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the world's scientific community lost one of its most brilliant and innovative members – Dr. Andrei Yakovlev. He was 63, and at the zenith of his intellectual powers. With his uncanny ability to persuade and to ignite interest in his ideas, he challenged and stimulated everyone around him through his keen scientific vision and unparalleled intellectual depth and rigor. He collaborated with dozens of researchers – mathematicians , statisticians, biologists, epidemiologists, medical doctors and engineers. For many of them, these collaborations were a life changing experience. His unflinching belief in the power of mathematics and his tireless pursuit of new biomedical knowledge were irresistibly infectious and inspiring to two generations of his students and hundreds of his colleagues. For more than three decades he passionately advocated his vision for the future of Biology and Medicine as disciplines built upon a solid foundation of mathematical theory and statistical methodology. He has always enjoyed and cherished those wonderful "moments of truth" when predictions based on his models were confirmed by biological experiments or provided a perfect fit to known biomedical data, and he shared his joy with his co-workers, students and friends. He was a natural leader, caring mentor, and paternal figure for everyone privileged to work under his tutelage. He led by his wisdom, honesty, dignity and championship for scientific truth. He was a towering moral authority for many of his friends and coworkers, always ready to help, offer advice and even, for better or for worse, make critical decisions for them. Andrei Yakovlev was born on November 30, 1944 in Len-ingrad (now St. Petersburg), Russia. The period of his adolescence and youth fell upon the era of the post-WWII revival of mathematics and natural sciences in the Soviet Union. The combination of good education, professional opportunity and relative freedom from the oppressive communist ideology attracted thousands of talented and daring young people to mathematics, theoretical and experimental physics, chemistry, biology and medicine. Andrei took full advantage of this unique historical chance and received an M.D. from the 1st Medical Institute in Leningrad (1967), a Ph.D. in cell biology from the Pavlov Institute of Physiology of the Academy of Sciences of the USSR (1973) and a Doctor of Science degree (which is the highest scientific degree in Russia) in Mathematics from Moscow State University (1981). With such a formidable background he was uniquely positioned to transcend the boundaries of the four …


Background
The HIV-1 accessory protein, viral protein R (Vpr), has been shown to be important in viral cytopathicity during virus infection and can kill cells independently when experimentally expressed [1,2]. It is highly conserved, specifically packaged into virions, and facilitates HIV-1 replication in nondividing cells [3], while removal of its homologues in simian immunodeficiency virus results in attenuation of virus replication and disease in rhesus monkeys [4]. The lethal capability of Vpr correlates with its ability to induce cell cycle blockade in G 2 ,M [2,[5][6][7][8][9][10]. Although multiple mechanisms have been proposed to explain how Vpr arrests the cell cycle, there is no unifying theory. For example, the DNA damage sensing kinase, ATR, the damaged DNA binding protein 1 component of an E3 ubiquitin ligase, and the scaffolding protein, 14-3-3, have all been shown to mediate Vpr-induced G 2 ,M cell cycle arrest [11][12][13][14][15][16][17]. Since the majority of these studies were performed in non-lymphocyte human cell lines, a better mechanistic understanding of how Vpr blocks the cell cycle in lymphocytes could help illuminate the mechanism of T cell killing during HIV-1 infection. We thus investigated the role of 14-3-3 in Vpr arrest in T lymphocytes and determined how its function is altered by Vpr and HIV-1 infection.
Progression through the mitotic cell cycle in mammalian cells is controlled by a group of serine/threonine protein kinases termed cyclin-dependent kinases (Cdks) that are activated as a heterodimeric complex with a partner from the family of cyclin proteins. Cdk1-CyclinB is responsible for regulating several events during the G 2 ,M transition and progression through mitosis, including chromosome condensation, Golgi network fragmentation, and breakdown of the nuclear lamina. Specific functional substrates include microtubule-binding proteins and proteins involved in translation, ubiquitin-dependent proteolysis, and Cdk1 regulation (e.g. Cdc25 phosphatases). Several negative and positive regulators of both Cdk1 and Cyclins affect kinase activity under normal cycling and cellular stress conditions. For example, Wee1 and Myt1 kinases inhibit Cdk1 activity through phosphorylation of threonine and tyrosine residues, while Cdc25 phosphatases restore activity by dephosphorylation of these residues. Phosphorylation of CyclinB1 is also important for Cdk1-CyclinB activity, as this is required for rapid nuclear entry of CyclinB1 at the end of prophase preceding nuclear envelope breakdown [18]. This gives Cdk1-CyclinB access to key nuclear mitotic substrates. The 14-3-3 scaffolding proteins have also been implicated in regulation of the G 2 ,M phase of the cell cycle. These small, acidic polypeptides of 28-32 kD are highly conserved, expressed in all eukaryotic organisms, and consist of seven different isoforms encoded by distinct genes in mammals with a variety of biological functions. Acting as either hetero-or homodimers, 14-3-3 proteins bind to specific phosphoserine and phosphothreonine sequence motifs and regulate target protein function by compartmental sequestration, altered enzymatic activity, or inhibition/promotion of protein-protein interactions. 14-3-3 regulates the G 2 ,M transition by at least two mechanisms: cytoplasmic sequestration of the Cdk1 activating phosphatase, Cdc25C, during most of the natural cell cycle [19,20], as well as cytoplasmic sequestration of Cdk1 itself following DNA damage [21,22]. Recent evidence demonstrated that Vpr-induced G 2 ,M arrest is mediated by Vpr binding to 14-3-3, which allows 14-3-3 to bind and inhibit unphosphorylated Cdc25C, preventing Cdk1 activation [11]. As these studies were based on overexpression systems and are conducted in yeast or non-T cell lines, extension of these findings to a more physiological system would help improve our understanding of this important event.

