title: Palbociclib, a selective inhibitor of cyclin-dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile α motif and HD domain-containing protein-1 (SAMHD1) activity.
abstract: BACKGROUND
  Sterile α motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication.
  
  
  METHODS
  Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4 T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method.
  
  
  RESULTS
  Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 replication was blocked by AT7519 (66.4 ± 3.8%; n = 4), roscovitine (47.3 ± 3.9%; n = 4) and purvalanol A (55.7 ± 15.7%; n = 4) at subtoxic concentrations. Palbociclib, a potent and selective CDK6 inhibitor, blocked SAMHD1 phosphorylation, intracellular dNTP levels, HIV-1 reverse transcription and HIV-1 replication in primary macrophages and CD4 T lymphocytes. Notably, treatment of macrophages with palbociclib led to reduced CDK2 activation, measured as the phosphorylation of the T-loop at the Thr160. The antiviral effect was lost when SAMHD1 was degraded by Vpx, providing further evidence for a role of SAMHD1 in mediating the antiretroviral effect.
  
  
  CONCLUSIONS
  Our results indicate that SAMHD1-mediated HIV-1 restriction is controlled by CDK as previously suggested but point to a preferential role for CDK2 and CDK6 as mediators of SAMHD1 activation. Our study provides a new signaling pathway susceptible for the development of new therapeutic approaches against HIV-1 infection.
authors.affiliation: aAIDS Research Institute-IrsiCaixa and AIDS Unit, Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain bResearch Group on Neuromuscular and Mitochondrial Disorders, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona and Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Valencia, Spain. *Eduardo Pauls and Roger Badia contributed equally to this work.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Pauls
authors.forename: Roger
authors.initials: R
authors.lastname: Badia
authors.forename: Javier
authors.initials: J
authors.lastname: Torres-Torronteras
authors.forename: Alba
authors.initials: A
authors.lastname: Ruiz
authors.forename: Marc
authors.initials: M
authors.lastname: Permanyer
authors.forename: Eva
authors.initials: E
authors.lastname: Riveira-Muñoz
authors.forename: Bonaventura
authors.initials: B
authors.lastname: Clotet
authors.forename: Ramón
authors.initials: R
authors.lastname: Marti
authors.forename: Ester
authors.initials: E
authors.lastname: Ballana
authors.forename: José A
authors.initials: JA
authors.lastname: Esté
mesh_terms: D019380:Anti-HIV Agents / Q000378:metabolism*; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D020559:Monomeric GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D010879:Piperazines / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D048348:Reverse Transcription; D000076106:SAM Domain and HD Domain-Containing Protein 1
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C495894:CDK2 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human; D020559:Monomeric GTP-Binding Proteins; C500026:palbociclib