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J Virol. Oct 2007; 81(19): 10822–10830.
Published online Jul 11, 2007. doi:  10.1128/JVI.01380-07
PMCID: PMC2045451

DDB1 and Cul4A Are Required for Human Immunodeficiency Virus Type 1 Vpr-Induced G2 Arrest[down-pointing small open triangle]

Abstract

Vpr-mediated induction of G2 cell cycle arrest has been postulated to be important for human immunodeficiency virus type 1 (HIV-1) replication, but the precise role of Vpr in this cell cycle arrest is unclear. In the present study, we have shown that HIV-1 Vpr interacts with damaged DNA binding protein 1 (DDB1) but not its partner DDB2. The interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by short interfering RNA (siRNA) treatment. The Vpr mutant (Q65R) that was defective for DCAF1 interaction also had a defect in DDB1 binding. However, Vpr binding to DDB1 was not sufficient to induce G2 arrest. A reduction in DDB1 or DDB2 expression in the absence of Vpr also did not induce G2 arrest. On the other hand, Vpr-induced G2 arrest was impaired when the intracellular level of DDB1 or Cullin 4A was reduced by siRNA treatment. Furthermore, Vpr-induced G2 arrest was largely abolished by a proteasome inhibitor. These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G2 arrest.

Vpr is a virion-associated accessory protein of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) (11, 60, 62). SIVmac with Vpr mutants replicate efficiently in peripheral blood mononuclear cells and macrophages in vitro (60) and are pathogenic in monkeys (17); however, in HIV type 1 (HIV-1), Vpr mutants have been shown to be less competent for replication in various systems (reviewed in references 3 and 27). A number of functions have been reported for Vpr, including mediating nuclear import of the viral preintegration complex (12, 15, 16, 20, 28, 41, 42, 56), inducing G2 cell cycle arrest (5, 14, 19, 24, 45) and apoptosis (5, 10, 23, 39, 46, 50, 52, 53, 66), decreasing the viral mutation rate during reverse transcription by recruiting uracil DNA glycosylases into viral particles, and partially neutralizing the antiviral function of cytidine deaminase APOBEC3G by degrading uracil DNA glycosylases (8, 9, 36, 48).

Although the biological relevance of cell cycle G2 arrest has yet to be elucidated, G2 arrest has been suggested to allow for efficient HIV-1 transcription, a possibility that correlates with the observation that Vpr-induced G2 arrest results in a high level of HIV-1 viral replication in T lymphocytes (18). The ability to elicit G2 arrest appears to require the phosphorylation of Vpr (1, 65), and G2 cell cycle arrest mutants tend to have mutations clustered in helix 3 of the Vpr C-terminal region. The Vpr-induced G2 cell cycle arrest phenomenon appears to be mediated by inactivation of the cyclin-dependent kinase 1/cyclin B complex (19, 45). To date, Vpr has been reported to either affect the activity of or associate with Wee-1 kinase, cdc25 phosphatase, ataxia telangiectasia-mutated, ataxia telangiectasia-Rad3-related protein, and protein phosphatase 2, which are known to be upstream regulators of the cdk1/cyclin B complex. In addition, Vpr may exert its inhibitory effects on the cell cycle by affecting the functions of other known cell cycle regulators, such as 14-3-3, p53, and p21 (reviewed in references 2, 3, 27, and 63).

Interaction of HIV-1 Vpr with DDB1.

HIV-1 Vpr has recently been shown to associate with Cullin 1 and Cullin 4A (Cul4A) as well as targeting UNG2 and SMUG1 for proteasomal degradation (48). Cul4A has been shown to associate with DNA binding protein 1 (DDB1) and Roc1, forming a functional E3 ubiquitin ligase complex to degrade the important cell cycle regulators cdt1 and p27Kip1 (4, 6, 21, 25, 30, 40, 43, 49). It is interesting to note that the association of paramyxovirus simian virus 5 V protein with the DDB1/Cul4A complex results in delayed cell cycle progression (32). Since Vpr is known to cause G2 cell cycle arrest, we hypothesized that it may associate with DDB1 to recruit Cul4A, leading to the degradation of important cell cycle proteins and causing G2 cell cycle arrest.

In order to determine whether Vpr can interact with DDB1, 293T cells were transfected with a Vpr-hemagglutinin (HA) or Vpr-myc expression vector, followed by coimmunoprecipitation analysis. Cell lysates were prepared 48 h after transfection and subjected to immunoprecipitation using an anti-HA antibody conjugated to agarose beads as previously described (59). The anti-HA affinity matrix (Roche) immunoprecipitated HA-tagged Vpr proteins from lysates of transfected 293T cells (Fig. (Fig.1A,1A, lane 1). As expected, Vpr-myc was not immunoprecipitated by the anti-HA affinity matrix (Fig. (Fig.1A,1A, lane 2). In repeated experiments, endogenous DDB1 coprecipitated with Vpr-HA (Fig. (Fig.1A,1A, lane 1). DDB1 was not detected in the samples containing Vpr-myc (Fig. (Fig.1A,1A, lane 2), attesting to the specificity of the interaction between Vpr and DDB1.