Assays for viral production, cell viability, and cell cycle
HIV-1 cytopathicity was assessed by flow cytometric forward and side light scatter or forward scatter and propidium iodide (PI; Sigma) profiles of at least 10,000 live cells daily throughout the course of infection (FACScalibur, ® Becton Dickinson). All FACS data analysis was performed using FlowJo software (Tree Star, Inc.). Simultaneously, these samples were measured for HIV-1 provirus expression by GFP fluorescence. Infected viable cell counts were measured by collection for a constant period of time (50 seconds) per sample. To measure DNA content, cells were fixed in 1% paraformaldehyde (in PBS) for 10 minutes, washed once in PBS, resuspended in 70% ethanol while gently mixing, incubated at -20°C for 30 minutes or overnight, washed in PBS, and resuspended in 200 μl DNA staining solution (5 μg/ml PI, 50 μg/ml RNase, 0.45 mg/ ml sodium citrate, in PBS) for 30 minutes at 25°C. Alternatively, cells were stained with 10 μM of the cell-permeable DNA dye, DRAQ5 (Axxora, San Diego), for 5 min at 37°C according to the manufacturer's instructions. Stained cells were processed on a FACScalibur ® or FACScan (Becton Dickinson) and using the doublet discriminator module (DDM) to exclude cells with DNA signal width greater than the bulk cell population.
For immunoprecipitation (IP) experiments, cells were lysed with a mild Nonidet P-40-based IP buffer (0.2% Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.5) for 30 minutes with gentle mixing, normalized for protein concentration quantitated by the BCA™ Protein Assay Kit (Pierce), pre-cleared with Protein G-Agarose (Roche) for 30 minutes, and immunoprecipitated with 3-5 μg antibody for 3 to 6 hours. All steps were performed at 4°C with gentle mixing. Generally, 2-4 mg lysate was used for each IP experiment and an input sample was set aside following the pre-clear step. IP pellets were washed three times in lysis buffer and eluted with SDS loading buffer and separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) as above.
Cross-linking experiments were performed by treatment of live cells with 1 or 5 mM disuccinimidyl suberate (DSS, Pierce) for 30 minutes at 25°C followed by quenching by addition of 50 mM glycine for 15 minutes at 25°C. Cells were washed in phosphate buffered saline (PBS) twice followed by lysis in IP buffer for 30 minutes at 4°C. Lysates were cleared by centrifugation at 2,000 × g for 10 minutes and 120 μg protein was separated on 3-8% Tris-acetate gel (Invitrogen) run in Tris-acetate running buffer (Invitrogen) on ice. Protein was transferred to a nitrocellulose membrane using NuPAGE transfer buffer (Invitrogen) for two hours and blotted as described below.

Centrosome isolation
Centrosomes were isolated as previously described [27] with minor adaptations. Briefly, 50 × 10 6 Jurkat T cells were treated with 1 μg/ml cytochalasin D and 0.2 μM nocodazole for 1 hour at 37°C then washed twice in PBS followed by lysis in 5 ml 0.5% NP40 lysis buffer (1 mM Tri1mM Tris-HCl pH 8.0, 0.5% NP40, 0.5 mM MgCl 2 , protease inhibitor, 0.1% 2-mercaptoethanol, phosphatase inhibitor) for 5 minutes on ice. Nuclei and chromatin were pelleted at 2,500 × g for 10 minutes and the supernatant was filtered through a 70 μm mesh nylon cell strainer (BD Falcon). PIPES buffer was added to 10 mM and DNase I (Pharmacia) added at 2 units/ml and lysates incubated 30 minutes on ice. 3.5 ml of lysate (~20 mg) was loaded onto a discontinuous sucrose gradient (0.5 ml 70%, 0.3 ml 50%, and 0.3 ml 40% sucrose) in SW55 Ti 5 ml ultracentrifuge tubes (Beckman). All sucrose solutions were prepared w/w in 10 mM PIPES, 0.1% TritonX-100, 0.1% BME. Samples were centrifuged at 120,000 × g for 1 h at 4°C. The bottom of the tube was gently pierced with an 18-guage needle and fractions were harvested dropwise collecting ~200 μl per fraction for 7 fractions. Fractions were diluted into 1 ml 10 mM PIPES and incubated 30 minutes at 4°C with gentle mixing to dissolve the sucrose. Centrosomes were recovered by centrifugation at 16,000 × g for 10 minutes.

14-3-3 and Chk1 mediate Vpr G 2 ,M cell cycle block in CD4+ T cells
The mechanism by which HIV-1 Vpr blocks the cell cycle remains unclear. It was previously shown that the σ and η isoforms of 14-3-3 bind HIV-1 Vpr and play an important role in mediating the G 2 ,M cell cycle arrest in the human non-T cell lines, HeLa, HEK 293, and HCT116 [11]. We therefore began our investigation by testing whether siRNA targeting of the various isoforms of 14-3-3 could mitigate Vpr-induced G 2 ,M blockade in the CD4+ Jurkat T cell leukemia line. The viral strains used to carry out these studies are shown in Fig. 1A. Since 14-3-3 σ is not expressed in T cells and 14-3-3 θ (or τ) was initially identified as an isoform that uniquely bound active but not inactive Vpr mutants [11,28], we focused on the θ isoform and observed a partial reduction (50-60%) in Vprinduced G 2 ,M arrest upon 14-3-3 θ knockdown as indicated by the decreased ratio of G 2 ,M to G 1 cells (Fig. 1B,C). Nonspecific siRNA, used as a negative control, did not impair the arrest activity. Silencing of Chk1, the DNA damage checkpoint kinase involved in Vpr cell cycle blockade [17], attenuated arrest to the same degree as 14-3-3 θ (Fig. 1B,C). Immunblotting showed a significant reduction of the 14-3-3 θ and Chk1 proteins after RNAi knockdown (Fig. 1D). Similar results were obtained when either virion-delivered Vpr (Vpr v, from virus particles that enter cells but cannot establish a productive infection) or endogenously expressed proviral Vpr (NL4-3 Vif-) was used to test Vpr activity. 14-3-3 silencing was generally less potent against proviral Vpr-induced arrest, most likely due to greater Vpr abundance in actively infected cells (data not shown and Fig. 2A). A Vif-deficient strain of NL4-3 was used to assay the activity of Vpr expressed in infected cells because Vif has been shown to independently arrest cells in G 2 ,M [26]. However, 14-3-3 θ knockdown did protect against HIV-1-mediated G 2 ,M block when both Vif and Vpr were intact, although to a lesser extent than that observed in the absence of Vif. This suggests that Vif likely causes G 2 ,M accumulation by a mechanism that does not Decreased expression of 14-3-3 θ, β, γ, and Chk1 protects against Vpr v -and HIV-1 (vif-)-induced cell cycle arrest   A rely on 14-3-3 θ or Chk1. Thus there are apparently redundant mechanisms for the virus to exert this prominent effect on the cell cycle. We corroborated the G 2 ,M arrest alleviation by measuring cell proliferation kinetics (Fig.  1E). While Vpr v severely halted cell division in nonspecific siRNA cultures over time, 14-3-3 θ and Chk1 knockdown partially rescued cell expansion, consistent with reduced G 2 ,M accumulation in Fig. 1B. Since 14-3-3 can function as self-dimers or heterodimers with other 14-3-3 isoforms [29][30][31], and additional isoforms have been shown to bind active Vpr [11], we determined whether other 14-3-3 isoforms play a role in Vpr cell cycle blockade in T cells. Targeting β, η, γ, and ζ isoforms by RNAi revealed that 14-3-3 β and γ knockdown also partially rescued Vpr-induced G 2 ,M arrest, although to a lesser degree than 14-3-3 θ knockdown (data not shown). Hence, the θ, β, and γ isoforms of 14-3-3 as well as the checkpoint kinase, Chk1, are important for mediat-ing the G 2 ,M arrest caused by Vpr, implicating the DNA damage checkpoint response. Attempts to silence Chk1 and 14-3-3 θ simultaneously impaired knockdown efficiency for 14-3-3 θ (Fig. 1F), but the lack of any further decrease in the G 2 ,M/G 1 ratio relative to silencing of either gene alone suggests that Chk1 and 14-3-3 θ likely mediate Vpr cell cycle block through the same molecular pathway (Fig. 1G).

