterisk indicates fully conserved residues; colon represents amino acid conservation with strongly similar properties; period designates amino acid conservation with weakly similar properties. Highlighted residues mark alpha helices and yellow stars mark residues Q65 and R80.

Determinants required for induction of G2 arrest

It was previously suggested that the C-terminal unstructured region of HIV-1 Vpr was required to interact with a putative G2 arrest-related cellular factor (DeHart et al., 2007; Di Marzio et al., 1995; Le Rouzic et al., 2007). In support of the previous notion, the point mutant Vpr(R80A), although capable of interacting with DCAF1, was unable to induce G2 arrest, and behaved as a dominant-negative protein by competing with wild-type Vpr for DCAF1 binding (DeHart et al., 2007).

We first constructed two truncations in HIV-1 Vpr (Vpr1-80 and Vpr1-84; Figure 1A) encoded by lentiviral vectors (Verrier et al., 2011). HeLa cells were then transduced with VSV-G-pseudotyped lentivirus vectors (Supplemental Figure 1) encoding HIV-1 Vpr, HIV-1 Vpr(R80A) or each of the indicated truncations. As shown in Figure 2A and 2B, HIV-1 Vpr, but not HIV-1 Vpr(R80A), HIV-1 Vpr(1–80), or HIV-1 Vpr(1–84) induced cell cycle arrest. The percentages of cells transduced with the corresponding lentivirus vectors are shown in Supplemental Figure 2A.

Figure 2.

Figure 2

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G2 arrest determinants are found in the C-terminal domains of HIV-1 Vpr and SIVagm Vpr. A. Cell cycle analysis of Hela cells transduced with VSV-G-pseudotyped lentiviral vectors for HIV-1 Vpr, HIV-1 Vpr(R80A), HIV-1 Vpr(1–80) or HIV-1 Vpr(1–84). 48h post transduction, DNA staining (propidium iodide) was used to quantify the cell cycle by flow cytometry. B. Positive values are above the mean percent of cells in G2 from 3 negative control repeats plus 3 times the standard deviation (line marked with an asterisk) corresponding to a 99.7% interval of confidence. C. Cell cycle analysis of Hela cells transduced with VSV-G-pseudotyped lentiviral vectors for HIV-1 Vpr, SIVagm Vpr and each of the chimeras. These experiments were performed at least three times.

While HIV-1 Vpr is able to induce cell cycle arrest in human and non-human primate cells, SIVagm Vpr arrests AGM, but not human cells (Planelles et al., 1996). We asked whether transposition of the C-terminal domain (HIV-1 Vpr residues 78–96) onto SIVagm Vpr (Ch1) would confer upon SIVagm Vpr a de novo ability to induce G2 arrest in human cells. As shown in Figure 2C and 2D, Ch1 was able to induce G2 arrest in human cells. Therefore, the inability of SIVagm Vpr to function in human cells can be overcome by a determinant within the C-terminal domain of HIV-1 Vpr.

Using the inactive truncation HIV-1 Vpr(1–80) as the recipient, we asked whether grafting the C-terminus of SIVagm Vpr (Ch2) would enable induction of G2 arrest in human cells. Hela cells transduced with the Ch2 also underwent cell cycle arrest (Figure 2C and 2D). The C-terminal domain of SIVagm Vpr is intrinsically capable of recruiting the target leading to G2 arrest in human cells when in the context of HIV-1 Vpr. One possible explanation for the previous observation is that, while DCAF1 is highly conserved across primate lentiviruses (Berger et al., 2014), the target protein may be variable. In addition, these observations suggest that the interaction of Vpr with the target may be dependent, in part, on determinants that lie upstream of the C-terminal domain of Vpr.

In contrast with the above findings, transposition of the N-terminal unstructured region of HIV-1 Vpr to SIVagm Vpr (Ch3) did not confer upon this chimera the ability to induce G2 arrest in human cells (Figure 2C and 2D). The reciprocal exchange (Ch4), which contained most of HIV-1 Vpr with the N-terminus of SIVagm Vpr, was still capable of inducing arrest in human cells (Figure 2C and 2D). The above results indicate that the N-terminal unstructured region of HIV-1 Vpr is not required to induce cell cycle arrest.