FIG. 1.
Vpr does not interact with DDB2, but interacts with DDB1 through DCAF1. (A) Interaction between endogenous DDB1 and Vpr-HA. 293T cells were transfected with the Vpr-HA or Vpr-myc expression vector. Cell lysates from transfected cells were immunoprecipitated ...

To confirm the interaction between Vpr and DDB1, 293T cells were cotransfected with the Vpr-myc expression vector plus DDB1-V5, DDB2-V5, or a green fluorescent protein (GFP) expression vector. Cell lysates were prepared 48 h after transfection and immunoprecipitated with anti-V5 antibody (Invitrogen). Coprecipitated samples were analyzed by immunoblotting with anti-myc (to detect Vpr-myc) (Upstate), anti-V5 (to detect DDB1-V5 or DDB2-V5), or anti-Cul4A antibody. Reciprocal immunoprecipitation of DDB1-V5 (Fig. (Fig.1B,1B, lane 1) but not the negative control GFP (Fig. (Fig.1B,1B, lane 3) coprecipitated Vpr-myc.

During the preparation of this paper, the interaction between Vpr and DDB1 was also reported by other groups (26, 46). In addition to its role as a component of the Cul4A E3 ubiquitin ligase complex, DDB1 was originally identified as playing an important role in nucleotide excision repair (22, 31, 44, 57). In response to DNA damage caused by UV light, DDB1 is translocated into the nucleus through an association with nuclear import protein DDB2, forming the UV-DDB repair complex, which exhibits a high affinity for UV-damaged DNA (33, 54, 57, 58). The simian virus 5 V protein and hepatitis B virus X protein have both been shown to compete directly with DDB2 for binding to DDB1 (7, 29). Consistent with the idea that Vpr and DDB2 competitively bind DDB1, we observed that DDB1, but not DDB2, interacted with Vpr (Fig. (Fig.1B,1B, lanes 1 and 2). As expected, both DDB1 and DDB2 interacted with Cul4A (Fig. (Fig.1B,1B, lanes 1 and 2).

Interaction of Vpr with DDB1 requires VprBP (DCAF1).

Although Vpr has recently been shown to associate with both DDB1 and DCAF1 (26, 46), the molecular assembly of the Vpr/DDB1/DCAF1 complex has yet to be resolved. In order to address this issue, we transfected 293T cells with DCAF1 (Dharmacon) or control short interfering RNA (siRNA) for 24 h, followed by transfection with the same siRNA, along with either Vpr-HA or Vpr-myc expression vector. Cell lysates were prepared 72 h after the second transfection and immunoprecipitated with anti-HA affinity matrix. Coprecipitated samples were analyzed by immunoblotting using anti-HA (to detect Vpr-HA) (Covance), anti-DDB1 (Zymed), and anti-DCAF1 (Shanghai Genomics) antibodies. We showed that Vpr-HA was able to coprecipitate with both DDB1 and DCAF1 in the presence of control siRNA (Fig. (Fig.1C,1C, lane 3). In contrast, in the presence of DCAF1 siRNA knockdown, Vpr-HA was unable to immunoprecipitate DDB1 and DCAF1 (Fig. (Fig.1C,1C, lane 1). DCAF1 siRNA efficiently reduced endogenous DCAF1 expression, as indicated by immunoblotting and quantitative real-time PCR (Fig. 1C and D). This result suggests the model in which Vpr interaction with DDB1 occurs through DCAF1 (Fig. (Fig.5A).5A). During the revision of the manuscript, DeHart et al. also reported similar findings suggesting that Vpr recruitment of DDB1 requires DCAF1 (13).

FIG. 5.
HIV-1 Vpr acts as a substrate receptor in a DDB1/DCAF1/Cullin containing E3 ubiquitin ligase. (A) Vpr interacts with DDB1 through DCAF1, forming an E3 ubiquitin ligase complex to target the unknown cellular factors for polyubiquitination and proteasomal ...

Interaction with DDB1 is not sufficient for Vpr-induced G2 arrest.

It has been previously reported that VprQ65 residue is important for binding to DCAF1 (26). Since it has been postulated that Vpr recruitment of DDB1 requires DCAF1, we predict that VprQ65 mutants that are defective for DCAF1 binding will also show defects in DDB1 binding. In comparison to wild-type (WT) Vpr interaction with DDB1 (Fig. (Fig.2B,2B, lane 1), the VprQ65R mutant, which did not interact with DCAF1 (Fig. (Fig.2C,2C, lane 3), also lost interaction with DDB1 (Fig. (Fig.2B,2B, lane 2). In addition, the VprQ65A mutant, which has reduced interaction with DCAF1 (26), also showed reduced interaction with DDB1 (Fig. (Fig.2B,2B, lane 3). The Vpr mutation analysis (summarized in Fig. Fig.2A)2A) further supports the model in which Vpr recruits DDB1 through DCAF1, as Vpr mutants which retained interaction with DCAF1 also retained interaction with DDB1 (Fig. 2D and E), while VprQ65 mutants with defective DCAF1 interactions displayed similar defects in DDB1 interactions (Fig. 2B and C).

