Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Dec 28.
Published in final edited form as: AIDS. 2012 Nov 28;26(18):2295–2306. doi: 10.1097/QAD.0b013e32835a5c2f

Concurrent Measures Of Total And Integrated HIV DNA Monitor Reservoirs And Ongoing Replication In Eradication Trials

Angela M Mexas 1, Erin H Graf 1, Matthew J Pace 1, Jianqing J Yu 1, Emmanouil Papasavvas 2, Livio Azzoni 2, Michael P Busch 3, Michele Di Mascio 5, Andrea S Foulkes 6, Stephen A Migueles 4, Luis J Montaner 2, Una O’Doherty 1
PMCID: PMC4692807  NIHMSID: NIHMS443616  PMID: 23014521

Introduction

Recent clinical reports have rekindled an interest in developing a cure for HIV [1–3]. This renewed interest is fueling several studies aimed at targeting treatment resistant reservoirs in patients with HIV [4–7]. While these studies have already yielded interesting and informative results, more work is needed to determine the efficacy of new therapeutic approaches in clinical trials. In many studies, measuring changes in reservoir size and detecting the onset of viral replication would help evaluate the effects of each therapeutic approach, their efficacy in individual patients, and the mechanisms involved in effecting a cure.

HIV persists in the cells of patients on antiretroviral therapy (ART), creating a reservoir that resurfaces if therapy is discontinued [8–3]. Currently, the infectious units per million (IUPM) assay remains the best accepted method to measure actual HIV reservoirs [14] but PCR measurements of either total or integrated cellular HIV DNA have been proposed as surrogate measures of reservoir size [6]. Although the IUPM assay is considered the gold-standard, this method requires large numbers of viable cells and has a high variance [14] making it difficult to reliably measure small differences in replication competent virus. Measurements of total and integrated HIV DNA require fewer cells and produce results with smaller errors [15, 16]. However, these methods overestimate reservoir size because they include proviurses that do not contribute to viremia. Furthermore, whether the proportion of replication incompetent DNA is constant over time is unclear. Total HIV DNA further overestimates reservoir size because it includes unintegrated HIV DNA species with variable half lives. Therefore, it remains unclear whether total or integrated DNA would be a better surrogate for measuring the HIV reservoir and how these two measures differ in specific clinical settings.

During viral replication, most of the cell-associated HIV DNA is full-length, linear, and unintegrated [10]. Only a fraction of total HIV DNA is integrated and a smaller fraction still is capable of producing infectious virus [10]. After completion of reverse transcription, unintegrated HIV DNA accumulates in cells if integration does not occur. Since the majority of unintegrated HIV DNA may degrade and contributes little to viral production [17–20], measuring integrated HIV DNA could be a more reliable surrogate of replication-competent virus than measuring total HIV DNA.

It has been previously reported that untreated HIV+ patients (progressors and elite-suppressors) exhibit significantly different levels of total compared to integrated HIV DNA in PBMC [10, 15, 16]. The difference between these measures in patients with ongoing replication [21, 22] denotes that there is detectable unintegrated HIV DNA and that total and integrated HIV DNA are not interchangeable in the presence of ongoing replication. However, since the majority of unintegrated HIV DNA appears to have a short half-life in vitro, it is reasonable to expect that most of the HIV DNA will be integrated over time when replication is completely halted [23]. Therefore, if effective ART controls ongoing replication, the levels of total and integrated HIV DNA should be similar on ART because the majority of unintegrated HIV DNA should disappear over time [23, 24]. Thus, measures of total and integrated HIV DNA have been suggested to be interchangeable surrogates for the reservoir size in PBMC in patients on ART [23, 25, 26]. However, therapies that target reservoirs may induce ongoing replication making it important to determine which measure is a more reliable marker for reservoir size and how often measurements of total and integrated HIV DNA are unequal in patients on ART.

Here, we apply a unique, robust, and sensitive assay for measurements of total and integrated HIV DNA to patient samples from several cohorts in order to compare these surrogates for reservoir size in PBMC in different clinical settings. We evaluated samples from patients before and after ART initiation, samples from patients on effective ART (i.e. patients with < 50 copies/ml of plasma) longitudinally and cross-sectionally, and samples from a recent clinical trial aimed at targeting viral reservoirs. The results of the latter trial are reported in detail elsewhere (currently under review). Briefly, patients on stable ART with undetectable viremia (< 50 copies/ml of plasma) were treated with Peg-IFN-Alpha-2A (IFN-α) plus ART for 5 weeks followed by IFN-α monotherapy during a 12 week ART interruption. A fraction of patients in the study (9/20, 45%) maintained plasma viremia levels below 400 copies/mL while on IFN-α monotherapy for a period of 12 weeks after ART interruption and were termed “responders”. Previous attempts at structured treatment interruptions without concurrent immunotherapy had resulted in control of viremia at this level (<400 copies/ml) in less than 9% of patients, suggesting immunotherapy with IFN-α may have affected reservoir size [27–29]. Here, we studied the relationship between measures of total and integrated HIV DNA in the responders after IFN-α monotherapy and found a decrease in the levels of integrated but not total HIV DNA. Furthermore, both measures together suggested residual HIV expression and de novo entry and reverse transcription had occurred during IFN-α monotherapy, suggesting an immune mechanism for the reduction in reservoir size. Therefore, measuring multiple viral intermediates may also be useful in other clinical trials aimed at reducing reservoir size.