Induction of a multiprotein complex containing 14-3-3 θ and G 2 ,M regulatory proteins by Vpr and HIV-1 infection
Several cell cycle regulatory functions have been described for 14-3-3 proteins, including cytoplasmic sequestration of the G 2 ,M regulatory proteins, Cdc25C and Cdk1 (Cdc2), which limits entry into mitosis [19][20][21][22]. To determine if 14-3-3 interactions were altered by Vpr or HIV-1 infection, we examined the association of 14-3-3 θ with Cdk1, CyclinB1, and Cdc25C by co-immunoprecipitation (IP). Vpr v significantly increased the amount of Cdk1 and Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV-and Vpr-induced G 2 ,M arrest CyclinB1 associated with 14-3-3 θ ( Fig. 2A, right panels, IP). Moreover, the Cdk1 associated with 14-3-3 θ included a substantial amount of inactive Cdk1 phosphorylated on tyrosine 15 (P-Y15) ( Fig. 2A). Similarly, inactive Cdc25C phosphorylated on serine-216 (P-S216) showed increased association with 14-3-3 θ upon Vpr v delivery. Actual HIV-1 infection (RT+) had the same effect, although this result was variable and occasionally less Cdk1 and CyclinB1 were recruited to 14-3-3 θ compared to Vpr v treatment ( Fig. 2A, HIV lane). This could be due to opposing effects of other HIV-1 proteins that alter the cell cycle, such as Vif or Tat [26,32]. The difference, however, cannot be explained by insufficient Vpr protein since its expression was markedly greater in HIV-infected cells ( Fig.  2A, input). Moreover, the G 2 ,M blockade was comparable in the Vpr v and HIV-infected cultures (Fig. 2B). Note that cells exposed to Vpr v are not GFP positive because they do not express a provirus due to an inactive RT and there is not sufficient GFP in the inoculating virions to "donate" fluorescence. Intriguingly, proportionately more high molecular weight CyclinB1, presumably hyperphosphorylated "active" CyclinB1 [18,33], was associated with 14-3-3 θ in HIV-infected samples ( Fig. 2A, IP). This upper band was more apparent in the IP than in the whole cell lysate, and thus likely represents a small fraction of total cellular CyclinB1. Thus the virus may drive the active form of CyclinB1 into complex with 14-3-3. This same event could also occur with Vpr v but the levels may be too low to detect.
Vpr was also readily observed in the 14-3-3 θ IP ( Fig. 2A, HIV) confirming their association [11]. In contrast to HIV infection, Vpr protein delivered by the virion, i.e. Vpr v , was generally low or undetectable in the 14-3-3 θ IP ( Fig.  2A, IP, Vpr v ). Thus, the Vpr-14-3-3 θ complex is not obviously stoichiometric with respect to the quantity of Vpr bound to 14-3-3. The augmented levels of Vpr in the complex during HIV infection conditions may reflect Vpr oligomers forming during infection when the protein is more abundant (Fig. 2A, input) [34], as Vpr self-association may be concentration-dependent. In summary, Vpr appears to spark the formation of a multiprotein complex containing 14-3-3 θ, Cdk1, Cdc25C, CyclinB1, and itself. However, the possibility that 14-3-3 θ associates independently with one or more of these proteins rather than as a holo-complex is also a formal possibility.

Localization of cell cycle regulatory proteins is not altered by Vpr or HIV infection
Although initial work describing 14-3-3 interaction with Vpr suggested that 14-3-3 binding and cytoplasmic sequestration of Cdc25C became more promiscuous in the presence of Vpr [11], we did not observe any gross changes in the cytoplasmic and nuclear distribution of multiple cell cycle proteins, including CyclinB1, Cdk1, Cdk1-P.Y15, Cdc25C-P.S216, and 14-3-3 θ [Additional file 1A]. As expected, inactive Cdc25C-P.S216 was predominantly cytoplasmic, presumably due to 14-3-3 sequestration during normal cell cycling through G1 phase [35]. Total Cdc25C appeared to have the same localization pattern as Cdc25C-P.S216, but possible residual signal from the phospho-probe made these data difficult to interpret (data not shown). The cytoplasmic purity was confirmed by absence of the nuclear protein, Poly(ADP-ribose) polymerase (PARP). Thus, recruitment to 14-3-3 does not alter the nucleocytoplasmic distribution of CyclinB1, Cdk1, or phosphorylated Cdc25C.
Although nuclear levels did not change, Vpr v , but not HIV-1 infection, significantly increased cytoplasmic expression specifically of CyclinB1 and Polo-like kinase 1 (Plk1), but not Cdc25C and Cdk1. Plk1, a centrosome-associated mitotic kinase, participates in several mitotic processes including centrosome maturation, Cdc25C activation, inactivation of the Cdk1 inhibitory kinase, Myt1, activation of CyclinB1, and degradation of mitotic Cyclins [36][37][38][39][40]. Furthermore, as we also found in the whole cell lysates in Fig. 2A, the level of Vpr in the cells following virion delivery, Vpr v, is markedly less than during HIV-1 infection and barely detectable by western blotting [Additional file 1A]. Nevertheless, Vpr v causes G 2 ,M blockade as profound as that observed in infected cells (Fig. 2B). Thus, Vpr v has additional biological effects on CyclinB1 and Plk1 cytoplasmic protein levels, possibly through regulation of their synthesis, subcellular distribution, or cytoplasmic half-life. It is also possible that the increase in cytoplasmic CyclinB1 and Plk1 reflects protein delivered into cells by HIV-1 virions rather than endogenously produced protein as discussed earlier. To explore this hypothesis, we examined the contents of HIV-1 (NL4-3 RT-) virions by Western blot analysis. Both CyclinB1 and Cdk1 were present [Additional file 1B], but Plk1 was not detected (data not shown). Moreover, the level of these proteins in the virions was unaltered by the presence of Vpr making it unlikely that virion-delivered protein accounted for the elevation. Rather, the elevated CyclinB1 and Plk1 are most likely derived from endogenous sources in the target cell. Since both CyclinB1 and Plk1 are present in mammalian centrosomes and Vpr causes abnormal centrosome duplication [41][42][43], these findings could also reflect augmented protein associated with centrosomes. However, the centrosomal marker, centrin, was not similarly increased by Vpr v (data not shown).