In an effort to test whether the chimeras maintained the capacity to fold correctly, we verified their abilities to interact with DCAF1. HIV-1 Vpr, SIVagm Vpr and chimeras Ch1 to Ch4 were able to Co-IP with DCAF1 (Figure 3, lanes 1 and 4). In contrast, the mutant HIV-1 Vpr(Q65R), did not efficiently co-IP with DCAF1 (Figure 3)

Figure 3.

Figure 3

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Chimeras Co-IP with endogenous DCAF1. HEK293FT cells were transfected with HIV-1 Vpr, HIV-1 Vpr(Q65R), SIVagm.gri Vpr or each of the chimeras. 48h post-transfection cells were lysed and subjected to immunoprecipitation using magnetic beads coated with anti-HA antibody.

The amino terminal domain of SIVagm Vpr is required for degradation of agmSAMHD1

SIVagm Vpr is bi-functional in AGM cells, where it can arrest cells in G2 and induce degradation of agmSAMHD1. However, SIVagm Vpr, when expressed in human cells, is unable to perform either function (Lim and al., 2012). We wished to analyze which domain(s) in SIVagm Vpr may be important for targeting agmSAMHD1 for degradation. To that end, we tested the chimeras between SIVagm Vpr and HIV-1 Vpr. AgmSAMHD1 was degraded by SIVmac Vpx and SIVagm Vpr (Figure 4A, lanes 5 and 6, respectively) but not by HIV-1 Vpr (Figure 4, lane 4) as previously reported (Hrecka et al., 2011). Addition of epoxomycin to cells transfected with SIVagm Vpr prevented the degradation of agmSAMHD1 (compare lanes 6 and 11), confirming the involvement of the UPS.

Figure 4.

Figure 4

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The N-terminal unstructured domain of SIVagm Vpr is required for agmSAMHD1 degradation. 293FT cells were co-transfected with HA-agmSAMHD1 and with vectors encoding HA-HIV-1 Vpr, HA-SIVagm Vpr, HA-SIVmac Vpx or each of the chimeras. 48h post-transfection cells were harvested and the level of ectopic agmSAMHD1 was analyzed by Western blot using anti-HA. Results are representative of three independent experiments.

To examine the potential role of the C-terminal domain of SIVagm Vpr in degradation of agmSAMHD1, we tested Ch1. As shown in Figure 4A, lane 7, Ch1 remained capable of inducing degradation of agmSAMHD1. Therefore, the C-terminal domain of SIVagm Vpr is dispensable for the degradation of SAMHD1. The reciprocal chimera (Ch2) in which the C-terminus of SIVagm Vpr was transferred onto HIV-1 Vpr (lane 8) failed to degrade agmSAMHD1. These results suggest that the C-terminal unstructured region of SIVagm Vpr is not sufficient to confer upon HIV-1 Vpr the ability to degrade agmSAMHD1.

The N-terminal domain of SIVmac Vpx was previously shown to be required to overcome SAMHD1 restriction in myeloid cells (Ahn et al., 2012; DeLucia et al., 2013). To confirm this notion, we tested Ch3. As shown in Figure 4A, lane 9, Ch3 failed to induce degradation of agmSAMHD1. We interpret these data to mean that the loss of the native N-terminal domain of SIVagm Vpr in Ch3 ablated the ability to target agmSAMHD1 for degradation. Previous findings for the related protein, SIVmac Vpx, demonstrated that its amino-terminal domain is required to induce hSAMHD1 degradation (Ahn et al., 2012; DeLucia et al., 2013). Therefore, we speculate, by analogy, that the amino-terminal domain of SIVagm Vpr is also required for degradation of agmSAMHD1.

Ch4, which contains the N-terminus of SIVagm Vpr (residues 1–26) followed by HIV-1 Vpr (residues 17–96), also failed to trigger degradation of agmSAMHD1 (Figure 4A, lane 10). Therefore, we conclude that the N-terminal domain of SIVagm Vpr, although required (see above), is not sufficient for this function. Recently, a crystal structure of a complex of DCAF1/SIVsmVpx/smSAMHD1 demonstrated that residues between the 2nd and 3rd alpha helices of SIVsm Vpx interact with smSAMHD1 (Schwefel et al., 2014) Therefore, by analogy, it is possible that a second determinant required for degradation of agmSAMHD1 might lie in the homologous location of SIVagm Vpr. Schwefel et al. identified two residues in SIVsm Vpx, Met62 and Ser63, which establish a hydrophobic interaction and a hydrogen bond, respectively, with Lys622 and Phe621 in SAMHD1. SIVagm Vpr does not have significant conservation in this area and, specifically, equivalent residues to Met62 and Ser63 are not present in SIVagm Vpr.