Methods

Study Subjects/Samples

Frozen PBMC and DNA samples were obtained from subjects recruited by the Clinical Research Center, NIH (Bethesda, MD), the Hospital at the University of Pennsylvania (Philadelphia, PA), or as part of the IFN-α clinical trial (clinicaltrials.org/NCT00594880, Philadelphia, PA). Subjects signed informed consent forms approved by each Institutional Review Board (IRB). The University of Pennsylvania’s IRB approved the transfer and use of materials to our laboratory for this research. Patient information was anonymized and all measures for confidentiality were followed. Table 1 summarizes patients’ CD4 counts, viral loads, treatment regimens and months on therapy. DNA from participants in the IFN-α clinical trial was isolated using TRI Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. For all other samples genomic DNA was extracted using the Blood and Cell Culture Maxi Prep Kit (Qiagen, Valencia, CA).

Table 1.

Patient Data

Figure Patient No Age Gender Year of Dx ART CD4 count (cells per ul) Months with VL <50 on ART
(at time-points measured)
1a 1 41 M 2003 atazanavir, abacavir, lamivudine 558–869 na
2 52 M 1995 fosamprenavir, ritonavir, lamivudine, zidovudine 516–833 na
1b 1 dna dna 1989 lamivudine, stavudine, indinavir 1234–2112 36
2 dna dna 1995 lamivudine, stavudine, atazanivir 1409 156
3 dna dna 1993 lamivudine, stavudine, atazanavir 386–864 48
4 dna dna 1985 dna 312–780 dna
2 1 54 M 1999 tenofovir, kivexa, sustiva 490 46
2 53 M 1985 kivexa, nevirapine, kaletra 650 112
3 50 M 1982 truvada, prezista, intelence 920 71
4 33 M 2002 truvada, nevirapine 180 63
5 50 M 2004 kivexa, sustiva 540 76
6 42 M 2001 truvada, isentress, celsentri 810 40
7 51 M 1986 truvada, kaletra, nevirapine dna 660 66
8 M dna 630 dna
9 58 M 1987 atripla 330 123
10 57 M 1989 atripla, integrase 820 142
11 59 M 1993 isentress, kaletra, sustiva 350 138
12 39 M 1993 prezista, ritonavir, isentress, intelence 410 33
13 40 M 1999 truvada, nevirapine 730 132
14 47 M 1989 kivexa, prezista, ritonavir, intelence 580 160
15 51 M 1992 380 34
16 57 M 1987 tenofovir, lamivudine, nevirapine 910 107
17 30 M 2002 kivexa, kaletra, integrase 800 92
18 41 M 1997 truvada, nevirapine 400 47
19 48 M 1997 truvada, isentress 510 15
20 41 M 1998 atripla 540 dna
3/4 1 43 F 2003 emtricitabine, tenofovir, efavirenz 545–925 na
2 47 M 2002 tenofovir, emtricitabine, ritonavir, atazanavir 383–542 na
3 50 M 1994 lopinavir, ritonavir, efavirenz, abacavir, lamivudine 440–1191 na
4 28 M 2002 lopinavir, ritonavir, tenofovir, emtricitabine 509–937 na
5 39 F 1994 fosamprenavir, abacavir, lamivudine 584–908 na
6 42 M 1998 emtricitabine, tenofovir, efavirenz 710–1358 na
7 45 M 2001 fosamprenavir, ritonavir, abacavir, lamivudine, tenofovirÊ 649–1263 na
Open in a new tab

na=not applicable

dna=data not available

PCR Assays

Total and integrated HIV DNA assays were performed according to previously published methods [16, 30–33]. HIV specific primers directed at the RU5 region alone, were used to amplify all forms of HIV DNA, annotated here as total HIV DNA. For total HIV DNA measurements, DNA was assayed in duplicates or triplicates at two to four dilutions for RU5 depending on the quantity of cells. We performed qPCR for the cellular DNA sequences encoding albumin to determine the number of cells per ml of extracted DNA. These DNA assays were performed in triplicates at 3-dilutions to rule out the presence of inhibitors. HIV DNA primers designed to detect host-genome repetitive motifs (Alu) were paired with HIV-gag specific primers to specifically quantify integrated HIV DNA in patient samples as described [30–33]. Our integration standard controls for differences in PCR amplification caused by variations in the distance between the integration sites and host-genome motifs. Thus, we can directly measure total and integrated forms and indirectly detect unintegrated HIV DNA by comparing the levels of total to those of integrated HIV DNA. If a significant difference between total and integrated HIV DNA was detected, we then assayed the samples for 2-LTR circles. Our 2-LTR assay detects 1.4 copies in 150,000 genomes 77% of the time, with a coefficient of variation of <0.30 [16]. 2-LTR assays were negative in all samples measured here. For the 2-LTR assay, DNA samples were measured using 1e6 cells in duplicate wells and 3.3×105 cells in two additional duplicate wells.

Low-level Plasma Viral Load (Residual viremia)

Individual collaborators measured the levels of plasma RNA in patient samples using previously described methods [34–36].

Statistical Analysis

To determine the ratio at which measures of total and integrated HIV DNA are significantly different (i.e unintegrated HIV DNA is detectable), we repeatedly measured total and integrated HIV DNA using an “integration only” standard diluted to 100 HIV DNA copies per million genomes. We measured the levels of total and integrated HIV DNA in our standard 7 times (42 Alu-gag replicates per integration measurement, and 10 replicates at three dilutions for each measurement of total HIV DNA), and calculated the possible ratios of all measured combinations [15]. In other words, we determined the error in our measurements of total and integrated HIV DNA and the error in the ratio of the two values. Using the most conservative estimates, we calculated that a significant excess would be present when the ratio is greater than 2.1 (i.e 2.58 standard deviations above the mean of ratios measured in seven independent experiments, p=0.01). This ratio conservatively provides the threshold when unintegrated HIV is present as long as the level of integration is greater than 0.0001 HIV copies per PBMC, which was the case in all tested samples.

