- Open Access
Episomal HIV-1 DNA and its relationship to other markers of HIV-1 persistence
© The Author(s) 2018
- Received: 9 October 2017
- Accepted: 19 January 2018
- Published: 30 January 2018
Reverse transcription of HIV-1 results in the generation of a linear cDNA that serves as the precursor to the integrated provirus. Other classes of extrachromosomal viral cDNA molecules can be found in acutely infected cells including the 1-LTR and 2-LTR circles of viral DNA, also referred as episomal HIV-1 DNA. Circulating CD4+ T-cells of treatment-naïve individuals contain significant levels of unintegrated forms of HIV-1 DNA. However, the importance of episomal HIV-1 DNA in the study of viral persistence during antiviral therapy (ART) is debatable. 2-LTR circles are preferentially observed in the effector memory CD4+ T cell subset of long-term treated subjects. Treatment intensification of standard regimens has been used to determine if more potent ART can impact viral reservoir activity. Adding a potent antiretroviral drug to a stable triple-drug regimen has no measurable impact on plasma HIV-1 RNA levels, suggesting that ongoing cycles of HIV-1 replication are not a major mechanism driving persistent plasma viremia during triple-drug ART. However, in randomized clinical trials of HIV-1-infected adults on apparently effective ART, the addition of an integrase inhibitor (raltegravir) to stable regimens resulted in a transient increase in 2-LTR circles in some patients, suggesting a pre-intensification steady-state in which the processes of virion generation and de novo infection were occurring. Mathematical modeling of 2-LTR production during integrase inhibitor intensification suggests the coexistence, at different levels, of ongoing de novo infection and de novo replication mechanisms, specifically in inflamed lymphoid drug sanctuaries. Most reports looking into potential changes in 2-LTR circles in interventional clinical studies have simultaneously assessed other potential surrogate markers of viral persistence. Transient increases in 2-LTR circles have been correlated to decreases in CD8+ T-cell activation, transient CD45RA−CD4+ T-cell redistribution, and decreases in the hypercoagulation biomarker D-dimer in ART-intensified individuals. It is difficult, however, to establish a systematic association because the level of correlation with different types of markers differs significantly among studies. In conclusion, despite suppressive ART, a steady-state of de novo infection may persist in some infected individuals and that this may drive immune activation and inflammation changes reflecting residual viral reservoir activity during otherwise apparently suppressive ART.
Integration into host-cell DNA is an essential step in the life cycle of all retroviruses, including HIV-1. Once integrated, the provirus is replicated as an integral element of the host genome, efficiently transcribing viral DNA into new copies of the viral genome and mRNAs that encode viral proteins . Integration also is important to viral persistence. By integrating within host cell DNA, the virus essentially usurps the life span of the infected cell. Therefore, integration within long lived cells such as memory CD4+ T cells and macrophages contributes to HIV-1 persistence in the host. Furthermore, during mitosis, proviruses are duplicated in each daughter cell and thus homeostatic proliferation of infected cells provides an additional mechanism for proviral persistence [2–7].
Reverse transcription results in the generation of a linear cDNA that serves as the precursor to the integrated provirus. In addition, other classes of extrachromosomal viral cDNA molecules can be found in acutely infected cells including (1) 1-long terminal repeat (1-LTR) circle, which is most likely the result of homologous recombination between the LTRs of the linear DNA molecule; (2) 2-LTR circles, whose structure is consistent with the ligation of the two ends of the linear precursor, often with deletions or insertions of a few nucleotides at the “circle junction” . The 1-LTR and 2-LTR closed circular DNA are also referred as episomal HIV-1 DNA. However, unlike episomal DNA molecules of herpesviruses such as Epstein Barr Virus (EBV) that contain elements allowing autonomous episomal replication, episomes generated by HIV-1 cannot replicate autonomously.
