title: Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals.
abstract: BACKGROUND
  Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR.
  
  
  RESULTS
  In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation.
  
  
  CONCLUSIONS
  Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Kateřina
authors.initials: K
authors.lastname: Trejbalová
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Denisa
authors.initials: D
authors.lastname: Kovářová
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Blažková
authors.affiliation: Department of Infectious Diseases, Third Faculty of Medicine, Charles University and Hospital Na Bulovce in Prague, Budínova 67/2, CZ-18081 Prague 8, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Machala
authors.affiliation: Department of Infectious, Tropical and Parasitic Diseases, First Faculty of Medicine, Charles University in Prague and Hospital Na Bulovce, Budínova 67/ 2, CZ-18081 Prague 8, Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Jilich
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Weber
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Kučerová
authors.affiliation: Department of Mathematical Analysis and Applications of Mathematics, Faculty of Science of the Palacky University in Olomouc, Olomouc, CZ-77146 Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Vencálek
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic ; Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ-16610 Prague 6, Czech Republic ; Faculty of Science, Department of Genetics and Microbiology, Charles University in Prague, Viničná 5, CZ-12844 Prague 2, Czech Republic ; Inserm, Centre de Recherche en Cancérologie de Marseille (CRCM), F-13273 Marseille, France ; Institut Paoli-Calmettes, F-13009 Marseille, France ; Aix-Marseille Univ., F-13284 Marseille, France ; CNRS, UMR7258, CRCM, F-13009 Marseille, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Hirsch
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Hejnar
mesh_terms: D019380:Anti-HIV Agents / Q000627:therapeutic use; D023241:Antiretroviral Therapy, Highly Active; D002460:Cell Line / Q000821:virology; D019175:DNA Methylation; D005260:Female; D015658:HIV Infections / Q000188:drug therapy / Q000235:genetics* / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells / Q000821:virology; D008297:Male; D011533:Proviruses / Q000235:genetics* / Q000502:physiology; D013997:Time Factors; D017735:Virus Latency / Q000235:genetics* / Q000502:physiology
chemical_list: D019380:Anti-HIV Agents
keywords: Chromatin conformation; DNA methylation; HIV-1; HIV-1-infected individuals; Latent HIV-1 provirus reactivation; Latent reservoir