Spearman coefficients were calculated to evaluate the correlations between levels of total and integrated HIV DNA and low-level viremia (residual HIV). Differences between total and integrated HIV DNA between patients with VL<1 or >1 copies/ml were determined using a two-tailed Wilcoxon sum rank test. Differences between time points for viremia, total and integrated HIV DNA in patients from the IFN-α study were determined using a two-tailed Wilcoxon signed rank test.

Results

In order to confirm that the majority of unintegrated HIV DNA would dissapear upon ART initiation [23], we analyzed longitudinal samples from patients starting ART and calculated the ratio of total to integrated HIV DNA at each time-point (Fig. 1a). A ratio greater than 2.1 denotes a statistically significant difference between total and integrated HIV DNA levels, indicating the presence of detectable unintegrated HIV DNA as explained in the methods and [15]. The calculated ratio of total to integrated HIV DNA in two patients starting ART exhibited a decline after ART initiation and approached 1 within 12 months, consistent with previously published results [23, 25]. We found a significant excess of total HIV DNA at time 0 and 6 months post ART initiation, indicating that there is detectable unintegrated HIV DNA. We also found a significant reduction in integrated HIV DNA 6 months after starting ART in both patients (p= 5.01×10−7 and 1.18×10−6 respectively). We interpret this ~2-fold change to represent the reduction in the frequency of activated CD4+ cells that contain higher levels of integrated HIV that occurs with the initiation of ART.

Figure 1. The Ratio of Total to Integrated HIV DNA in patients on ART.

Figure 1

Open in a new tab

a: The Ratio of Total to Integrated HIV DNA Approaches 1 Within One Year of ART Initiation. The top bar graphs depict the total and integrated HIV DNA in PBMC samples from 2 HIV+ patients during the first year of ART. Samples were collected at time 0 (Off-ART), and following 6 months, and 12 months of ART. Black bars indicate the copies of total HIV DNA per µl and gray bars indicate the copies of integrated HIV DNA per µl. The numbers in the graph indicate the ratio of total to integrated HIV DNA at each time point. A ratio greater than 2.1 indicates the presence of unintegrated HIV DNA; marked with an asterisk (*). The bottom line graphs depict the CD4+ T cell count per µl at each time-point tested.

b: Measurements of Total and Integrated HIV DNA in Longitudinal samples from 4 patients on ART. PBMC from patients who maintained good control of plasma viremia were obtained for concurrent measurements of total and integrated HIV DNA. Patients had maintained viremia at <50 copies per mL of plasma when monitored every three months for more than one year prior to inclusion in the study. The CD4+ T cell count is plotted against the right y-axis for each patient. Asterisks (*) denote samples in which the ratio of total to integrated HIV DNA is greater than 2.1, indicating the presence of unintegrated HIV DNA. Viral load remained below 50 copies/ml blood in all patients three of four patients. In one patient who had one episode of viremia (823 copies/mL) on 1/03 (V) two samples exhibited a ratio of total to integrated HIV DNA >2.1 (*). Arrows denote two of the time points (4/7/03 and 6/30/03) at which viremia was undetectable after the one viremic episode.

Next, we measured both total and integrated HIV DNA longitudinally in 4 well suppressed patients on ART (Fig. 1b). As predicted, three of four patients showed consistently similar levels of total and integrated HIV DNA. In contrast, one patient had detectable unintegrated HIV DNA (ratio of 43) coinciding with an episode of viremia (Fig. 1b, 822 HIV RNA copies per ml of plasma on 1/03). In this patient, viremia was undetectable (<50 copies/ml) when sampled twice before the next apheresis (arrows). At this apheresis there was detectable unintegrated HIV DNA (ratio of 218), in the absence of plasma viremia (Fig. 2b, 8/03). Notably, we did not detect 2-LTR circles during either episode of unintegrated HIV DNA so the persistence of circular DNA does not appear to explain the excess (or apparent long half-life) of unintegrated HIV DNA in this patient. Overall, the data from these patients suggests measures of total and integrated HIV DNA are often similar in patients that are well suppressed on ART, whereas viremic episodes correlate with an increase in the ratio of total to integrated HIV DNA, consistent with the appearance of unintegrated HIV DNA.

Figure 2. Patients with higher levels of both total and integrated HIV DNA have statistically higher low level plasma viremia in a cross-sectional cohort of patients on ART.

Figure 2

Open in a new tab

Total and integrated HIV DNA was measured in CD4+ T cells from patients on ART. Residual plasma viremia was measured by ultra-sensitive assays to levels between 1 and 50 copies/mL. Higher plasma viremia levels also correlated with higher levels of integrated HIV DNA, but not total HIV DNA. The ratio of total to integrated HIV DNA was not statistically different in the presence and absence of viremia.

We then investigated the association between the residual viremia (<50 copies) and total or integrated HIV DNA as well as the correlation of residual viremia with the presence of unintegrated HIV DNA in samples from a different, cross-sectional cohort of patients on ART who had undetectable viremia (<50 copies/ml) by standard methods (Fig 2). This cohort consisted of 10 patients with residual viremia (<50 copies per ml of plasma) and 10 patients with undetectable viremia (<1 copy per ml of plasma). There was significantly more total (p=0.02) and integrated (p=0.02) HIV DNA in patients with low level viremia compared to those with undetectable viral load, suggesting that higher integration levels may be associated with higher low-copy plasma viral loads in patients on ART (Fig. 2a,b). Furthermore, when the viral load was >1 but <50, there was a correlation between residual plasma viral load and integrated HIV DNA (rho=0.62, p=0.03), but not with total HIV DNA (rho=0.06, p=.44), (Fig. 2 c,d). Finally, if we included all of the patients with undetectable viral loads (data not shown), a significant correlation existed between viral load and integrated HIV DNA (rho=0.61, p=0.002) and between viral load and total (rho=.53, p=0.008), as suggested by others [36, 37]. Thus, integration is more strongly correlated with low level viremia on ART than total HIV DNA measurements.