Induction of a 14-3-3 θ complex is specific to G 2 ,M arresting Vpr
Since cell cycle perturbations are exerted by Vif, Vpr [26], and possibly other more complicated events during a full HIV infection, we focused on the virion-delivered Vpr v system to study the Vpr-specific effect on 14-3-3 binding partners. We first compared Vpr mutants for their ability to drive mitotic proteins into the 14-3-3 θ complex. We found that the well-established R80A point mutant of Vpr, which lacks G 2 ,M arrest activity [44], failed to increase Cdk1 and CyclinB1 association with 14-3-3 θ (Fig. 3A, IP; Fig. 3B, top row). Similar results were obtained with the inactive S79A mutant (data not shown). By contrast, both wild-type Vpr and the point mutant, I70S, which does not affect G 2 ,M-blocking function [34] (Fig. 3B, top row), increased the complex of Cdk1, CyclinB1, as well as Plk1, with 14-3-3 θ. Western blots showed that similar amounts of mutant and wildtype Vpr protein were delivered (Fig. 3A, input). There was a marked increase in CyclinB1 and Plk1 total protein during G 2 ,M arrest by Vpr (Fig. 3A, input, wt and 70S), likely due to enhanced expression of cyclic proteins in premitotic cells, whereas Cdk1 expression was relatively stable. Thus a general increase in protein level could account for augmented 14-3-3 θ association with Cyclin B1 and Plk1 but not Cdk1. To determine if the recruitment was specific, we probed the 14-3-3 θ IP for additional cell cycle regulatory proteins. However, we were unable to detect Chk1, Wee1, or Myt1 (data not shown). Intriguingly, the Cdk1 activating kinase, Cdk7 [45], clearly associated with 14-3-3 θ (Fig. 3A, IP), suggesting that 14-3-3 θ may function as a platform for Cdk1 modification by binding multiple mitotic regulators. Since Cdk7 levels in the IP were unaltered by Vpr v -induced G 2 ,M arrest, though, the increased recruitment of Cdk1, CyclinB1, and Plk1 to 14-3-3 θ does not extend to all 14-3-3-binding partners.
To distinguish whether Vpr induces a high molecular weight (MW) complex(es) or multiple individual complexes containing cell cycle proteins we used the chemical cross-linker disuccinimidyl suberate (DSS) to preserve protein-protein interactions in live cells prior to lysis and SDS-PAGE. The results were complicated. Strikingly, CyclinB1 existed in several higher MW complexes upon wild-type but not inactive R80A Vpr v  We next attempted to detect 14-3-3 θ, Vpr, and Cdc25C in high MW complexes by western blot, but these experiments were unsuccessful. The chemical cross-linking process may have impaired detection of these proteins by altering or masking epitopes. Nevertheless, these proteins still remain strong candidates for association with CyclinB1 and Cdk1 based on our immunoprecipitation experiments. Similar analyses of cells undergoing HIV-1 infection-induced G 2 ,M arrest did not exhibit any increase in CyclinB1 high MW bands [Additional file 2E, lane 6] consistent with its tenuous enhanced association with 14-3-3 during infection ( Fig. 2A and 6F To determine if 14-3-3 θ binding to cell cycle regulatory proteins is a general characteristic of the G 2 ,M phase of the cell cycle or specific to Vpr arrested cells, we examined synchronized Jurkat cells using DNA polymerase inhibition by aphidicolin [47]. Upon release from the drug, most cells were in G 1 with diploid DNA content (Fig. 3B, bottom row, 0h). By 9 hours, the culture progressed to predominantly G 2 ,M phase, characterized by tetraploid DNA, and exhibited a DNA profile similar to cells arrested by Vpr v (Fig. 3B, top row, wt). However, co-IP of Cdk1 and CyclinB1 with 14-3-3 θ was not enhanced in these cells (Fig. 3C, IP, 6 and 9 h), despite a slight increase in abundance of these proteins at 6 and 9 hours after release (Fig.  3C, input). Treatment with several chemical inhibitors of mitosis that cause G 2 ,M blockade by targeting the cytoskeleton, including demecolcine, nocodazole, and paclitaxel (taxol) also failed to enhance CyclinB1 or Cdk1 association with 14-3-3 θ (data not shown). Thus, the induction of a complex between 14-3-3 θ, CyclinB1, and Cdk1 is specific to Vpr-induced cell cycle blockade.
To confirm that Vpr binding to 14-3-3 θ correlates with its cell cycle arrest activity, we tested this association in infected cells expressing endogenous Vpr because the Vpr interaction with 14-3-3 θ is easier to observe in infected cells than in Vpr v -arrested cells ( Fig. 2A). Specifically, we mutated Vpr within the physiological context of the full virus HIV-1 NL4-3 e-n-GFP to the inactive R80A point mutant. These experiments were conducted in a Vif-deficient strain of NL4-3 to eliminate the confounding influence of the G 2 ,M arrest activity of Vif [26]. Unexpectedly, R80A mutant Vpr co-immunoprecipitated with 14-3-3 θ to a similar extent as wild-type Vpr (Fig. 3D, top panel), even though Vpr G 2 ,M cell cycle blockade was abrogated (Fig. 3D, bottom panel). Thus the reduced function of R80A cannot be explained by its inability to bind 14-3-3 θ. Rather, other components normally present in a 14-3-3 θ complex may be eliminated by this inactivating Vpr mutant. It is also interesting to note that the Vpr co-IP with 14-3-3 θ was weaker in the absence of Vif in HIVinfected cells, suggesting that Vif enhances this interaction. This may partly explain the better association of Vpr to 14-3-3 θ in HIV than in Vpr v treated cells ( Fig. 2A).

DNA damage induces a similar 14-3-3 θ complex
Multiple components of the DNA damage checkpoint are involved in Vpr-induced cell cycle blockade, including Chk1, ATR, and BRCA1 [17,48]. To determine whether elevated 14-3-3 θ binding to Cdk1 and CyclinB1 may be a shared feature of Vpr-induced cell cycle blockage and the mitosis-inhibitory DNA damage response in T lym-phocytes, we examined Jurkat cells arrested in G 2 ,M by adriamycin [49]. 14-3-3 σ, an isoform not expressed in lymphocytes, binds Cdk1 during G 2 ,M arrest caused by adriamycin in the colorectal cancer cell line HCT116 [21], and thus there is precedent for such an interaction. Indeed, the association between 14-3-3 θ and both Cdk1 and CyclinB1 increased dramatically following adriamycin treatment for 24, 48 or 72 hours (Fig. 4A, IP). Plk1 binding to 14-3-3 θ was also enhanced, indicating that mitotic regulatory proteins are specifically elevated in the induced complex. By contrast, the centrosomal resident protein, centrin, was constitutively associated with 14-3-3 θ. Adriamycin powerfully activated the DNA damage checkpoint so that essentially all Jurkat cells accumulated in G 2 ,M with 4N DNA content within 24 hours of treatment (Fig. 4B, inset). Furthermore, cell viability decreased from 96% to 80% by 72 hours, indicating emerging toxicity to the DNA damage stimulus (Fig. 4B, dot plot). The magnitude and slow kinetics of adriamycin-induced death are similar to cell death caused by virion-delivered or transfected Vpr, which is usually not apparent until 24-48 hours after initiation of the G 2 ,M arrest [2,34,50]. Thus cell cycle dysregulation by both adriamycin and Vpr share several key features: 1) enhanced 14-3-3 θ binding to Cdk1, CyclinB1, and Plk1, 2) profound G 2 ,M arrest, and 3) cytotoxicity.

