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Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death.

ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr.

SUBMITTER: Barnitz RA

PROVIDER: S-EPMC3174981 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death.

Barnitz R Anthony RA Chaigne-Delalande Benjamin B Bolton Diane L DL Lenardo Michael J MJ

PloS one 20110916 9

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M bl ...[more]

PMID: 21949789

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Project description:Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.

Project description:UnlabelledThe accessory gene vpr, common to all primate lentiviruses, induces potent G2/M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vpr in vivo.ImportanceThe Vpr protein of HIV-1 and related viruses is essential for the virus in vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replication in vivo.

Project description:Background:Dihydroartemisinin (DHA), a derivate of artemisinin, is an effective antimalarial agent. DHA has been shown to exert anticancer activities to numerous cancer cells in the past few years, while the exact molecular mechanisms remain to be elucidated, especially in esophageal cancer. Methods:Crystal violet assay was conducted to determine the cell viability of human esophageal cancer cell line Eca109 treated with DHA. Tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Soft agar and crystal violet assays were used to measure the tumorigenicity of Eca109 cells. Flow cytometry was performed to evaluate ROS or cell cycle distribution. GFP-LC3 plasmids were delivered into Eca109 cells to visualize autophagy induced by DHA under a fluorescence microscope. The mRNA and protein levels of each gene were tested by qRT-PCR and western blot, respectively. Results:Our results proved that DHA significantly reduced the viability of Eca109 cells in a dose- and time-dependent manner. Further investigation showed that DHA evidently induced cell cycle arrest at the G2/M phase in Eca109 cells. Mechanistically, DHA induced intracellular ROS generation and autophagy in Eca109 cells, while blocking ROS by an antioxidant NAC obviously inhibited autophagy. Furthermore, we found that telomere shelterin component TRF2 was down-regulated in Eca109 cells exposed to DHA through autophagy-dependent degradation, which could be rescued after autophagy was blocked by ROS inhibition. Moreover, the DNA damage response (DDR) was induced obviously in DHA treated cells. To further explore whether ROS or autophagy played a vital role in DHA induced cell cycle arrest, the cell cycle distribution of Eca109 cells was evaluated after ROS or autophagy blocking, and the results showed that autophagy, but not ROS, was essential for cell cycle arrest in DHA treated cells. Conclusion:Taken together, DHA showed anticancer effect on esophageal cancer cells through autophagy-dependent cell cycle arrest at the G2/M phase, which unveiled a novel mechanism of DHA as a chemotherapeutic agent, and the degradation of TRF2 followed by DDR might be responsible for this cell phenotype.

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Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death.

Publications

Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death.

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