There was no significant difference in the ratio of the levels of total over integrated HIV DNA in the presence and absence of viremia (Fig. 2e). These results suggest integration measures correlate better with residual viremia and there are different mechanisms associated with viral blips and the presence of unintegrated HIV DNA. For example, a viral blip may reflect stimulation of transcription of the viral reservoir while a new excess of unintegrated HIV DNA likely indicates at least one new round of viral entry and reverse transcription.

Finally, we measured both total and integrated HIV DNA in an IFN-α trial to determine the relevance of quantifying both intermediates in a clinical setting. The results of the clinical trial are detailed elsewhere [38]. Briefly, 23 HIV-infected subjects, virologically suppressed on ART (CD4 count >450 cells/ml) were randomized to receive either 90 or 180 µg of IFN-α for 5 weeks while receiving ART and then for up to 12 weeks during ART interruption. Study endpoints included rebounding viremia (>400 copies/ml), adverse effects, or completion of the 12 weeks off ART. Total and integrated cellular HIV DNA and plasma viremia (<50 copies/ml) were measured while receiving conventional ART (labeled On ART), after 5 weeks of adding IFN-α to the regular ART regimens (labeled ART and IFN), and after they received IFN-α monotherapy (IFN only) for the next 12 weeks. Since only 9 of 20 primary end-point participants were able to stay on IFN-α monotherapy for 12 weeks while off ART (termed responders), we only measured both HIV intermediates in those patients. Additionally, we had insufficient DNA sample to measure integration in 2 responders, leaving us with 7 patients to report herein. In responders, the levels of integrated HIV DNA decreased during therapy with IFN-α (as described elsewhere), while measurements of total HIV DNA did not decrease over time (Fig. 3). Furthermore, treatment with IFN-α (either alone or with ART) led to an increase in the number of patients with a ratio of total to integrated HIV DNA greater than 2.1 (Fig. 4), suggesting that IFN-α treatment resulted in increased low level replication.

Figure 3. Total and Integrated HIV DNA in patients from IFN-α trial.

Figure 3

Open in a new tab

PBMC from patients that maintained low levels (<400 copies/ml) of plasma viremia during 12 weeks of monotherapy with IFN-α were analyzed for levels of total and integrated HIV DNA at baseline on ART (On ART), on ART plus IFN-α (ART and IFN), and after monotherapy with IFN-α (IFN only). The levels of total HIV DNA did not change over time, but there was a significant decrease in the level of integrated HIV DNA in patients who responded to IFN-α.

Figure 4. Changes in residual viremia mimic changes in the ratio of total to integrated HIV DNA in pateints responding to IFN-α.

Figure 4

Open in a new tab

The number of patients with and without a ratio of total to integrated HIV DNA greater than 2.1 is shown for each time-point (a). The increase in the number of patients with detectable unintegrated HIV DNA corresponded to changes in plasma viremia as measured by ultra-sensitive methods at similar time-points (b).

Discussion

The mechanisms underlying the maintenance of HIV reservoirs in patients treated with effective ART are the subject of intense study [6, 39–47]. Here we provide data to suggest that measuring integrated HIV DNA may serve as a more reliable surrogate of reservoir size than measuring total HIV DNA in certain patient populations, especially if ongoing replication occurs. In addition, we suggest that analysis of total and integrated HIV DNA in combination with plasma viremia levels may contribute to our understanding of reservoir maintenance and help determine the mechanisms of action in trials aimed at targeting viral reservoirs.

Current dogma suggests that measuring total HIV DNA should be sufficient to monitor HIV cellular reservoirs given the short half-life of the predominant form of unintegrated HIV DNA. As such, it is reasonable to assume that accurate measurements of total and integrated HIV DNA would eventually become equal in most patients starting antiretroviral therapy. While our data is consistent with this hypothesis (Figure 1A), it appears as though it may take more than six months for the majority of unintegrated HIV DNA to disappear in patients starting antiretrovirals for reasons that remain to be determined [23, 48]. There are at least two potential explanations for the slow decay of unintegrated HIV DNA: (1) the in vivo half-life of unintegrated HIV DNA maybe much longer than expected from in vitro experiments [23] and (2) ongoing replication may persist during the initial period of therapy due to insufficient drug levels in sanctuary sites [48–50] or intermittent adherence to therapy. In either case, cells with unintegrated DNA may migrate to the peripheral blood from other anatomical sites [48–50]. Thus, an excess of unintegrated HIV DNA in the absence of plasma viremia may be seen if cells from these other sites traffic into circulation carrying unintegrated HIV DNA. Notably, the half-life of circular forms also continues to be debated, but is clearly longer than for unintegrated HIV DNA [51–57]. Understanding why unintegrated HIV DNA decays slower in vivo than in vitro after initiating ART may lead to a better understanding of reservoir persistence.

We were next interested in how often total and integrated HIV DNA were different over time in patients on long term ART. Our data suggest that in most well controlled patients, total and integrated HIV DNA were similar at many time points (Figure 1b). Interestingly, we were able to capture a difference in total and integrated HIV DNA at the same time an episode of detectable plasma viremia appeared. This indicates concurrent measurements of total and integrated HIV DNA may help identify periods of viral replication in patients on ART. Consistent with our previous results (Figure 1a), unintegrated HIV DNA was still detected approximately four months after plasma viremia returned to undetectable levels. This presence of unintegrated HIV DNA may support the notion that unintegrated HIV could have a prolonged half-life in vivo perhaps related to the presence of circular forms. Alternatively, it may reflect a second burst of viral replication that was not detected by monitoring plasma viremia every 3 months, which is also reasonable since there should be more rapid clearance of viral RNA than viral DNA [58]. This second burst could be due to intermittent adherence.