Centrosomal proteins dissociate from 14-3-3 θ during HIVinduced G 2 ,M arrest
Given the growing connection between the centrosome and G 2 ,M regulation [42,51,52], we considered the possibility that the altered association between 14-3-3, which localizes to the centrosome [53], and other cell cycle regulatory proteins might reflect disrupted centrosomal localization of these proteins. To test this model, we isolated centrosomes from cytosolic proteins by discontinuous sucrose gradients and determined the abundance of Cdk1, CyclinB1, Plk1, and 14-3-3 θ. Examination of HIV-1-infected G 2 ,M arrested cells showed a similar centrosomal protein profile to that of asynchronous and adri-Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G 2 ,M arrest induced by adriamycin   amycin-treated cells ( Fig. 5A and 5C). Plk1, CyclinB1, and Cdk1 were all highly enriched in the centrosome fraction (lane 3), characterized by the abundance of centrosomal proteins, γ-tubulin and centrin. 14-3-3 θ, by contrast, was primarily found in the cytosol, as it remained in the top of the gradient (lane 7). However, at darker exposures, 14-3-3 θ was also detected in the centrosome fraction in HIVinfected, asynchronous, and adriamycin-arrested cells ( Fig. 5A and 5C, "D" exposure, lane 3). These data suggest that 14-3-3 θ does in fact localize to the centrosome, as has been described for other 14-3-3 isoforms [53,54]. Centrosomal location of the θ isoform may be specific to cells in the G 2 ,M phase, however, consistent with previous reports of regulated 14-3-3 centrosome localization during cell division [53]. Control experiments showed that the infection was robust (85% of the culture infected) and caused cell cycle arrest and death (Fig. 5B). Similarly, the adriamycin-treated culture accumulated in G 2 ,M (Fig.  5D). These data suggest that the general composition of centrosomes in HIV-infected cells does not differ from asynchronous or adriamycin-arrested cells with respect to the cell cycle regulatory proteins Plk1, CyclinB1, Cdk1, or 14-3-3 θ. Vif and Vpr were detected throughout the gradient (lanes 3-7), which suggests that a small fraction of these accessory proteins may localize to the centrosome (Fig. 5A). Thus, enhanced association between 14-3-3 θ and cell cycle proteins during Vpr arrest cannot be explained by failure of these proteins to localize to the centrosomes.
We therefore considered the possibility that the configuration of 14-3-3 and cell cycle regulatory proteins with centrosomal proteins is altered by the presence of Vpr, and this inhibits normal mitotic activation of Cdk1/CyclinB1 within the centrosome. We tested this hypothesis by co-IP and found markedly decreased centrin associated with 14-3-3 θ upon infection with wild-type HIV-1 (Fig. 6A). Plk1, which also localizes to the centrosome [55], showed a similar striking reduction in 14-3-3 θ association after HIV infection. This contrasted with our findings with Vpr v and adriamycin (Figs. 3 and 4). These data indicate that the normal binding of 14-3-3 θ to centrosomal proteins is reduced during HIV-1 infection and that viral components besides Vpr may alter the composition of a 14-3-3 θ complex. To determine if impaired 14-3-3 θ binding to centrin and Plk1 in HIV-1-infected T cells correlated with cell cycle blockade, we examined HIV-1 mutants lacking the G 2 ,M arresting proteins, Vpr and Vif. Elimination of either of these genes alone had only modest effects, but the combined mutant virus lacking both Vpr and Vif was severely attenuated in G 2 ,M blocking function (Fig. 6B) [26]. While wild-type HIV-1 infection resulted in a massive decrease in 14-3-3 θ-associated centrosomal proteins, infection with vif-vpr-mutant HIV-1completely restored Plk1 and centrin association with 14-3-3 θ to levels observed in uninfected cells (Fig. 6A). Moreover, vpr and vif single mutant virus infections, which caused little or no attenuation in G 2 ,M arrest activity, respectively, also reduced Plk1 and centrin association with 14-3-3 θ, although less than wild-type virus. Infection efficiencies were matched between the mutant viral strains with 70-75% of the culture expressing the HIV-1 provirus (Fig. 6C, inset) and therefore this difference was not due to inefficient viral replication of the mutant HIV-1 strains. Taken together, these results show that the impaired association between 14-3-3 θ and centrosomal proteins was specific to HIV-1 infection and directly correlated with the extent of the G 2 ,M block. Moreover, adriamycin treatment did not cause a similar dissociation between 14-3-3 θ and centrin (Fig. 4A), and thus this effect is not a general feature of G 2 ,M arrested cells. We therefore conclude that cell cycle blockade by the Vif and Vpr proteins during HIV-1 infection disrupted protein associations within the centrosome in a manner that correlated with cell cycle blockade.

Importin β binding to CyclinB1, Cdk1, and 14-3-3 θ is elevated by Vpr
To determine what other cellular proteins bind 14-3-3 θ and therefore might affect the activity of cell cycle proteins associated with the scaffold protein during HIV-1 infection, we performed mass spectrometry on 14-3-3 θ immunoprecipitates from untreated Jurkat whole cell lysates. A matched isotype control antibody was used to distinguish non-specific bands in the immunoprecipitate. Intriguingly, importin β was identified in the 14-3-3 θ-associated fraction (data not shown). To confirm whether 14-3-3 θ and importin β form a complex, we measured importin β in 14-3-3 θ immunoprecipitates and observed low levels in untreated (mock-infected) cells (Fig. 6D, right panel, "m"). The association between these proteins was dramatically increased during HIV-1 infection, however (Fig.  6D), while Vpr v caused a milder increase in binding. The G 2 ,M arrest by Vpr v and HIV-1 was comparable in this experiment (Fig. 6E), and therefore the difference in complex induction cannot be explained by the magnitude of cell cycle block. As in Fig. 2A, the amount of Vpr delivered by Vpr v was very low and, in this case, undetectable (Fig.  6D, input), whereas endogenously produced protein in productively infected cells was apparent in the cell lysates and 14-3-3 θ IP. Together, these data suggest that a multiprotein complex consisting of Vpr, 14-3-3 θ, and importin β accumulates in infected cells as well as during Vpr v arrest. And since importin β localizes to centrosomederived spindle poles during the early stages of mitosis [56], it is conceivable that these complexes are derived from centrosomal material.
To test whether CyclinB1 is part of an importin β complex and if Vpr alters the association between them, we immu-Cell cycle regulatory proteins reside in the centrosome during G 2 ,M arrest induced by HIV-1 infection and adriamycin   Vpr v adr noprecipitated importin β in cells blocked in G 2 ,M by Vpr v . Elevated levels of CyclinB1, Plk1, and Cdk1 were bound to importin β compared to untreated cells (Fig. 6F, right panel). In addition, Vpr was present in the importin β IP, consistent with importin β-mediated docking of HIV-1 pre-integration complexes [57]. Since importin β mediates nuclear entry of CyclinB1-Cdk1 [58,59], increased binding among these proteins could simply be a consequence of G 2 ,M status, during which the mitotic kinase must be shuttled into the nucleus. However, arrest by adriamycin did not cause increased importin β-Cdk1 association, despite the more pronounced G 2 ,M block than that by Vpr v and the enhanced association of CyclinB1 and Plk with importin β (Fig. 6G and 6F). The increased binding between Cdk1 and importin β was observed only in cells arrested in G 2 M by Vpr v , suggesting that this complex may prevent mitosis possibly by sequestering Cdk1. Therefore, the CyclinB1-importin β complex may be a general characteristic of cells in G 2 ,M, while Vpr uniquely induces a Cdk1-importin β complex that could disrupt mitotic progression. Since CyclinB1 is present in both the 14-3-3 θ and importin β IPs, which also co-IP with one another, these proteins likely form a large holocomplex.