FIG. 2.
Interaction of Vpr mutants with DDB1. (A) Characterization of Vpr G2 arrest mutant constructs with their DDB1 and DCAF1 binding abilities. “+” indicates substantial interaction, “−” indicates lack of interaction, ...

As proposed by Schröfelbauer et al., it is plausible that Vpr binds to DDB1 and interferes with DDB1's normal cellular function in triggering G2 arrest (46). In such a case, all G2 arrest-defective Vpr mutants would be expected to have lost the ability to bind DDB1. Alternatively, Vpr could recruit DDB1 as a copartner to form an E3 ubiquitin ligase complex, thereby destroying a cell cycle regulator that induces G2. If this second scenario is correct, then Vpr would require at least two functional domains: one to recruit DDB1-containing E3 ligase components and another to recognize the substrate. We therefore tested several Vpr mutants known to be defective in inducing G2 arrest (summarized in Fig. Fig.2A),2A), comparing their interaction with DDB1 to that of WT Vpr by coimmunoprecipitation analysis. The comparison to WT Vpr showed that VprA30L, VprR73A, VprG75A, and VprR90A all maintained the ability to bind to DDB1 (Fig. (Fig.2D).2D). VprC76A showed a slight reduction in DDB1 binding compared to WT Vpr (Fig. (Fig.2D).2D). The Vpr mutants VprA30L, VprR73A, VprG75A, and VprR90A, which were able to bind DDB1 (Fig. (Fig.2D),2D), were also able to retain efficient interaction with DCAF1 (Fig. (Fig.2E).2E). Therefore, although Vpr may disrupt the formation of normal cellular DDB1 complexes by binding to DDB1 (46), its interaction with DDB1 is not sufficient to induce G2 arrest.

DDB1 is important for Vpr-induced G2 arrest.

To further examine the role of DDB1 in Vpr-induced G2 arrest, we determined the effect on G2 arrest of silencing DDB1 with siRNA (Dharmacon) in the absence and presence of Vpr. Using a Vpr-inducible expression system comprised of a 293VE-632 cell line stably transfected with an inducible Vpr expression vector, we were able to study the effects of Vpr induction on G2 cell cycle arrest (64). The 293VE-632-inducible Vpr cells were transfected with either DDB1 or control siRNA for 3 days, followed by the induction of Vpr expression with 1 μM muristerone A (Invitrogen) for 3 days. In the absence of Vpr induction (ethanol control), both the control siRNA- and the DDB1 siRNA-transfected cells exhibited similar G2/G1 ratios of 0.49 and 0.51, respectively (Fig. (Fig.3A).3A). DDB1 siRNA efficiently reduced endogenous DDB1 expression, as indicated by immunoblotting and quantitative real-time PCR (Fig. 3D and F). These results indicated that DDB1 knockdown in 293VE-632 cells did not cause G2/M arrest. Thus, although DDB1 knockdown has been shown to induce G2 arrest in HeLa cells (34, 46), 293 cells provide a unique system to study the role of DDB1 in Vpr-induced G2 arrest.

FIG. 3.
DDB1 is required for Vpr-induced G2 arrest. (A) Effect of DDB1 siRNA shutdown in the presence or absence of Vpr induction. A Vpr-inducible 293VE-632 cell line was transfected for 72 h with either DDB1 or control siRNA for 3 days, followed by the addition ...

As expected, the induction of Vpr expression (Fig. (Fig.3C)3C) in control siRNA-transfected 293VE-632 cells produced an increased G2/G1 ratio of 0.92 (compared to a G2/G1 ratio of 0.49 in the absence of Vpr [P = 0.008]) (Fig. 3A and B), consistent with the previously reported Vpr-induced G2 arrest in 293VE-632 cells (64). However, Vpr-induced G2 arrest was significantly inhibited in 293VE-632 cells transfected with DDB1 siRNA, with a G2/G1 ratio of 0.66; the G2/G1 ratio was 0.51 in the control cells (Fig. (Fig.3A).3A). In repeated experiments, Vpr-induced G2 arrest was inhibited by 60 to 75% in the presence of DDB1 siRNA, compared to the control siRNA (P value = 0.026) (Fig. (Fig.3B).3B). Quantitative real-time PCR analysis indicated that DDB1 siRNA reduced the level of DDB1 mRNA by approximately 61% (Fig. (Fig.3F),3F), consistent with the incomplete inhibition of Vpr-induced G2 arrest by DDB1 siRNA. Thus, DDB1 is apparently required for Vpr-induced G2 arrest.

