CpG Methylation and HIV Proviral DNA Latency
A recent study published by Antony Fauci group (1) casts doubts on the role of CpG methylation in vivo in the maintenance and stability of HIV latency. DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression. Different results were published 3 years ago by Ivan Hirsch group (2), in this article we try to understand why such differences were found between the 2 studies.
CpG methylation, an epigenetic transcriptional silencing mechanism critical for organismal development and cell differentiation, has been implicated in suppressing the expression of various endogenous and/or infectious retroviruses, such as HIV. Recently, CpG methylation has been proposed as an important restriction factor contributing to the maintenance and stability of HIV latency (2). It has been suggested that the degree of methylation in the promoter/enhancer region, 5’ long terminal repeat (LTR), of HIV DNA negatively correlates with the level of viral expression following stimulation of chronically infected Jurkat cell lines and in vitro infection models.
In this study published ahead of print on the 15 February 2012 in the Journal of Virology (1) the authors analyzed 11 patients on ART with undetectable plasma viremia. The authors isolated resting CD4+ T cells from these patients and found a median frequency of methylated CpG dinucleotides within the HIV 5’ LTR of 2.4% (range 0-10%).
These results are quite different from those of Ivan Hirsch group (2). In this previous study, the 5′ LTR of HIV-1 in six patients without detectable plasma viremia contained 20%, 30%, 48%, 71%, 96%, and 100% of methylated CpGs compared with <0.1% of methylated CpGs in HIV-1 promoters in a control group of viremic patients (p = 0.0012).
The Fauci group concludes that "HIV DNA carrying high levels of 5’ LTR methylation is rare in latently infected, resting CD4+ T cells of aviremic infected individuals. Thus, the latent viral reservoir is likely maintained predominantly by mechanisms other than methylation of HIV 5’ LTR."
Of note, the first author in the 2 papers seems to be the same at a 3 years' distance, that's why we asked to Professor Ivan Hirsch his explanations. Here are the answers:
Jana Blazkova studied in our Marseille laboratory and later on in Jiri Hejnar’s laboratory in Prague, resistance of latent HIV-1 provirus to reactivation signals. These results were published in PLoS Pathogens, 2009. We used for this study Jurkat-Lat cell system harboring LTR-tat-GFP HIV-1 mini-provirus, developed in Eric Verdin laboratory. We showed that in the analyzed Jurkat cell lines, harboring latent provirus, the CpG dinucleotides in HIV-1 promoters have not been methylated. However, by consecutive repeated stimulations and selections of cells harboring activation-resistant proviruses (simulation of establishment of latent reservoir in vitro), we obtained clones with heavily methylated HIV-1 promoters. We concluded that CpG methylation is not required for the initial provirus silencing but for the resistance of latent provirus against repeated reactivation signals.
The development of in vitro model based on Jurkat-Lat cell lines published in PLoS Pathogens was complemented by methylation analysis of HIV-1-infected individuals. We found that HIV-1 promoters harbored in memory CD4+ T cells from 6 individuals receiving ART, whose virus load was <50 HIV RNA copies/ml of plasma for at least 2 years, were methylated at 20-100% of CpG residues, while HIV-1 promoters of viremic controls were methylated at <0.1% of CpGs. We interpreted the patient’s data, in agreement with the data obtained from the Jurkat cell lines, as the result of a long-lasting selection of latent proviruses resistant to repeated reactivations of the immune system.
In the recent study performed in NIH (J Virol. 2012 Feb 15.), Jana Blazkova et al. studied the methylation status of HIV 5’LTRs in resting CD4+ T cells from 11 HIV-1-infected individuals without detectable plasma viremia. They used the same technique of methylation analysis and the same system of controls based on the Jurkat clones, developed in the PLoS Pathogens study. They did not find significant methylation of 5’LTR in any of them.
The major source of differences between both studies obviously can originate in patients’ variability and in differences in ART. The patients from Marseille’s study with the most heavily methylated 5’LTRs were long-term infected individuals (infected for 11, 12 and 16 years, with a median of 10 years of ART). Some of them were treated by inefficient therapy at the beginning (see Supplementary Table 3 in the PloS Pathog. at: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000554#s5). In contrast, the longest treatment period in J.Virol study was 6.6 years with a median of 2.9 years. Another difference between both studies is the population of analyzed CD4+ T cells: HIV-1 proviruses analyzed in the PloS Pathog. study were extracted from memory CD4+ T cells, while HIV-1 proviruses analyzed in J. Virol. paper were obtained from resting CD4+ T cells. More stringent mechanisms of maintenance of HIV-1 latency, including CpG methylation, may be needed in the memory CD4+ T cells, which are more prone to proliferate than the resting CD4+ T cells.
Thus, substantially longer period of selection of latent proviruses in the memory cell population (PLoS Pathog. study), can result in accumulation of HIV-1 proviruses with methylated promoters in comparison to non-methylated HIV-1 proviruses harbored in the shorter-term-selected population of resting cells (J. Virol. Study).
Evidently further studies will be necessary to clarify these points. A cohort of HIV-1-infected individuals that will be followed prospectively to study dynamics of CpG methylation of HIV-1 promoters in latent reservoir is constructed by Katerina Trejbalova in Jiri Hejnar’s laboratory.
Ivan Hirsch, CRCM, INSERM U1068, Marseille
Jiri Hejnar, IMG, CAS, Prague
1-Blazkova J, et al. Paucity of HIV DNA Methylation in Latently Infected, Resting CD4+ T Cells from Infected Individuals Receiving Antiretroviral Therapy. J Virol 2012; Feb 15. [Epub ahead of print]. doi:10.1128/JVI.00040-12
2-Blazkova J, et al. CpG methylation controls reactivation of HIV from latency. PLoS Pathog 2009; 5(8):e1000554.
Key words: HIV latency, HIV persistence, HIV reservoirs.