As recent studies noted a correlation between levels of total HIV DNA and low-level viremia measured by ultrasensitive measures [36, 37], we wanted to determine whether the same was true for integrated HIV DNA. Therefore, we examined a cross-sectional sampling of patients on ART (Figure 2). Interestingly, integrated but not total HIV DNA measures significantly correlated with the level of plasma viremia in this patient cohort (Figure 2c,d). This discrepancy with the literature is likely due to the number of patients studied [36, 37]. To the best of our knowledge, our report is the first to show a better correlation between the level of integrated HIV DNA and low-level plasma viremia.

Measuring total and integrated HIV DNA concurrently may also provide important mechanistic information about HIV persistence. For example, total and integrated HIV DNA together, along with viral load, may help distinguish expression of latent proviruses from ongoing replication [12]. For instance, complete control of viral replication may result in the extinction of unintegrated HIV DNA over a period of time (Fig. 1a), making the ratio of total and integrated HIV DNA closer to one during a period of complete latency (Fig. 1b). At this point, the production of viral particles upon cellular stimulation could result in detectable RNA levels in plasma, without changing the ratio of total to integrated HIV DNA in cells. However, the presence of ongoing replication results in synthesis of intracellular unintegrated DNA leading to an increase in the ratio of total to integrated HIV DNA (Fig. 3). Identifying this process early in patients undergoing therapeutic changes could be valuable for understanding HIV persistence. For example, histone deacetylase inhibitors added to ART may result in viral expression without ongoing replication or they might stimulate new rounds of replication. These two possibilities could be distinguished using these methods but may not be distinguishable by viremia measures alone.

While our data indicate integrated HIV DNA correlates with low level viremia, another important question is the relationship between integration and replication competent proviruses (IUPM). Current estimates suggest that roughly one percent of integrated HIV DNA is replication competent at any time [10], but it remains unclear if the fraction of replication competent virus is constant. In other words, it is unclear if integrated HIV DNA is a strong surrogate for replication competent virus. There are three reasons why integrated HIV DNA may not give rise to replication competent virus: (1) the viral promoter may be so mutated that no transcription occurs (2) epigenetic silencing may prevent HIV expression or (3) the provirus may be mutated so that it does not produce infectious virus but may still express viral proteins. Integrated HIV DNA in setting 1 is unlikely to be relevant to treatment of HIV infection. On the other hand, integrated HIV DNA in settings 2 and 3 may be relevant. Epigenetic changes may occur over time and thus release replication competent virus; in addition, replication incompetent mutated HIV proviruses might express HIV proteins that may have toxicity for cells or may make the cells susceptible to immune pressure. Given that the proportion of integrated HIV DNA that exists in the three possibilities mentioned is unclear, the relevance of changes in integrated HIV DNA remains unclear. Unfortunately, we did not have enough cells to examine the relationship between integration and IUPM here. Nonetheless, our data suggest integrated HIV DNA can provide useful insights into the latent reservoir.

Finally, we examined the usefulness of measuring both total and integrated HIV DNA in a clinically relevant setting. To do this, we measured total and integrated HIV DNA in patients participating in a clinical trial where ART was interrupted in the presence of IFN-α immunotherapy. As expected, these patients initially exhibited similar levels of total and integrated HIV DNA while on ART. However, when IFN-α was administered for 5 weeks with ART and for 12 weeks after ART interruption there was a decrease in the level of integrated HIV DNA in responders. These patients exhibited a decrease in integrated HIV DNA per CD4+ T cell (also per PBMC and per ul of blood), without a similar decrease in total HIV DNA (Figure 3). Rather than contradicting our integration data, total HIV DNA instead provides important insight into the decline in integrated HIV DNA and suggests an immune mechanism.

As integration alone decreased, our data suggest a mechanism by which integrated HIV DNA would be preferentially removed. Immune clearance of cells expressing HIV proteins meets this criteria. Thus, IFN-α might enhance immune mediated killing of cells that contain integrated HIV DNA. First, studies have demonstrated that integrated HIV DNA is more effectively expressed than unintegrated HIV [59, 60]. Furthermore, we have recently shown that latently infected cells can express HIV proteins without leading to spreading infection [61]. Thus, it is reasonable that integrated HIV DNA would be preferentially cleared by an immune response. Finally, it is possible that IFN-α either enhances target susceptibility to CTL (e.g. IFN-α could increase HIV presentation) or enhances effector function. In fact, IFN-α has previously been shown to augment both CTL and NK effector function, [62–64].

One major concern with this hypothesis is how we managed to detect a decrease in integrated HIV DNA if most proviruses are defective. As previously described, it is possible that defective proviruses not capable of producing infectious virus might still be capable of expressing some viral proteins and thus be susceptible to an immune response. It is also possible that epigenetic silencing of proviruses may be reversed by IFN-α (allowing viral protein expression) but are not captured by the IUPM assay (Siliciano, R. 2012. “HIV Eradication: Understanding the magnitude of the problem.” Towards an HIV Cure Internationa AIDS Society’s Pre-Conference Symposium, Washington D.C. Ronald Reagan Building).

Why then did we not detect a decrease in total HIV DNA if in fact cells with integrated HIV DNA were cleared? There are two possibilities. First, it is possible that the decrease in integration levels occurred alongside a period of de novo infection (HIV entry and reverse transcription) in new target cells. Given that the patients were no longer taking ART, this hypothesis seems reasonable. Notably, viral RNA increased slightly during the treatment interruption and the ratio of total over integrated also increased (Figure 4), both of which are also consistent with the idea that a low level of de novo infection occurred after ART was stopped. A second possibility is that total in fact did decrease due to the drop in integrated HIV DNA, but the presence of unintegrated DNA before IFN-α made it difficult to detect with a total HIV DNA assay. However, our integration assay has a sufficiently small variance (error) that it can detect the difference as it specifically measures the HIV DNA intermediate that was cleared—integrated HIV DNA. It is also possible that both of these scenarios occurred in different patients. In any case, measuring integrated HIV DNA provided important information that total HIV DNA did not.