Schröfelbauer et al. proposed that Vpr-induced G2 arrest occurs through the disruption of the normal function of the DDB1/DDB2 complex via Vpr recruitment of DDB1 (46). Our data are more consistent with the model proposed by Le Rouzic et al. (26) in which Vpr-induced G2 arrest occurs through recruiting the DDB1/DCAF1 complex and not through the disruption of DDB1/DDB2 function. Our results lend support to the model of Vpr recruiting DDB1 for G2 arrest, as DDB2 siRNA knockdown (Fig. (Fig.3F)3F) in the absence of Vpr did not result in G2 arrest (Fig. (Fig.3E).3E). These data are also in agreement with a recent publication demonstrating that cells from xeroderma pigmentosum complementation group E (XPE), which lacks DDB2/XPE function, can still undergo Vpr-induced G2 arrest (13).

Cul4A is required for Vpr-induced G2 cell cycle arrest.

Since Vpr was shown to associate with both DDB1 and DCAF1, which are known to interact with Cul4A, we sought to investigate whether Cul4A is required for Vpr-induced G2 arrest. The 293VE-632-inducible Vpr cells were transfected with either Cul4A (Dharmacon) or control siRNA, followed by the induction of Vpr expression with 1 μM muristerone A. In the absence of Vpr induction, the control siRNA- and Cul4A siRNA-transfected cells exhibited G2/G1 ratios of 0.68 and 0.67, respectively (Fig. (Fig.4A).4A). Cul4A siRNA knockdown showed a moderate reduction in endogenous Cul4A expression, as indicated by immunoblotting and quantitative real-time PCR (Fig. 4C and D). The induction of Vpr expression by muristerone A (Fig. (Fig.4B)4B) in control siRNA-transfected 293VE-632 cells produced an increased G2/G1 ratio of 1.59; the G2/G1 ratio was 0.68 in the absence of Vpr (Fig. (Fig.4A).4A). We show for the first time that Vpr-induced G2 arrest was inhibited in 293VE-632 cells transfected with Cul4A siRNA, with a G2/G1 ratio of 1.01; the G2/G1 ratio was 1.59 for the control siRNA-transfected cells (Fig. (Fig.4A).4A). Both the Cul4A immunoblotting and the quantitative real-time PCR analysis indicated that Cul4A siRNA did not completely shut down endogenous Cul4A levels (Fig. 4C and D), consistent with the ~64% inhibition of Vpr-induced G2 arrest by Cul4A siRNA (Fig. (Fig.4A).4A). We hereby suggest that an E3 ubiquitin ligase, Cul4A, is involved in Vpr-induced G2 arrest.

FIG. 4.
Vpr-induced G2 arrest is dependent on Cul4A-mediated proteasome degradation. (A) Effect of Cul4A siRNA shutdown in the presence or absence of Vpr induction. The Vpr-inducible 293VE-632 cell line was transfected for 72 h with either Cul4A or control siRNA ...

To further support the hypothesis that Vpr recruitment of DDB1-containing E3 ubiquitin ligase complexes causes G2 arrest through the degradation of cell cycle regulatory proteins, we examined the effect of the proteasome inhibitor MG132 on Vpr-induced G2 arrest. In the absence of Vpr induction, the control (dimethyl sulfoxide [DMSO]) and MG132-treated cells exhibited similar G2/G1 ratios (Fig. (Fig.4E).4E). In the presence of Vpr induction and an intact functioning proteasome, control (DMSO-treated) cells had a G2/G1 ratio of 1.19, clearly demonstrating a significant increase in the percentage of cells arrested in G2, compared to a G2/G1 ratio of 0.78 in control cells without Vpr induction (Fig. (Fig.4E).4E). In contrast, the cells had a G2/G1 ratio of 0.60 in the presence of MG132 and Vpr induction; the ratio was 0.73 in cells without Vpr induction (Fig. (Fig.4E4E).

We are cautious regarding the interpretation of the data from MG132 treatment, as inhibition of the proteasome is known to interfere with the normal degradation of cell cycle control proteins, which will in turn affect cell cycle progression. Although there are caveats in the interpretation of the results from treating Vpr-expressing cells with MG132, we attempt to minimize the nonspecific effects of a proteasome inhibitor on cell cycle progression. We first induced Vpr expression for 56 h to promote G2 arrest before transient treatment with the proteasome inhibitor MG132 for 16 h to see whether MG132 could relieve Vpr-induced G2 arrest.

The collective results from proteasome inhibitor and Cul4A siRNA knockdown experiments suggest that Vpr-induced G2 cell cycle arrest is dependent on an intact Cul4A-mediated ubiquitin proteasome pathway. The data substantiate the hypothesis that Vpr recruits the DDB1/DCAF1/Cul4A-containing complex to facilitate the ubiquitination and subsequent proteasomal degradation of cell cycle regulatory proteins, resulting in G2 arrest.