Discussion
Our findings confirm that an association between 14-3-3 and Vpr occurs not only in yeast and transfected 293 cells as observed by Kino et al. [11], but also in HIV-1-infected Jurkat T cells. However, the σ isoform of 14-3-3, which was important for Vpr G 2 ,M arrest in 293 cells, is not expressed in lymphoid tissue. We have determined that in T lymphocytes, the θ, β, and γ isoforms are most relevant to Vpr function. Thus 14-3-3 isoforms may perform redundant functions in different cell types depending on the isoforms expressed.
In contrast to the yeast two hybrid and human 293 cell systems [11], we found that Vpr binding to 14-3-3 θ was independent of its ability to block the cell cycle in Jurkat cells. The cell cycle arrest Vpr mutant R80A associated with 14-3-3 θ almost as efficiently as wild-type Vpr in HIV-1 infected cells. Since 14-3-3 isoforms are known to function as homo-and heterodimers, poor interaction between R80A Vpr and 14-3-3 θ in yeast may be due to lack of an additional 14-3-3 isoform that stabilizes R80A binding in mammalian cells. And since 14-3-3 isoforms other than σ were not individually examined in 293 cells by Kino et al., these findings may not reflect Vpr binding interactions with other isoforms or in lymphoid cells which lack 14-3-3 σ. Rather than a 14-3-3 binding defect, we show that cell cycle arrest mutants are impaired for the ability to recruit G 2 ,M regulatory proteins to 14-3-3. In addition, this implicates residues other than phosphorylated S79 of Vpr in binding 14-3-3 θ since the R80A mutation prevents S79 phosphorylation [60]. Other 14-3-3 isoforms may bind this residue, though, and be responsible for transporting Cdk1, CyclinB1 in heterodimeric 14-3-3 complexes.
Vpr binding to 14-3-3 has previously been shown to increase cytoplasmic Cdc25C, presumably independent of the Cdc25C phosphoserine residue that usually mediates 14-3-3 binding [11]. However, we were unable to detect any redistribution of phospho-Cdc25C caused by Vpr v or HIV-1 infection. Since the prior work was based on overexpression of mutant Cdc25C lacking the phosphorylation site required for 14-3-3 binding, it is possible that Vpr affects only the unphosphorylated Cdc25C population, which was not examined in our studies. Vpr and HIV-1 infection did increase Cdc25C association with 14-3-3, consistent with the idea that Vpr influences 14-3-3-Cdc25C binding. Thus although the distribution of endogenous Cdc25C was not significantly affected by Vpr, altered Cdc25C-14-3-3 binding could impair Cdc25C activation of Cdk1 and prevent normal progression through mitosis.
The prevailing model of Vpr-induced G 2 ,M arrest entails inhibition of Cdk1, as evidenced by an increase in the phosphorylated inactive form of the enzyme and impaired kinase activity [5][6][7]. Despite numerous attempts, we were unable to detect an increase in phosphorylated, inactive Cdk1 in Jurkat cells blocked in G 2 ,M by either Vpr v or HIV-1 infection. Again, this discrepancy may be due to cell-type specific differences, as previous work on Cdk1 inactivation by Vpr was performed in HeLa epithelial cells. Cell cycle regulation in lymphoid tissue is distinct from that in other mammalian cells during embryogenesis, as hematopoietic cell proliferation defects in Cdk4 and Cdk6 double knockout mice are not exhibited in other cell types [61]. Thus, Cdk1 may play a different role in cell division in epithelial and lymphoid cells. Likewise, a Vpr-induced proliferation block in HeLa cells may be characterized by dramatic inhibition of Cdk1 while other Cdks are more significant in T cells. This is supported by the fact that overexpression of constitutively active Cdk1 blocks Vpr-induced G 2 ,M arrest in 293 fibroblasts [5], but not Jurkat cells (data not shown). It is also possible that only a small, critical subset of cellular Cdk1 is inactive, and thus examination of total cell lysates might not reveal this distinct population. In addition, since Jurkat cells are derived from a human T cell leukemia sample that was adapted for continuous growth in culture, its cell cycle regulatory machinery may not be representative of primary T cells. Thus it will be important to confirm these findings in primary cells.
Although we did not observe increased Cdk1 phosphorylation by Vpr or HIV-1 infection, the association of Cdk1 and CyclinB1 with 14-3-3 was significantly altered during Vpr-induced G 2 ,M arrest. Similar increases in Cdk1-14-3-3 binding during DNA damage induced G 2 ,M arrest are accompanied by cytoplasmic sequestration of Cdk1-CyclinB1 complexes [21]. However, this was not the case in Vpr-or HIV-1-induced G 2 ,M arrest. Hence, Vpr increases 14-3-3-Cdk1 binding, presumably as a result of Vpr associating with 14-3-3, but appears to attenuate its activity by a mechanism that does not rely on cytoplasmic sequestration. Since the association of several other G 2 ,M regulatory proteins with 14-3-3 also increased, Vpr may nucleate a large multi-protein complex that prevents proper activation of Cdk1. For example, Cdk1 inhibitor kinases, Wee1 or Myt1, could have greater activity on Cdk1 in the complex. This is supported by the apparent increase in phosphorylated, inactive Cdk1 associated with 14-3-3 θ. Furthermore, Wee1 also binds 14-3-3 [62][63][64], although we were unable to detect Wee1 in 14-3-3 immunoprecipitates (data not shown). The fact that the overall levels of phospho-Cdk1 were unaltered in Vpr-arrested cells could also indicate that the 14-3-3-Cdk1 complex dominantly interferes with unbound Cdk1. In particular, hyperphosphorylated, presumably active CyclinB1, which is required for Cdk1-CyclinB1 nuclear import, was enriched in the 14-3-3 complex in HIV-1-arrested cells, and thus might be unavailable for facilitating nuclear entry of other Cdk1 molecules. Alternatively, a CyclinB1 activating kinase may also be complexed with 14-3-3 and restrained from acting upon CyclinB1 not associated with 14-3-3. Such mechanisms would prevent Cdk1 from entering the nucleus and phosphorylating substrates required for mitotic progression.
It remains unclear why CyclinB1 binding to 14-3-3 θ during HIV-1 infection is variable and occasionally inconsistent with the enhancement induced by Vpr v , but other viral proteins may be involved [26,32]. Similarly, Vpr v but not HIV-1-infected cell Vpr expression generated high MW CyclinB1/Cdk1 complexes. Thus other HIV-1 proteins may interfere with or disrupt this activity. Alternatively, these complexes may originate from virus-producing cells, as many cell cycle regulatory proteins are packaged into HIV-1 virions. Enrichment of a cell cycle regulatory complex containing CyclinB1 within HIV-1 virions and subsequent delivery of this complex into target cells could substantially increase the amount of higher-order CyclinB1 in target cells. Since these complexes were not consistently amplified in HIV-1-blocked or synchronous G 2 ,M cells, they do not appear to be a general feature of G 2 ,M-phase cells. We therefore conclude that induction of CyclinB1 multi-protein complexes is specific to Vprinduced G 2 ,M blockade, and this may serve to negatively regulate CyclinB1 activity and maintain the G 2 ,M blockade.
The centrosome is emerging as an important cell cycle regulatory organelle where active Cdk1-CyclinB1 is first detected [42]. Moreover, 14-3-3 isoforms ε and γ localize to centrosomes [53], along with several other G 2 ,M regulatory proteins. Our data indicate that 14-3-3 θ binding to centrosomal proteins is significantly impaired during HIV-1-induced G 2 ,M arrest. This does not appear to reflect poor 14-3-3 localization to centrosomes, but rather a more subtle disruption in the specific binding of 14-3-3 to centrosomal proteins within the centrosome. This could have important implications for Cdk1-CyclinB1 activation and nuclear import. For example, binding of the 14-3-3 scaffold protein to centrosomal proteins may form a crucial platform that facilitates Cdk1-CyclinB1 biochemical modification and activation. Increased Cdk1, CyclinB1, or Vpr binding to 14-3-3 could impair the ability of 14-3-3 to interact with centrosomal proteins, which may be required for proper activation of Cdk1 or CyclinB1. Plk1, in particular, phosphorylates CyclinB1, although it is not clear how this affects CyclinB1 activity or how it could be regulated by centrin [42,65]. It is also possible that nuclear import of Cdk1-CyclinB1 occurs via the centrosome, which may serve as a cytoplasmic-nuclear shuttling vessel [52], and the loss of 14-3-3 binding to centrosomal proteins reduces transport efficiency.
Although the θ isoform of 14-3-3 had not previously been found to localize to the centrosome [53,54], 14-3-3 θ expression in the centrosome may be unique to certain phases of the cell cycle or to specific cell types. Together, these findings suggest that normal 14-3-3 activity within the centrosome is disturbed in HIV-1 infection-induced G 2 ,M arrest and may be responsible for impaired Cdk1 activation. Further evidence that Vpr causes rearrangement, rather than dislocation, of normal protein assemblies is the enhanced association of cell cycle regulatory proteins with importin β, which governs nuclear import and export as well as various functions related to the mitotic spindle.
Vpr is known to cause accumulation of multiple centrosome-like structures and aberrant mitotic spindles [43,66,67]. It is presently not clear how this relates to the reduced 14-3-3-centrosome binding observed in our studies. Abnormal centrosomal duplication during HIV-1 infection apparently requires Vpr [67], whereas HIV-1 lacking Vpr still disrupted 14-3-3 binding to centrosomal proteins, presumably due to Vif. Thus, these two effects on the centrosome are likely unrelated. Since DNA damageinduced G 2 ,M arrest increases association between 14-3-3 and the centrosomal kinase, Plk1, HIV-1 arrested cells are distinct from other G 2 ,M-arrested cell phenotypes.
Many of the changes in the association between 14-3-3 and cell cycle regulatory proteins in Vpr arrested cells were also observed upon G 2 ,M blockade caused by adriamycin-induced DNA damage. This supports the model of Vpr activation of the ATR DNA damage response [17], although it is not clear how this relates to 14-3-3. Consistent with this possibility, our data confirmed that the ATR substrate, Chk1, is important for Vpr-induced G 2 ,M arrest. In addition, we provide the first evidence that Chk1 as well as 14-3-3 are of central importance for Vpr cell cycle block in the context of actual HIV-1-infection. Future studies examining the combined effect of Chk1 and 14-3-3 loss will help to determine if these proteins are involved in separate or related mechanisms.