Estimates of the efficiency with which newly synthesized viral cDNA molecules complete the subsequent steps leading to integration are technically difficult to obtain. However, under favorable in vitro conditions, between 10 and 30% of the viral cDNA molecules synthesized in acutely infected permissive cells will ultimately become integrated [8, 9]. Therefore, unintegrated forms represent the largest fraction of HIV-1 cDNA in the nucleus. The suggested relative abundance is greater for unintegrated linear DNA followed by integrated provirus, 1-LTR circles (~ 10%), and finally 2-LTR circles (~ 1%) [9–11]. Kinetically, they seem to appear in the same order . While the linear molecule is the direct precursor to the integrated provirus, the circular forms appear to be dead-end by-products and do not serve as intermediates in the viral replication cycle. Since the free ends of linear viral DNA mimic double strand breaks of the chromosome and thus may provide a signal for apoptosis, circularization might be considered as a repair process to reduce such cellular danger signals . Interestingly, recent data suggest that 2-LTR circles can also be used as a reserve supply of genomes for proviral integration . However, this hypothesis has only been described in ex vivo experiments and its potential role in the overall HIV-1 replication cycle in vivo remains to be determined.
There are different PCR-based molecular methods for the specific detection and quantification of 2-LTR circles. A recent review has been devoted to precisely compare their properties and limitations . The more recently developed digital droplet PCR (ddPCR) technology  is replacing conventional PCR methods [16, 17]. Even if the relative abundance of unintegrated 1-LTR circles has been suggested to be tenfold greater than that of 2-LTR circles , methods for quantification of either 1-LTR circles or unintegrated viral linear DNA have only been developed in in vitro experiments , but not systematically applied to samples from clinical trials. As HIV-1 lacks necessary factors for the maintenance of episomal replication, it has been argued that these episomes are labile intermediates in the virus life cycle and as such, indicative of recent infection events . However, the lability of 2-LTR circles has been questioned by in vitro experiments in some HIV-1-infected cell lines [20–22]. This specific aspect will be further discussed in the next sections.
It has been suggested that unintegrated HIV-1 DNA molecules can be transiently transcribed and perhaps even support virus production, latency and immune responses, especially during direct infection of resting CD4+ T cells [23–25]. Moreover, it has been shown that Nef expressed from extrachromosomal DNA, downregulates CD4 surface expression on primary CD4+ T lymphocytes, albeit not as efficiently as integrating virus [25, 26]. It is, however, uncertain whether the accumulation of unintegrated viral DNA might have a role in clinical pathogenesis. It has also been suggested that abundant unintegrated viral DNA is a manifestation of high-multiplicity of infection  that could occur during cell-to-cell transmission in lymphoid tissues rather than in peripheral blood. This hypothesis would be compatible with the potential enrichment of 2-LTR circles in CD4+ T lymphocytes migrating from tissues to peripheral blood [28, 29].
The intensification experiments discussed above shared several key characteristics that can guide modeling efforts. The patients had been on stable antiviral therapy with suppressed plasma viremia for extended periods prior to intensification, so it is reasonable to assume that the dynamics were at steady-state prior to the intensification. The administration of integrase inhibitor was sustained for the duration of the experiment, so the perturbation was monotonic (i.e., there were no complicated dynamics introduced by the perturbation itself). Finally, the response in the measured 2-LTR dynamics was characterized by a sharp increase followed by a gradual decrease, with the final concentration of 2-LTR episomes often below the baseline level.
Modeling 2-LTR production during integrase inhibitor intensification
This pathway, however, is not capable of explaining the subsequent observed decrease in 2-LTR, integrase inhibitors have no suppressive effect on any step of this pathway (activation of quiescent infected cells, production of virus, viral entry and reverse transcription, formation of pre-integration complex). If we assume the presence of ongoing rounds of replication prior to intensification, illustrated (together with its characteristic response) in Fig. 2 as pathway 2, then a suppressive pathway exists where integrase inhibitor blocks the successful integration of HIV DNA, suppressing the population of virus-producing cells, suppressing the production of virus, cell entry, and formation of pre-integration complexes, and ultimately suppressing the production of 2-LTR episomes. An exhaustive search of possible monotone effect models [50, 51] has shown that only models that assume ongoing rounds of successful replication have suppressive pathways that are consistent with the known effects of integrase inhibitors . The observed increase in 2-LTR following intensification, together with the subsequent decrease, can only be explained by the presence of successful rounds of HIV replication prior to intensification.