The data presented here, and those published recently by others (13, 26, 46), indicate that an interaction occurs between HIV-1 Vpr and DDB1. Two models have been proposed to explain the role of this Vpr/DDB1/DCAF1 interaction in G2 cell cycle arrest. The first suggests that Vpr binding to DDB1 induces G2 arrest by interfering with the normal cellular functions of DDB1 (46); however, this scenario is unlikely to occur in 293 cells, since several Vpr mutants were as efficient as WT Vpr in binding DDB1 but were unable to induce G2 arrest (Fig. (Fig.2D).2D). Thus, binding to DDB1 is not sufficient for Vpr-induced G2 arrest. Furthermore, reducing DDB1 levels with siRNA did not induce G2 arrest in the absence of Vpr (Fig. (Fig.3A).3A). A similar siRNA treatment of DDB2, another component of the UV-induced DNA damage repair complexes, also did not induce G2 arrest in the absence of Vpr (Fig. (Fig.3E3E).

The second model proposes that Vpr recruits DDB1/DCAF1 to form an E3 ubiquitin ligase complex that can target cell cycle regulatory protein(s) for destruction and induce G2 arrest (13, 26). The data presented here support this model. Although we and others established that Vpr interaction with DDB1 requires DCAF1 (Fig. (Fig.5A)5A) (13), it is plausible that Vpr might interact with both DDB1 and DCAF1 (Fig. (Fig.5B).5B). In this case, like other WD40 repeat proteins, Vpr would act as a substrate receptor within the Cullin-DDB1 complex to dictate the substrate specificity of the Cullin-DDB1-DCAF1-Vpr E3 ligase. One class of Vpr targets that has already been identified is the uracil DNA glycosylases, such as UNG2 and SMUG1 (46). Our results suggest that Vpr-induced G2 arrest is mediated by Cullin-DDB1-DCAF1-Vpr E3 ligase activity; knocking down the DDB1 expression level impaired Vpr-induced G2 arrest (Fig. (Fig.3A).3A). More importantly, we demonstrated for the first time that proteasomal activity, which is a downstream effector of E3 ubiquitin ligases, and Cul4A, a putative E3 ubiquitin ligase in the Vpr/DDB1/DCAF1 complex, were found to be required for Vpr-induced G2 arrest (Fig. (Fig.4A).4A). Although Cul4A is shown to play a role in Vpr-induced G2 arrest, Cul4A knockdown did not completely inhibit Vpr-induced G2 arrest. It is plausible that other Cullins could also be recruited by the DDB1/DCAF1/Vpr complex to degrade target substrates, resulting in a G2 arrest phenotype.

HIV-1 Vif also recruits cellular Cul5, elongin B, and elongin C to form an E3 ubiquitin ligase that mediates APOBEC3G polyubiquitination and degradation (59). The C-terminal region of Vif contains a BC-box and an HCCH motif, which bind elongin B/elongin C and Cul5, respectively (35, 38, 61). The N-terminal region of Vif mediates APOBEC3G/APOBEC3F recognition (37, 47, 51, 55). Although a Vpr mutant (L64P) that has been identified to lose the ability to interact with DDB1 is defective for G2 arrest (46), our results indicate that other Vpr mutants that are defective for G2 arrest can still interact with DDB1 (Fig. (Fig.2D).2D). This suggests that the DDB1 association is important but not sufficient for Vpr-induced G2 arrest. By analogy to HIV-1 Vif, some of these Vpr mutants may have lost the ability to interact with the putative cellular target protein(s) whose destruction by proteasomes is critical for the induction of G2 arrest. The identification of these Vpr-targeted cellular proteins will not only further our understanding of Vpr-mediated G2 arrest but may also shed light on the normal regulation of the cell cycle.

Acknowledgments

We thank A. Morrot, I. Cockburn, P. Sarkis, and A. Niewiadomska for advice and technical assistance and L. Ratner (293VE-632 cell line), R. Zhao (Vpr antibody), N. Landau (pcDNA3-HA-Vpr), Y. Xiong (Cul4A antibody), J. Skowronski (pCG-myc-DCAF1 and pCG-HA-DCAF1), and J. Ford (pcDNA3-DDB1-V5 and pcDNA3-DDB2-V5) for critical reagents.

This work was supported by a grant from the NIH (AI062644) to X.-F. Yu.

Footnotes

[down-pointing small open triangle]Published ahead of print on 11 July 2007.