Degradation of Mus81 is independent of Vpr’s ability to induce G2 arrest

The SLX4 complex was recently proposed as a target for HIV-1 Vpr (Laguette et al., 2014). In the presence of HIV-1 Vpr the Mus81 protein, a constituent of the SLX4 complex, is ubiquitinated, contributing to the activation of the complex and leading to G2 arrest (Laguette et al., 2014). We observed that HIV-1 Vpr induces degradation of Mus81 as reported by Laguette et al. (Laguette et al., 2014), although to a modest degree (Figure 5A).

Figure 5.

Figure 5

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Degradation of Mus81 by HIV-1 Vpr is independent of G2 arrest induction. A. HEK293FT cells were co-transfected with HA-HIV-1 Vpr (from 1μg to 0.05 μg) and constant amount of V5-Mus81. 48h after transfection cells were lysed and the level of Mus81 was analyzed by Western blot using anti-V5 antibody. B. HEK293FT cells were cotransfected with V5-Mus81 and HIV-1 Vpr, or HIV-1 Vpr(R80A), or HIV-1 Vpr(Q65R). 18h prior to cell harvesting, Epoxomicin or MLN4924 were added to cells. Levels of exogenous Mus81 were measured by Western blot using anti-V5 antibody. C. Quantification of Mus81 normalized to β-actin from panel B. The line indicates the basal level of Mus81 in the presence of empty vector. Protein level below this line represents degradation of Mus81. D. HEK293FT cells were co-transfected with V5-Mus81 and HIV-1 Vpr or SIVagm Vpr or SIVmac Vpx or each of the chimeras. E. Quantification of Mus81 normalized to β-actin from panel D.

We then asked whether the degradation of Mus81 correlated with the ability of known HIV-1 Vpr mutants and homologues to induce G2 arrest. We conducted these experiments in the presence or absence of the neddylation inhibitor that blocks CRULs, MLN4924 (Soucy et al., 2009) or the proteasome inhibitor, Epoxomicin (Meng et al., 1999). Surprisingly, HIV-1 Vpr(R80A) (Figure 5B, compare lanes 3 and 11,) and HIV-1 Vpr(Q65R) (compare lane 3 and 15 and Supplemental Figure 3), two mutants that are deficient in G2 arrest induction, degraded Mus81 to a similar degree as did wild-type HIV-1 Vpr. Degradation of Mus81 for the above experiment was quantified by densitometry (Figure 5C). Vpr(Q65R) is deficient in binding to DCAF1 and, therefore, to degrade Mus81 it may utilize a different DCAF than that involved in induction of G2 arrest. Vpr(R80A) is thought to be unable to bind the putative G2 arrest-related target, but is still able to induce degradation of Mus81 (Figure 5B lane 11). Therefore, it appears that the molecular determinants of Mus81 degradation induced by Vpr are different from those required for induction of G2 arrest.

We then asked whether V5-Mus81 was degraded in the presence of chimeras 1–4 and also in the presence of Vpx and SIVagm Vpr. As depicted in Figure 5D, SIVmac Vpx and SIVagm Vpr did not induce degradation of Mus81 (lanes 5 and 6). Ch1, a construct that induces G2 arrest, did not target Mus81 for degradation (lane 7). Ch2, which also induced G2 arrest, was able to induce degradation of Mus81 (lane 8). Therefore, as we showed above with the Vpr point mutants Q65R and R80A, the ability to induce G2 arrest does not correlate with degradation of Mus81 when using our chimeric constructs. Furthermore, Ch2 is able to induce Mus81 degradation in the absence of HIV-1 Vpr’s unstructured C-terminal domain, pointing at another important difference between the requirements for G2 arrest and degradation of Mus81.