By fitting patient data to the model described above , we can determine how much of the virus involved in the de novo infection events was sourced from successful rounds of de novo replication, and how much came from sources unaffected by raltegravir intensification (including quiescent reservoir cells). We are also able to obtain estimates of the amount of viral replication prior to intensification necessary to produce the observed 2-LTR dynamics post-intensification. We have previously reported the simplest quantitative model capable of describing the 2-LTR behavior post-intensification . This model tracked virus-producing infected cells, which may be created either by new infection events or by the activation of quiescent infected cells, and 2-LTR-containing cells.
The relatively sparse number of time points available mean than only the data from the Buzon trial [43, 46] had sufficient power to provide meaningful estimates of these rates, and even these estimates gave broad confidence intervals for most parameters. Interestingly, however, the estimates for the infection success ratios were very tightly constrained by the data, with seven of the patients in the study having this ratio bounded between 99 and 100%. This means that almost all of the integration events occurring in these patients prior to intensification came from ongoing replication (pathway 2), with only a negligible amount coming from the activation of quiescent infected cells (pathway 1). By contrast, the other six patients with positive 2-LTR measurements following intensification did not have a tight constraint on the infection ratio, meaning that pathway 1 and pathway 2 explained the 2-LTR measurements equally well. The data from the Buzon trial  therefore provided evidence of at least ongoing de novo infection in 30% of the patients, and evidence of de novo replication in 15% of the patients. The amount of ongoing infection necessary to explain the data, while poorly constrained, was nevertheless quite high, with median estimates on the order of 107–108 infected cells per day in the eight patients described.
Unchanging plasma viremia implies sanctuary-site origin of 2-LTR
The high success ratios of viral infection found in the seven patients following intensification could not occur in the presence of suppressive levels of antiretrovirals. Furthermore, the levels of ongoing infection required to explain the observed 2-LTR dynamics in the thirteen patients would correspond to measurable level of plasma virus if it were evenly distributed through the body. Since plasma viral loads in the intensification trials remained below the standard limit of detection in all cases, it is necessary to assume that the 2-LTR formation (and the ongoing replication that produced it) are occurring in a site with non-suppressive concentrations of antiretrovirals, sufficiently isolated from the blood that transport of free virus and actively infected cells are greatly reduced, but sufficiently connected to the blood that transport of 2-LTR-containing cells is possible. There is growing experimental evidence that many lymphoid tissues may serve as sanctuary sites [53, 54], with biopsy studies demonstrating non-suppressive levels of antiretrovirals in the lymphoid tissues of apparently suppressed patients. In fact, preliminary viral genetic analysis suggests that cells sequestered in tissues, rather than circulating T cells, might be responsible for supporting virus replication [28, 29].
When multiple lymphoid sites with diameters larger than 0.2 mm, a total dispersed site volume over 30 mL, and sufficiently low antiviral activity are modeled, we find that ongoing infection rates as high as 107–108 infected cells per day are consistent with no measurable change in the plasma virus level. When the addition of integrase inhibitor to these sites is simulated, the dynamics of 2-LTR formation in the site and their migration to the plasma produce curves identical to those seen in the data from the integrase inhibitor intensification trials. The mathematical models demonstrate that ongoing replication in inflamed lymphoid drug sanctuaries is a feasible explanation for the 2-LTR dynamics observed in seven patients following intensification. Recent data also suggest that HIV-1 actively evolves within lymph nodes due to low antiretroviral drug penetration in patients with undetectable levels of virus in their bloodstream , highlighting how dynamic and spatial processes act together to permit HIV-1 to persist within the infected host.