REFERENCES

1. Agostini, I., S. Popov, T. Hao, J. H. Li, L. Dubrovsky, O. Chaika, N. Chaika, R. Lewis, and M. Bukrinsky. 2002. Phosphorylation of Vpr regulates HIV type 1 nuclear import and macrophage infection. AIDS Res. Hum. Retrovir. 18:283-288. [PubMed]
2. Amini, S., K. Khalili, and B. E. Sawaya. 2004. Effect of HIV-1 Vpr on cell cycle regulators. DNA Cell Biol. 23:249-260. [PubMed]
3. Andersen, J. L., and V. Planelles. 2005. The role of Vpr in HIV-1 pathogenesis. Curr. HIV Res. 3:43-51. [PubMed]
4. Angers, S., T. Li, X. Yi, M. J. MacCoss, R. T. Moon, and N. Zheng. 2006. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443:590-593. [PubMed]
5. Bartz, S. R., M. E. Rogel, and M. Emerman. 1996. Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. J. Virol. 70:2324-2331. [PMC free article] [PubMed]
6. Bondar, T., A. Kalinina, L. Khair, D. Kopanja, A. Nag, S. Bagchi, and P. Raychaudhuri. 2006. Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome. Mol. Cell. Biol. 26:2531-2539. [PMC free article] [PubMed]
7. Bontron, S., N. Lin-Marq, and M. Strubin. 2002. Hepatitis B virus X protein associated with UV-DDB1 induces cell death in the nucleus and is functionally antagonized by UV-DDB2. J. Biol. Chem. 277:38847-38854. [PubMed]
8. Chen, R., E. Le Rouzic, J. A. Kearney, L. M. Mansky, and S. Benichou. 2004. Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages. J. Biol. Chem. 279:28419-28425. [PubMed]
9. Chen, R., H. Wang, and L. M. Mansky. 2002. Roles of uracil-DNA glycosylase and dUTPase in virus replication. J. Gen. Virol. 83:2339-2345. [PubMed]
10. Cheng, X., M. Mukhtar, E. A. Acheampong, A. Srinivasan, M. Rafi, R. J. Pomerantz, and Z. Parveen. 2007. HIV-1 Vpr potently induces programmed cell death in the CNS in vivo. DNA Cell Biol. 26:116-131. [PubMed]
11. Cohen, E. A., E. F. Terwilliger, Y. Jalinoos, J. Proulx, J. G. Sodroski, and W. A. Haseltine. 1990. Identification of HIV-1 vpr product and function. J. Acquir. Immune Defic. Syndr. 3:11-18. [PubMed]
12. Connor, R. I., B. K. Chen, S. Choe, and N. R. Landau. 1995. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206:935-944. [PubMed]
13. DeHart, J. L., E. S. Zimmerman, O. Ardon, C. M. Monteiro-Filho, E. R. Arganaraz, and V. Planelles. 2007. HIV-1 Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system. Virol. J. 4:57. [PMC free article] [PubMed]
14. Di Marzio, P., S. Choe, M. Ebright, R. Knoblauch, and N. R. Landau. 1995. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J. Virol. 69:7909-7916. [PMC free article] [PubMed]
15. Fouchier, R. A., B. E. Meyer, J. H. Simon, U. Fischer, A. V. Albright, F. Gonzalez-Scarano, and M. H. Malim. 1998. Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex. J. Virol. 72:6004-6013. [PMC free article] [PubMed]
16. Gallay, P., V. Stitt, C. Mundy, M. Oettinger, and D. Trono. 1996. Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import. J. Virol. 70:1027-1032. [PMC free article] [PubMed]
17. Gibbs, J. S., A. A. Lackner, S. M. Lang, M. A. Simon, P. K. Sehgal, M. D. Daniel, and R. C. Desrosiers. 1995. Progression to AIDS in the absence of a gene for vpr or vpx. J. Virol. 69:2378-2383. [PMC free article] [PubMed]
18. Goh, W. C., M. E. Rogel, C. M. Kinsey, S. F. Michael, P. N. Fultz, M. A. Nowak, B. H. Hahn, and M. Emerman. 1998. HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo. Nat. Med. 4:65-71. [PubMed]
19. He, J., S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau. 1995. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J. Virol. 69:6705-6711. [PMC free article] [PubMed]
20. Heinzinger, N. K., M. I. Bukinsky, S. A. Haggerty, A. M. Ragland, V. Kewalramani, M. A. Lee, H. E. Gendelman, L. Ratner, M. Stevenson, and M. Emerman. 1994. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc. Natl. Acad. Sci. USA 91:7311-7315. [PMC free article] [PubMed]
21. Hu, J., C. M. McCall, T. Ohta, and Y. Xiong. 2004. Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage. Nat. Cell Biol. 6:1003-1009. [PubMed]
22. Hwang, B. J., J. C. Liao, and G. Chu. 1996. Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells. Mutat. Res. 362:105-117. [PubMed]