Transpositon of the N-terminus of HIV-1 Vpr into SIVagm Vpr (Ch3) did not lead to Mus81 degradation (Figure 5D, lane 9). Ch4, which contains the N-terminus of SIVagm Vpr (residues 1–26) followed by HIV-1 Vpr (residues 17–96) induced degradation of Mus81 (Figure 5D, lane 10). These results suggest that the N-terminal unstructured region of HIV-1 Vpr is also not critical for Mus81 degradation.

Inhibition of the proteasome with Epoxomicin caused stabilization of Mus81 in the presence of HIV-1 Vpr or HIV-1 Vpr(R80A) (Figure 5B, compare lanes 4 to 7 and 11 to 12, respectively) or HIV-1 Vpr(Q65R) (Supplemental Figure 3). Because Epoxomicin targets the proteasome, degradation of ubiquitinated proteins is inhibited (Meng et al., 1999). Blockade of Cullin activity by the neddylation inhibitor MLN4924 (Soucy et al., 2009) also stabilized Mus81 in the presence of HIV-1 Vpr, HIV-1 Vpr(R80A) (Figure 5B; compare lanes 4 to 8 and 11 to 13, respectively) and HIV-1 Vpr(Q65R) (Supplemental Figure 3). Interestingly, treatment with Epoxomicin but not with MLN4924, led to the stabilization of Mus81 (Figure 5B, compare lane 3 with 5 and 6). This result suggests that Mus81 is controlled naturally by the ubiquitin proteasome system but independently of a CRUL. The above data, taken together with the observation that Vpr(Q65R) is active at inducing degradation of Mus81, suggests that Vpr manipulates a CRUL not containing DCAF1, for targeting Mus81. We also observed that SIVagm Vpr, which causes G2 arrest in AGM cells, was unable to degrade agmMus81 (Supplemental Figure 4), supporting that also for SIVagm Vpr, degradation of Mus81 is independent of the induction of G2 arrest.

Discussion

The main conclusions from this study are as follows: (i) the C-terminal unstructured region of HIV-1 Vpr contains a determinant required for induction of G2 arrest; transfer of the C-terminus of HIV-1 Vpr onto SIVagm Vpr is sufficient to confer upon this chimera a de novo ability to induce G2 arrest in human cells; (ii) the carboxy-terminal unstructured region of SIVagm Vpr is intrinsically capable of recruiting the G2 arrest-related target in human cells when transferred onto HIV-1 Vpr, but not in its native configuration within SIVagm Vpr; (iii) the amino-terminal domain of SIVagm Vpr is required for agmSAMHD1 degradation, although it is not sufficient; (iv) targeting Mus81 for degradation and induction of G2 arrest are independent, separable functions of HIV-1 Vpr; and (v) degradation of Mus81 by HIV-1 Vpr is proteasome-and cullin-dependent, although it is likely that DCAF1 is not involved in this activity.

The amino-terminal domain of SIVagm.gri Vpr, like the homologous regions in HIV-2 and SIVmac (Ahn et al., 2012; DeLucia et al., 2013) was required for degradation of agmSAMHD1. The crystallographic data for SIVsm Vpx (Schwefel et al., 2014) showed that Glu residues 15 and 16 establish ionic interactions with Arg residues 609 and 617, respectively, in SAMHD1. While residues Glu 15 and 16 are conserved in HIV-2, SIVmac, SIVsm and SIVrcm Vpx, SIVagm Vpr only contains one Glu residue in the corresponding region (Glu 16). Therefore, it is tempting to speculate that Glu 16 from SIVagm Vpr may interact with either Arg 609 or 617 in SAMHD1.