Observed half-life of 2-LTR in the PBMCs is consistent with activated T-cell turnover
The stability of 2-LTR episomes in vivo has been a subject of some controversy [19–22, 59, 60]. In vitro assays have shown the 2-LTR to be stable on short time scales, but in vivo studies have demonstrated 2-LTR dynamics consistent with a high turnover rate. In the model of sanctuary site dynamics described above, the 2-LTR in the intensification trial are formed in T cells in an environment of high inflammation, which will likely result in a high rate of T-cell activation and proliferation. The vast majority of activated T-cells during a proliferative immune response have short lifespans, with only a minority surviving to revert to long-lived memory cells . The estimated half-life of the 2-LTR circles from these studies, when bias-corrected for the fact that measurements were made in the blood, not the sanctuary site , is fully consistent with the turnover rate of uninfected, activated, proliferating T-cells as estimated from patients with uncontrolled HIV infection [63, 64]. The observed 2-LTR turnover in this experiment can easily be explained either by native lability of the circles, or by the turnover rate of the host cells.
There is still significant controversy regarding to what extent either single or multiple markers of viral persistence can provide clinically meaningful information. Suggested interventions to reduce the level of viral reservoirs, residual viral replication or low-level viral replication do not necessarily translate into actual clinical benefit.
In the context of theoretically suppressive HIV-1 therapies, antiretroviral regimens based on PI might be less able to achieve the same level of viral suppression as suggested by the more frequent transient increase in 2-LTR circles when triple-drug therapies are intensified with raltegravir [46, 47]. However, the sole virologic success rates of subjects with continued virologic suppression under triple-drug suppressive regimens based on PI does not seem to be different from patients on other regimens.
Raltegravir, as the first clinically available integrase inhibitor, has been exhaustively explored in its potential to contribute to HIV-1 cure strategies. Elvitegravir and dolutegravir are running a little bit behind in such exploratory studies. Newer, still in-development antiretroviral drugs will potentially add novel possibilities to the design of HIV-1 eradication strategies. Such strategies will have to prove their efficacy not only in peripheral blood but also in multiple lymphoid tissues throughout the body where the vast majority of target cells for HIV-1 reside. In this regard, new methodologies that could allow the quantification of markers of viral persistence in intact lymphoid cells obtained from less accessible tissues might play a pivotal role [74, 75]. In fact, the specific detection of 2-LTR circles in tissue-resident lymphoid cells has not yet been reported, either in a descriptive way or in the setting of an interventional strategy to reduce viral persistence in otherwise well-treated individuals.
JM-P, RZ, MJB, MS contributed equally to this manuscript. All authors read and approved the final manuscript.
We thank S. Morón-López and M. Salgado for expert assistance with the figures preparation. J.M.P. group is supported by the Spanish Secretariat for Research through Grants SAF2016-80033-R and RTC-2016-5324-1, the European Commission under the FP7 program (Project HEALTH-602570), and the Foundation for AIDS Research amfAR (109552-61-RSRL). R.Z. group is supported in part by the National Institutes of Health (NIH) through Grant AI11028. M.J.B. group is supported by the NIH through Grant R21AI118411, the Spanish Secretariat of Science and Innovation and FEDER funds (Grant SAF2015-67334-R), the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RD16/0025/0007) and GeSIDA. MJB holds a Miguel Servet contract (CP17/00142) funded by the Spanish Health Institute Carlos III. MS group is supported in part by the NIH through Grants 12065631, 096109 and 093306.
M.P. group is partially supported by unrestricted grant support from Gilead, Merck Sharp and Dohme, and ViiV Healthcare. R.Z. group has previously received unrestricted grant support from Merck Sharp and Dohme. M.J.B. group is partially supported by unrestricted grant support from Bristol-Myers Squibb. The funders had no role in the preparation of this manuscript. R.Z. is named on a US patent application related to this work.
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