23. Jacotot, E., L. Ravagnan, M. Loeffler, K. F. Ferri, H. L. Vieira, N. Zamzami, P. Costantini, S. Druillennec, J. Hoebeke, J. P. Briand, T. Irinopoulou, E. Daugas, S. A. Susin, D. Cointe, Z. H. Xie, J. C. Reed, B. P. Roques, and G. Kroemer. 2000. The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J. Exp. Med. 191:33-46. [PMC free article] [PubMed]
24. Jowett, J. B., V. Planelles, B. Poon, N. P. Shah, M. L. Chen, and I. S. Chen. 1995. The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle. J. Virol. 69:6304-6313. [PMC free article] [PubMed]
25. Kim, Y., and E. T. Kipreos. 2007. The Caenorhabditis elegans replication licensing factor CDT-1 is targeted for degradation by the CUL-4/DDB-1 complex. Mol. Cell. Biol. 27:1394-1406. [PMC free article] [PubMed]
26. Le Rouzic, E., N. Belaidouni, E. Estrabaud, M. Morel, J. C. Rain, C. Transy, and F. Margottin-Goguet. 2007. HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle 6:182-188. [PubMed]
27. Le Rouzic, E., and S. Benichou. 2005. The Vpr protein from HIV-1: distinct roles along the viral life cycle. Retrovirology 2:11. [PMC free article] [PubMed]
28. Le Rouzic, E., A. Mousnier, C. Rustum, F. Stutz, E. Hallberg, C. Dargemont, and S. Benichou. 2002. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1. J. Biol. Chem. 277:45091-45098. [PubMed]
29. Leupin, O., S. Bontron, and M. Strubin. 2003. Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities. J. Virol. 77:6274-6283. [PMC free article] [PubMed]
30. Li, B., N. Jia, R. Kapur, and K. T. Chun. 2006. Cul4A targets p27 for degradation and regulates proliferation, cell cycle exit, and differentiation during erythropoiesis. Blood 107:4291-4299. [PMC free article] [PubMed]
31. Li, J., Q. E. Wang, Q. Zhu, M. A. El-Mahdy, G. Wani, M. Praetorius-Ibba, and A. A. Wani. 2006. DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and Cullin 4A ubiquitin ligase activity. Cancer Res. 66:8590-8597. [PubMed]
32. Lin, G. Y., and R. A. Lamb. 2000. The paramyxovirus simian virus 5 V protein slows progression of the cell cycle. J. Virol. 74:9152-9166. [PMC free article] [PubMed]
33. Liu, W., A. F. Nichols, J. A. Graham, R. Dualan, A. Abbas, and S. Linn. 2000. Nuclear transport of human DDB protein induced by ultraviolet light. J. Biol. Chem. 275:21429-21434. [PubMed]
34. Lovejoy, C. A., K. Lock, A. Yenamandra, and D. Cortez. 2006. DDB1 maintains genome integrity through regulation of Cdt1. Mol. Cell. Biol. 26:7977-7990. [PMC free article] [PubMed]
35. Luo, K., Z. Xiao, E. Ehrlich, Y. Yu, B. Liu, S. Zheng, and X. F. Yu. 2005. Primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin 5-E3 ligase through a HCCH motif to suppress APOBEC3G. Proc. Natl. Acad. Sci. USA 102:11444-11449. [PMC free article] [PubMed]
36. Mansky, L. M., S. Preveral, L. Selig, R. Benarous, and S. Benichou. 2000. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 in vivo mutation rate. J. Virol. 74:7039-7047. [PMC free article] [PubMed]
37. Marin, M., K. M. Rose, S. L. Kozak, and D. Kabat. 2003. HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat. Med. 9:1398-1403. [PubMed]
38. Mehle, A., J. Goncalves, M. Santa-Marta, M. McPike, and D. Gabuzda. 2004. Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation. Genes Dev. 18:2861-2866. [PMC free article] [PubMed]
39. Muthumani, K., D. S. Hwang, B. M. Desai, D. Zhang, N. Dayes, D. R. Green, and D. B. Weiner. 2002. HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells. J. Biol. Chem. 277:37820-37831. [PubMed]
40. Nishitani, H., N. Sugimoto, V. Roukos, Y. Nakanishi, M. Saijo, C. Obuse, T. Tsurimoto, K. I. Nakayama, K. Nakayama, M. Fujita, Z. Lygerou, and T. Nishimoto. 2006. Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. EMBO J. 25:1126-1136. [PMC free article] [PubMed]
41. Popov, S., M. Rexach, L. Ratner, G. Blobel, and M. Bukrinsky. 1998. Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex. J. Biol. Chem. 273:13347-13352. [PubMed]
42. Popov, S., M. Rexach, G. Zybarth, N. Reiling, M. A. Lee, L. Ratner, C. M. Lane, M. S. Moore, G. Blobel, and M. Bukrinsky. 1998. Viral protein R regulates nuclear import of the HIV-1 preintegration complex. EMBO J. 17:909-917. [PMC free article] [PubMed]
43. Ralph, E., E. Boye, and S. E. Kearsey. 2006. DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1. EMBO Rep. 7:1134-1139. [PMC free article] [PubMed]