While it may be tempting to attribute a decrease in integrated HIV DNA to an IFN-α mediated restriction to HIV infection as it is known to inhibit infection [65–68], it is difficult for this restriction to result in a reduction in integrated HIV DNA. Instead, a restriction based mechanism would likely result in similar levels of integration that did not increase after ART was stopped. For this reason, we favor an immune based mechanism for clearance of integrated HIV DNA. However, it remains possible that IFN-α’s ability to restrict HIV infection may play a role. Specifically, we envision that during the structured treatment interruption, IFN-α may restrict (or reduce) HIV replication and thereby may enhance the chance that a boosted immune response could control HIV before immune exhaustion prevents control of viremia.

A final possibility is that integrated HIV DNA did not decrease and lower levels in PBMC were due to the expansion of CD4+T and non CD4+T cells or to changes in trafficking. Given that total HIV DNA did not decrease along with integrated makes both of these scenarios less likely. These explanations would require significant differences in expansion or trafficking of cells with integrated compared to unintegrated HIV DNA. Furthermore, we found a significant reduction in integrated HIV DNA when we normalized to CD4+ cells, PBMC and to uL of blood, thus making selective expansion less likely. Finally, neither expansion nor trafficking differences should result in enhanced viral control as seen in these IFN-α responders.

Whether IFN-α enhances the effectiveness of the immune response, enhances the expression of HIV, or works by other means awaits future studies. In any case, concurrent measurements of total and integrated HIV DNA in these and other eradication trials may help to identify therapies that are effective against reservoirs and may provide clues to their mechanism of action.

Acknowledgments

We gratefully acknowledge Tae-Wook Chun, Steven Deeks, and Daria Hazuda, who contributed intellectually and/or provided data and samples used in this manuscript. We thank Kenneth M. Lynn, Karam Mounzer, Pablo Tebas, Jeffrey M. Jacobson, Ian Frank, and Jay Kostman for their support of the IFN-α study samples used. In addition, we wish to thank Audra Anastasi, Louise Showe, Audra Pompeani and Lindsay Lynch for technical expertise and data analysis.

Funding Sources: NIH/NIAID grants 5K08AI073102 to AM, K02 AI078766, R21 AI087461 to UO, U01AI065279 to LJM. Additional support provided by Merck, amfAR, PKC pharmaceuticals, Genentech/Roche, The Philadelphia Foundation (Robert I. Jacobs Fund), Henry S. Miller, Jr., J. Kenneth Niblett, AIDS funds from the Commonwealth of Pennsylvania and from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health, the Penn Center for AIDS Research (P30 AI 045008), and the Genomics Core from Cancer Center Grant (P30 CA10815).