Our observations suggest that the inability of SIVagm Vpr to induce G2 arrest in human cells is not intrinsic to this protein, because its C-terminal domain, when grafted onto HIV-1 Vpr, generates a chimera that is active. Because SIVagm Vpr is able to bind hDCAF1, the species-specificity of this protein likely can be explained based on the different ability to interact with SLX4 complex in the different species. Recently, it has been reported that interaction between HIV-1 Vpr or SIV Vpr and SLX4 correlates with induction of G2 arrest (Berger et al., 2014). Since Mus81 is targeted for ubiquitination by Vpr (Laguette et al., 2014) we decided to focus on this protein to investigate whether the requirements for induction of G2/M arrest and Mus81 degradation were the same. We found that HIV-1 Vpr induced degradation of Mus81 through the proteasome system, by manipulating CRUL, since addition of Epoxomicin or MLN4924 relieved this effect. Interestingly, HIV-1 Vpr(R80A) and HIV-1 Vpr(Q65R) degraded Mus81 in our hands. Therefore, our findings, at first sight, appear to be at odds with those of Berger et al., who reported that knockdown of SLX4 (of which Mus81 is an integral component) abolished the cell cycle arrest by the lentiviral proteins (Berger et al., 2014). One possible explanation that might reconcile our findings with those of Berger et al. (Berger et al., 2014) could be if the modest degradation of Mus81 that we and Laguette et al. (Laguette et al., 2014) observe is simply not essential for induction of G2 arrest. It would still remain to be explained why Vpr(R80A) is competent for interaction with the SLX4 complex (Berger et al., 2014; Laguette et al., 2014), but incapable of inducing G2 arrest.

Materials and Methods

Cell Lines and Transfections

HeLa cells and HEK293FT (Invitrogen) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% of fetal bovine serum (FBS) and 2mM of L-glutamine (Invitrogen). HEK293FT cells were transfected by Calcium Phosphate method, as previously described (Zhu et al., 2001). HeLa cells were transfected using FuGENE HD (Promega) according to manufacturer’s instruction.

Plasmids

HA-HIV-1Vpr, HA-SIVagm.gri Vpr (from Grivet monkeys), HA-SIVmac Vpx, and each of the chimeras were PCR amplified and cloned into pFIN-EF1-GFP-2A-mCherry-HA-WPRE, a kind gift of Dr. Susan Semple-Rowland (Verrier et al., 2011) in place of mCherry-HA. The truncations HIV-1 Vpr(1–80) and HIV-1 Vpr(1–84) were generated on HIV-1Vpr using QuickChange Lightning (Agilent Technologies). Mammalian expressing vector encoding HA-agmSAMHD1 was a kindly provided by Dr. Michael Emerman. hMus81 cDNA was purchased from DNASU, PCR amplified, N-terminus tagged with V5 epitope cloned into pFIN vector (without mCherry) in place of GFP.

Immunopreciptation and Western blot

Cells were washed in PBS and lysed in NETN buffer, in the presence of phosphatase (PhosSTOP; Roche) and protease (Complete EDTA free tablets; Roche, Indianapolis, IN) inhibitors. The concentration of proteins were determined by PierceTM BCA (Thermo Scientific, Rockford, IL). Magnetic Beads (SIGMA-ALDRICH, St Louis, MO) were coated with anti-HA antibody (HA1.1, Covance) for 30min (RT). For immunoprecipitation, cell lysates were incubated with the beads coated with anti-HA for 1h at 4°C. Beads were washed 3 times with NETN buffer and proteins were eluted in Lamelli buffer and boil for 10min. Samples were subjected to SDS-PAGE on 4–12% acrylamide gel Criteron™ TGX gel (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (EMD Millipore, Billerica, MA). For degradation assay, cells were lysed using SET buffer (1% SDS, 50mM tris-HCl, pH 7.4, 1mM EDTA) and boiled for 20min. Samples were resolves by SDS-PAGE as described above. V5 antibody (Sigma Aldrich), β-actin (Sigma Aldrich), rabbit polyclonal DCAF1 antibody was kindly provided by Dr. Ling-Jun Zhao (Saint Louis University).

Cell cycle analysis

HeLa cells were treated with trypsin, washed with PBS and stained with Propidium Iodide (PI) using Hypotonic PI buffer (Sodium Citrate, Triton X-100, Propidium Iodide, RNAse). Samples were analyzed by flow cytometry for DNA content. FlowJo was then used to analyze the cell cycle.

Lentiviral vectors

Lentiviral were produced in HEK293FT cells. Briefly, transfer plasmid (12.5μg), packaging plasmid (12.5μg) and envelope (5μg) plasmid were co-transfected in HEK293FT cells by calcium phosphate. Supernatants were collected every 12h until monolayer died. Lentiviruses were concentrated by ultracentrifugation 25,000rpm for 2h at 4°C. Viruses were titrated in HeLa cells.

Drugs

Epoxomicin (Calbiochem) was solubilized in DMSO and used at 1μM. MLN4924 (MedChem Express) was diluted in DMSO and used at 1μM.