Conclusion
Although many different mechanisms have been proposed to explain Vpr G 2 ,M cell cycle blockade, the data presented herein provide strong support for a 14-3-3dependent process involving abnormal associations of cell cycle regulatory proteins. Silencing of 14-3-3 θ, in particular, decreased the arrest induced by both virion-associated Vpr and endogenous Vpr during HIV-1 infection in Jurkat T cells. Although 14-3-3 is known to negatively regulate mitosis through cytoplasmic sequestration of Cdk1 and Cdc25C, this mechanism does not appear to be involved in the Vpr arrest. Rather, multiple G 2 ,M regulatory proteins accumulate in a complex with 14-3-3 θ, including CyclinB1, Cdk1, and Cdc25C. Similar proteinprotein interactions were observed in cells arrested in G 2 ,M by DNA damage, but not synchronous G 2 ,M cells, suggesting that this process is likely the cause rather than a consequence of G 2 ,M status. Intriguingly, the binding of several centrosomal proteins to 14-3-3 θ was dramatically reduced in HIV-1-arrested cells, while importin β increased in association with CyclinB1, Cdk1, and 14-3-3 θ under Vpr arrest conditions. Combined, these data support a model in which mitotic proteins are inactive due to improper post-translational modification conditions in the centrosome or incorporation into a protein complex that prevents access to substrates.