References

  • 1.Wilen CB, Wang J, Tilton JC, Miller JC, Kim KA, Rebar EJ, et al. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog. 7:e1002020. doi: 10.1371/journal.ppat.1002020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. 2009;360:692–698. doi: 10.1056/NEJMoa0802905. [DOI] [PubMed] [Google Scholar]
  • 3.Xing S, Bullen CK, Shroff NS, Shan L, Yang HC, Manucci JL, et al. Disulfiram reactivates latent HIV-1 in a Bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation. J Virol. 85:6060–6064. doi: 10.1128/JVI.02033-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Trono D, Van Lint C, Rouzioux C, Verdin E, Barre-Sinoussi F, Chun TW, Chomont N. HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science. 329:174–180. doi: 10.1126/science.1191047. [DOI] [PubMed] [Google Scholar]
  • 5.Dahl V, Josefsson L, Palmer S. HIV reservoirs, latency, and reactivation: prospects for eradication. Antiviral Res. 2010;85:286–294. doi: 10.1016/j.antiviral.2009.09.016. [DOI] [PubMed] [Google Scholar]
  • 6.Lewin SR, Rouzioux C. HIV cure and eradication: how will we get from the laboratory to effective clinical trials? AIDS. 25:885–897. doi: 10.1097/QAD.0b013e3283467041. [DOI] [PubMed] [Google Scholar]
  • 7.Richman DD, Margolis DM, Delaney M, Greene WC, Hazuda D, Pomerantz RJ. The challenge of finding a cure for HIV infection. Science. 2009;323:1304–1307. doi: 10.1126/science.1165706. [DOI] [PubMed] [Google Scholar]
  • 8.Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nature Medicine. 1999;5:512–517. doi: 10.1038/8394. [DOI] [PubMed] [Google Scholar]
  • 9.Wong JK, Hezareh M, Günthard HF, Havlir DV, Ignacio CC, Spina CA, Richman DD. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science. 1997;278:1291–1295. doi: 10.1126/science.278.5341.1291. [DOI] [PubMed] [Google Scholar]
  • 10.Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature. 1997;387:183–188. doi: 10.1038/387183a0. [DOI] [PubMed] [Google Scholar]
  • 11.Han Y, Lassen K, Monie D, Sedaghat AR, Shimoji S, Liu X, et al. Resting CD4+ T Cells from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals Carry Integrated HIV-1 Genomes within Actively Transcribed Host Genes. J. Virol. 2004;78:6122–6133. doi: 10.1128/JVI.78.12.6122-6133.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Shen L, Siliciano RF. Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection. J Allergy Clin Immunol. 2008;122:22–28. doi: 10.1016/j.jaci.2008.05.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proceedings of the National Academy of Sciences USA. 1997;94:13193–13197. doi: 10.1073/pnas.94.24.13193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Siliciano JD, Siliciano RF. Enhanced culture assay for detection and quantitation of latently infected, resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals. Methods Mol Biol. 2005;304:3–15. doi: 10.1385/1-59259-907-9:003. [DOI] [PubMed] [Google Scholar]
  • 15.Agosto LM, Liszewski MK, Mexas A, Graf E, Pace M, Yu JJ, et al. Patients on HAART often have an excess of unintegrated HIV DNA: Implications for monitoring reservoirs. Virology. 2011;409:46–53. doi: 10.1016/j.virol.2010.08.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Graf EH, Mexas AM, Yu JJ, Shaheen F, Liszewski MK, Di Mascio M, et al. Elite Suppressors Harbor Low Levels of Integrated HIV DNA and High Levels of 2-LTR Circular HIV DNA Compared to HIV+ Patients On and Off HAART. PLoS Pathog. 2011;7:e1001300. doi: 10.1371/journal.ppat.1001300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bukrinsky MI, Stanwick TL, Dempsey MP, Stevenson M. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science. 1991;254:423–427. doi: 10.1126/science.1925601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zhou Y, Zhang H, Siliciano JD, Siliciano RF. Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells. J Virol. 2005;79:2199–2210. doi: 10.1128/JVI.79.4.2199-2210.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zack JA, Haislip AM, Krogstad P, Chen ISY. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. Journal of Virology. 1992;66:1717–1725. doi: 10.1128/jvi.66.3.1717-1725.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Pierson TC, Zhou Y, Kieffer TL, Ruff CT, Buck C, Siliciano RF. Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J Virol. 2002;76:8518–8531. doi: 10.1128/JVI.76.17.8518-8531.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Mens H, Kearney M, Wiegand A, Shao W, Schonning K, Gerstoft J, et al. HIV-1 Continues to Replicate and Evolve in Patients with Natural Control of HIV infection. J Virol. 2010 doi: 10.1128/JVI.00387-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.O'Connell KA, Brennan TP, Bailey JR, Ray SC, Siliciano RF, Blankson JN. Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus. J Virol. 2010;84:7018–7028. doi: 10.1128/JVI.00548-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Koelsch KK, Liu L, Haubrich R, May S, Havlir D, Gunthard HF, et al. Dynamics of total, linear nonintegrated, and integrated HIV-1 DNA in vivo and in vitro. J Infect Dis. 2008;197:411–419. doi: 10.1086/525283. [DOI] [PubMed] [Google Scholar]
  • 24.Dickover RE, Donovan RM, Goldstein E, Cohen SH, Bolton V, Huth RG, et al. Decreases in unintegrated HIV DNA are associated with antiretroviral therapy in AIDS patients. J Acquir Immune Defic Syndr. 1992;5:31–36. [PubMed] [Google Scholar]
  • 25.Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab B, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med. 2009;15:893–900. doi: 10.1038/nm.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Brenchley JM, Hill BJ, Ambrozak DR, Price DA, Guenaga FJ, Casazza JP, et al. T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis. J Virol. 2004;78:1160–1168. doi: 10.1128/JVI.78.3.1160-1168.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.El-Sadr WM, Lundgren JD, Neaton JD, Gordin F, Abrams D, Arduino RC, et al. CD4+ count-guided interruption of antiretroviral treatment. N Engl J Med. 2006;355:2283–2296. doi: 10.1056/NEJMoa062360. [DOI] [PubMed] [Google Scholar]
  • 28.Papasavvas E, Kostman JR, Mounzer K, Grant RM, Gross R, Gallo C, et al. Randomized, controlled trial of therapy interruption in chronic HIV-1 infection. PLoS Med. 2004;1:e64. doi: 10.1371/journal.pmed.0010064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Davey RT, Bhat N, Yoder C, Chun T-W, Metcalf JA, Dewar R, et al. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:15109–15114. doi: 10.1073/pnas.96.26.15109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Liszewski MK, Yu JJ, O'Doherty U. Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods. 2009;47:254–260. doi: 10.1016/j.ymeth.2009.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Yu JJ, Wu TL, Liszewski MK, Dai J, Swiggard WJ, Baytop C, et al. A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients. Virology. 2008;379:78–86. doi: 10.1016/j.virol.2008.05.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.O'Doherty U, Swiggard WJ, Jeyakumar D, McGain D, Malim MH. A sensitive, quantitative, assay for Human Immunodeficiency Virus type 1 integration. J Virol. 2002;76:10,942–910,950. doi: 10.1128/JVI.76.21.10942-10950.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Butler SL, Hansen MST, Bushman FD. A quantitative assay for HIV DNA integration in vivo. Nature Medicine. 2001;7:631–634. doi: 10.1038/87979. [DOI] [PubMed] [Google Scholar]