Supplementary Material

Supplemental figures

Supplemental Figure 1. Schematic representation of the pFIN-EF1-GFP-2A-HA-Vpr-WPRE vector. The EF1 promoter drives the expression of GFP and HA-Vpr as a single polyprotein. Co-translational cleavage within the 2A site (between amino acid residues 18 and 19) results in equimolar amounts of both proteins. LTR (Long Term Repeat); EF-1 elongation factor 1 alpha promoter; GFP, green fluorescent protein; 2A-like cleavage peptide from porcine Teschovirus; WPRE (woodchuck hepatitis virus posttranscriptional regulatory element). Bacterial sequences for this plasm id are not shown.

Supplemental Figure 2. Transduction efficiencies in Hela cells. A and B. The efficiency transduction was measured by GFP expression from the lentiviral vector. Further cell cycle experiments were only performed when the efficiency of transduction was at least 45%. Shown is a representative experiment of 3 independent repeats.

Supplemental Figure 3. HIV-1 Vpr(Q65R) degrades Mus81 in a proteasome- and Cullin-dependent manner. A. 293FT cells were transfected with human V5-Mus81 in the presence of HIV-1 Vpr(WT) or HIV-1 Vpr(Q65R). 48h post transfection cells were harvested and the level of Mus81 was analyzed using anti-V5 antibody. B. Bar graph representation of the level of hMus81. The amount of Mus81 was normalized to the β-actin. Representative of three independent experiments.

Supplemental Figure 4. agmMus81 is not degraded by SIVagm Vpr. A. 293FT cells were transfected with human V5-Mus81 (hMus81) or V5-agmMus81 in the presence of HIV-1 Vpr or SIVagm Vpr. 48h post transfection cells were harvested and the level of Mus81 was analyzed using anti-V5 antibody. B) Bar graph representation of the level of hMus81 (dark grey) and agmMus81 (light grey). The amount of Mus81 was normalized to the β-actin.

Acknowledgments

We would like to thank Drs. Stillman and Fujinami and for expert guidance and helpful suggestions. This work was supported by the National Institute of Health grants R01 AI087508 and R01 AI49057 to V.P.

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Associated Data

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Supplementary Materials

Supplemental figures

Supplemental Figure 1. Schematic representation of the pFIN-EF1-GFP-2A-HA-Vpr-WPRE vector. The EF1 promoter drives the expression of GFP and HA-Vpr as a single polyprotein. Co-translational cleavage within the 2A site (between amino acid residues 18 and 19) results in equimolar amounts of both proteins. LTR (Long Term Repeat); EF-1 elongation factor 1 alpha promoter; GFP, green fluorescent protein; 2A-like cleavage peptide from porcine Teschovirus; WPRE (woodchuck hepatitis virus posttranscriptional regulatory element). Bacterial sequences for this plasm id are not shown.

Supplemental Figure 2. Transduction efficiencies in Hela cells. A and B. The efficiency transduction was measured by GFP expression from the lentiviral vector. Further cell cycle experiments were only performed when the efficiency of transduction was at least 45%. Shown is a representative experiment of 3 independent repeats.

Supplemental Figure 3. HIV-1 Vpr(Q65R) degrades Mus81 in a proteasome- and Cullin-dependent manner. A. 293FT cells were transfected with human V5-Mus81 in the presence of HIV-1 Vpr(WT) or HIV-1 Vpr(Q65R). 48h post transfection cells were harvested and the level of Mus81 was analyzed using anti-V5 antibody. B. Bar graph representation of the level of hMus81. The amount of Mus81 was normalized to the β-actin. Representative of three independent experiments.

Supplemental Figure 4. agmMus81 is not degraded by SIVagm Vpr. A. 293FT cells were transfected with human V5-Mus81 (hMus81) or V5-agmMus81 in the presence of HIV-1 Vpr or SIVagm Vpr. 48h post transfection cells were harvested and the level of Mus81 was analyzed using anti-V5 antibody. B) Bar graph representation of the level of hMus81 (dark grey) and agmMus81 (light grey). The amount of Mus81 was normalized to the β-actin.

NIHMS695217-supplement-Supplemental_figures.pdf (2.9MB, pdf)

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