Author's response
We agree that using the Jurkat cell line as a model system for studying regulation of the cell cycle introduces impor-tant limitations due to its origins as a leukemia sample adapted for continuous growth in culture. We have elaborated on this point in the conclusions and adjusted the terminology when referring to Jurkat cells as suggested by the reviewer.
2. There is no consideration of statistics in the manuscript. Error bars are not supplied. Consequently, I looked for consistency in the results presented. For instance, in Figure 1C siRNA for 14-3-3 theta clearly decreased the G2 arrest induced by infection with NL4-3 vif-, but in Figure  1F siRNA for 14-3-3 theta induced only a slight change in the G2 arrest induced by infection with NL4-3 vif-.
Author's response The reviewer expresses concern over the level of variation between two siRNA knockdown experiments shown in different panels within Figure 1. These differences result from varying levels in the efficiency of gene knockdown and HIV-1 infection. Since the experiment in Fig. 1F was less representative of multiple replicates, and for reasons described in point #4 below, we removed this panel. As stated in the figure legend, Fig. 1B and 1C are representative of six independent experiments. For example, Fig. 1C and Fig. 1G (formerly Fig. 1I) are highly consistent.
Author's response Although the amount of 14-3-3 θ appears slightly reduced in the Chk1 knockdown in Fig. 1D, this was not observed in multiple repeats of the experiment, including Fig. 1F, and was thus likely a blotting defect.
4. In Figure 1, the targeting of the various isoforms of 14-3-3 by siRNA does not appear to have produce much specificity in effect. For instance, in Figure 1G knockdown of beta has decreased both beta and theta expression. Inhibition of tau has decreased theta expression. Inhibition of gamma appears to have cause a decrease in Chk1. You have indicated that the antibodies to the nu and zeta isoforms crossreact with other isoforms. It may be best to reconsider these data. I am not sure that they add much to the manuscript.

Author's response
We agree that the knockdown data of 14-3-3 isoforms in Fig. 1F and 1G are complicated by the possible poor siRNA specificity. We have removed these panels as suggested by the reviewer.
5. I do not understand why there are no GFP positive events in Figure 2B, middle panel, since the Vpr-v vector has GFP.
Author's response Samples treated with Vpr v simply receive virion-associated proteins and do not express any genes encoded by the viral genome due to the lack of a functional RT protein.
There is insufficient GFP packaged in the viral particle to cause recipient cells to fluoresce. We have added this explanation to the results for clarity.
6. On page 20, you suggest that the monoclonality of the antibody to Chk1 argues against cross-reaction. However, I believe that argument is not correct. Monoclonal antibodies are more prone to cross-reaction than polyclonal antibodies. All antibodies are cross-reactive because the structure of the antibody binding site allows for different structures to be bound. The cross-reactions of different antibodies would not expect to be the same so cross-reaction for polyclonal antibodies is not likely to cause a problem. For monoclonal antibodies cross-reactions can be a problem because all the antibody present has the same cross-reaction.

Author's response
We agree with reviewer's point and removed the comment regarding the monoclonal origin of the antibody.
7. In Figure 3D, the flow cytometric panels should be labeled more clearly.
Author's response Due to space constraints, it is not possible to add additional text to label the histograms in Fig. 3D. We clarified in the figure legend that the numbers within each histogram correspond to the lane numbers in the blots directly above.

Reviewer's report 2 Nathaniel R. Landau, New York University School of Medicine
The Vpr accessory protein of HIV-1 and other lentiviruses has been found to associate with several cellular proteins and it is not yet clear which of these is physiologically relevant. Bolton et al. show that Vpr can form a complex with 14-3-3, a series of protein scaffolds that directs the cellular localization of cyclin dependent kinases. Vpr caused 14-3-3 to associate more strongly with Cdk1, Cdc25C and cyclin B1. In addition, Vpr caused 14-3-3 to bind less well to Plk1 and centrin in the centrosome. These findings shed light on how Vpr affects the cell cycle. It will be of interest to understand whether the effects of Vpr on 14-3-3 are caused by the association of Vpr with DDB1 containing E3 ubiquitin ligases or whether this is an independent function for Vpr. It will also be of interest to understand how these associations contribute to virus replication and pathogenesis.
There is a lot of good work described in the study. It is well written, thorough and novel. However, there are some weaknesses that need to be addressed. The manuscript is too long. There is a tremendous amount of data presented in the 7 figures, each of which contains several panels. The results, discussion and figure legends, although scholarly, are lengthy. Even the aficionados may not want to read them. The authors should decide on a few panels that can be deleted and a few that can be moved to the Supplement. The results and discussion should be shortened to make a few major points. While the authors have done a huge amount of work, if the paper is too long, its impact will be like a tree falling in the forest. There needs to be a clear take-home message conveyed by the manuscript.

Author's response
The reviewer suggests shortening the length of the Results and Discussion sections to better focus the manuscript. We have eliminated a few figure panels and moved some to the supplemental material. We have also edited the text of the Results and Discussion to be more concise.
Secondly, there were recently a set of important papers published on Vpr that show its interaction with a DDB1 containing E3 ubiquitin ligase. The authors make no mention of this work. This may leave the impression that the authors are somewhat disconnected from the rest of the Vpr field. The authors may want to at least mention this work.

Author's response
We have added a comment to the introduction mentioning the recent published body of work on Vpr binding to DDB1 and its relevance to the cell cycle block activity of Vpr.
Thirdly, the authors portray the early work on Vpr that shows its role in nuclear import as established fact. It is not. The authors may want to soften their statements about this model for Vpr function, as it is not generally accepted in the field.

Author's response
As suggested by the reviewer, we have softened the tone of any statements regarding the role of Vpr in mediating import of the virus into the nucleus.

Reviewers' report 3 Yan Zhou, Johns Hopkins University School of Medicine
In "14-3-3 θ binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr", Bolton et al. investigate the role of scaffold protein 14-3-3 in Vpr-mediated cell cycle arrest in Jurkat T cells. A previous study using the yeast twohybrid system and non-lymphocyte cell lines suggested that Vpr directly binds to 14-3-3 and facilitates the cytoplasmic sequestration of Cdc25C. The siRNA knockdown experiment in this study convincingly demonstrates that endogenous 14-3-3, particularly 14-3-3 θ, plays an important role in Vpr-mediated cell cycle arrest.
The authors performed immunoprecipitation experiments to probe the components of 14-3-3 θ complex formed upon Vpr-induced cell cycle arrest. In addition to single-round viral infection, the authors took the elegant approach of introducing virion associated Vpr (Vpr v  The direct binding of Vpr to 14-3-3 θ would suggest an initiating role for the multi-protein complex in Vpr-mediated cell cycle arrest. The authors could not prove this in cells arrested by Vpr v . Instead, they found that both active and inactive Vpr bound to 14-3-3 θ in infected cells. Therefore, it remains possible that the complex executes rather than initiates Vpr-induced G 2, M arrest.
studies suggested the involvement of damaged DNA binding protein 1 and Cul4A in Vpr-induced cell cycle arrest. It will be interesting to determine the spatial and temporal relationship between ubitquitin-ligase complex, Vpr, and the 14-3-3 θ complex described in this study. It will also be important, as the authors point out at the end of this manuscript, to confirm all of these findings in primary CD4 + T cells.