44. Rapi ć Otrin, V., I. Kuraoka, T. Nardo, M. McLenigan, A. P. Eker, M. Stefanini, A. S. Levine, and R. D. Wood. 1998. Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A. Mol. Cell. Biol. 18:3182-3190. [PMC free article] [PubMed]
45. Re, F., D. Braaten, E. K. Franke, and J. Luban. 1995. Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B. J. Virol. 69:6859-6864. [PMC free article] [PubMed]
46. Schröfelbauer, B., Y. Hakata, and N. R. Landau. 2007. HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1. Proc. Natl. Acad. Sci. USA 104:4130-4135. [PMC free article] [PubMed]
47. Schröfelbauer, B., T. Senger, G. Manning, and N. R. Landau. 2006. Mutational alteration of human immunodeficiency virus type 1 Vif allows for functional interaction with nonhuman primate APOBEC3G. J. Virol. 80:5984-5991. [PMC free article] [PubMed]
48. Schröfelbauer, B., Q. Yu, S. G. Zeitlin, and N. R. Landau. 2005. Human immunodeficiency virus type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA glycosylases. J. Virol. 79:10978-10987. [PMC free article] [PubMed]
49. Shiyanov, P., A. Nag, and P. Raychaudhuri. 1999. Cullin 4A associates with the UV-damaged DNA-binding protein DDB. J. Biol. Chem. 274:35309-35312. [PubMed]
50. Shostak, L. D., J. Ludlow, J. Fisk, S. Pursell, B. J. Rimel, D. Nguyen, J. D. Rosenblatt, and V. Planelles. 1999. Roles of p53 and caspases in the induction of cell cycle arrest and apoptosis by HIV-1 vpr. Exp. Cell Res. 251:156-165. [PubMed]
51. Simon, V., V. Zennou, D. Murray, Y. Huang, D. D. Ho, and P. D. Bieniasz. 2005. Natural variation in Vif: differential impact on APOBEC3G/3F and a potential role in HIV-1 diversification. PLoS Pathogens 1:e6. [PMC free article] [PubMed]
52. Stewart, S. A., B. Poon, J. B. Jowett, and I. S. Chen. 1997. Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. J. Virol. 71:5579-5592. [PMC free article] [PubMed]
53. Stewart, S. A., B. Poon, J. B. Jowett, Y. Xie, and I. S. Chen. 1999. Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells. Proc. Natl. Acad. Sci. USA 96:12039-12043. [PMC free article] [PubMed]
54. Tang, J., and G. Chu. 2002. Xeroderma pigmentosum complementation group E and UV-damaged DNA-binding protein. DNA Repair 1:601-616. [PMC free article] [PubMed]
55. Tian, C., X. Yu, W. Zhang, T. Wang, R. Xu, and X. F. Yu. 2006. Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F. J. Virol. 80:3112-3115. [PMC free article] [PubMed]
56. Vodicka, M. A., D. M. Koepp, P. A. Silver, and M. Emerman. 1998. HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev. 12:175-185. [PMC free article] [PubMed]
57. Wakasugi, M., A. Kawashima, H. Morioka, S. Linn, A. Sancar, T. Mori, O. Nikaido, and T. Matsunaga. 2002. DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair. J. Biol. Chem. 277:1637-1640. [PubMed]
58. Wittschieben, B. B., and R. D. Wood. 2003. DDB complexities. DNA Repair 2:1065-1069. [PubMed]
59. Yu, X., Y. Yu, B. Liu, K. Luo, W. Kong, P. Mao, and X. F. Yu. 2003. Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302:1056-1060. [PubMed]
60. Yu, X. F., M. Matsuda, M. Essex, and T. H. Lee. 1990. Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein. J. Virol. 64:5688-5693. [PMC free article] [PubMed]
61. Yu, Y., Z. Xiao, E. S. Ehrlich, X. Yu, and X. F. Yu. 2004. Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines. Genes Dev. 18:2867-2872. [PMC free article] [PubMed]
62. Yuan, X., Z. Matsuda, M. Matsuda, M. Essex, and T. H. Lee. 1990. Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res. Hum. Retrovir. 6:1265-1271. [PubMed]
63. Zhao, R. Y., and R. T. Elder. 2005. Viral infections and cell cycle G2/M regulation. Cell Res. 15:143-149. [PubMed]
64. Zhou, Y., and L. Ratner. 2001. A novel inducible expression system to study transdominant mutants of HIV-1 Vpr. Virology 287:133-142. [PubMed]
65. Zhou, Y., and L. Ratner. 2000. Phosphorylation of human immunodeficiency virus type 1 Vpr regulates cell cycle arrest. J. Virol. 74:6520-6527. [PMC free article] [PubMed]
66. Zhu, Y., H. A. Gelbard, M. Roshal, S. Pursell, B. D. Jamieson, and V. Planelles. 2001. Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes. J. Virol. 75:3791-3801. [PMC free article] [PubMed]

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