  • 34.Hatano H, Hayes TL, Dahl V, Sinclair E, Lee TH, Hoh R, et al. A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response. J Infect Dis. 203:960–968. doi: 10.1093/infdis/jiq138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, Polis M, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. Journal of Clinical Microbiology. 2003;41:4531–4536. doi: 10.1128/JCM.41.10.4531-4536.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Chun TW, Murray D, Justement JS, Hallahan CW, Moir S, Kovacs C, Fauci AS. Relationship Between Residual Plasma Viremia and the Size of HIV Proviral DNA Reservoirs in Infected Individuals Receiving Effective Antiretroviral Therapy. J Infect Dis. 204:135–138. doi: 10.1093/infdis/jir208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, Duan L, et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis. 202:1553–1561. doi: 10.1086/656722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Azzoni L, Foulkes A, Papasavvas E, Mexas A, Lynn K, Mounzer K, Tebas P, Jacobson J, Frank I, Busch M, Deeks S, Carrington M, O'Doherty U, Kostman J, Montaner L. Pegylated Intergeron-a2A mono-therapy results in suppression of HIV-1 replication and decreased cell-associated HIV DNA integration. Journal of Infectious Diseases in press. doi: 10.1093/infdis/jis663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ramratnam B, Ribeiro R, He T, Chung C, Simon V, Vanderhoeven J, et al. Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication. J Acquir Immune Defic Syndr. 2004;35:33–37. doi: 10.1097/00126334-200401010-00004. [DOI] [PubMed] [Google Scholar]
  • 40.Pomerantz RJ, Otero M, Nunnari G, Malin A, Coates C, Dascenzo C, et al. Residual HIV-1 Disease Eradication (RHIDE) Trial: an HIV-1 eradication approach in humans using a novel and stimulatory intensification therapy. Seattle, WA: Poster 405-T; 2002. [Google Scholar]
  • 41.Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, Hare CB, et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS. 2010;24:2451–2460. doi: 10.1097/QAD.0b013e32833ef7bb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Havlir DV, Strain MC, Clerici M, Ignacio C, Trabattoni D, Ferrante P, Wong JK. Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years. J Virol. 2003;77:11212–11219. doi: 10.1128/JVI.77.20.11212-11219.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Dinoso JB, Kim SY, Wiegand AM, Palmer SE, Gange SJ, Cranmer L, et al. Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc Natl Acad Sci U S A. 2009;106:9403–9408. doi: 10.1073/pnas.0903107106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Chun TW, Nickle DC, Justement JS, Large D, Semerjian A, Curlin ME, et al. HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir. J Clin Invest. 2005;115:3250–3255. doi: 10.1172/JCI26197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.McMahon D, Jones J, Wiegand A, Gange SJ, Kearney M, Palmer S, et al. Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy. Clin Infect Dis. 50:912–919. doi: 10.1086/650749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Gandhi RT, Zheng L, Bosch RJ, Chan ES, Margolis DM, Read S, et al. The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial. PLoS Med. 7 doi: 10.1371/journal.pmed.1000321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Julg B, Pereyra F, Buzon MJ, Piechocka-Trocha A, Clark MJ, Baker BM, et al. Infrequent recovery of HIV from but robust exogenous infection of activated CD4(+) T cells in HIV elite controllers. Clin Infect Dis. 2010;51:233–238. doi: 10.1086/653677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Coiras M, Lopez-Huertas MR, Perez-Olmeda M, Alcami J. Understanding HIV-1 latency provides clues for the eradication of long-term reservoirs. Nat Rev Microbiol. 2009;7:798–812. doi: 10.1038/nrmicro2223. [DOI] [PubMed] [Google Scholar]
  • 49.Lafeuillade A, Stevenson M. The search for a cure for persistent HIV reservoirs. AIDS Rev. 2011;13:63–66. [PubMed] [Google Scholar]
  • 50.Palmer S, Maldarelli F, Wiegand A, Bernstein B, Hanna GJ, Brun SC, et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy. Proc Natl Acad Sci U S A. 2008;105:3879–3884. doi: 10.1073/pnas.0800050105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Sharkey M, Triques K, Kuritzkes DR, Stevenson M. In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA. J Virol. 2005;79:5203–5210. doi: 10.1128/JVI.79.8.5203-5210.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Sharkey ME, Teo I, Greenough TC, Sharova N, Luzuriaga K, Sullivan JL, et al. Persistence of episomal HIV-1 intermediates in patients on highly active anti-retroviral therapy. Nature Medicine. 2000;6:76–81. doi: 10.1038/71569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Butler SL, Johnson EP, Bushman FD. Human Immunodeficiency Virus cDNA Metabolism: Notable Stability of Two-Long Terminal Repeat Circles. Journal of Virology. 2002;76:3739–3747. doi: 10.1128/JVI.76.8.3739-3747.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Pierson TC, Kieffer TL, Ruff CT, Buck C, Gange SJ, Siliciano RF. Intrinsic stability of episomal circles formed during Human Immunodeficiency Virus Type-1 replication. Journal of Virology. 2002;76:4138–4144. doi: 10.1128/JVI.76.8.4138-4144.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Murray JM, McBride K, Boesecke C, Bailey M, Amin J, Suzuki K, et al. Integrated HIV DNA accumulates prior to treatment while episomal HIV DNA records ongoing transmission afterwards. AIDS. 2012;26:543–550. doi: 10.1097/QAD.0b013e328350fb3c. [DOI] [PubMed] [Google Scholar]
  • 56.Zhu W, Jiao Y, Lei R, Hua W, Wang R, Ji Y, et al. Rapid turnover of 2-LTR HIV-1 DNA during early stage of highly active antiretroviral therapy. PLoS One. 6:e21081. doi: 10.1371/journal.pone.0021081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Sharkey M, Babic DZ, Greenough T, Gulick R, Kuritzkes DR, Stevenson M. Episomal viral cDNAs identify a reservoir that fuels viral rebound after treatment interruption and that contributes to treatment failure. PLoS Pathog. 7:e1001303. doi: 10.1371/journal.ppat.1001303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Ramratnam B, Bonhoeffer S, Binley J, Hurley A, Zhang L, Mittler JE, et al. Rapid production and clearance of HIV-1 and hepatitis C virus assessed by large volume plasma apheresis. Lancet. 1999;354:1782–1785. doi: 10.1016/S0140-6736(99)02035-8. [DOI] [PubMed] [Google Scholar]
  • 59.Nakajima N, Lu R, Engelman A. Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines. J Virol. 2001;75:7944–7955. doi: 10.1128/JVI.75.17.7944-7955.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Sakai H, Kawamura M, Sakuragi J, Sakuragi S, Shibata R, Ishimoto A, et al. Integration is essential for efficient gene expression of human immunodeficiency virus type 1. Journal of Virology. 1993;67:1169–1174. doi: 10.1128/jvi.67.3.1169-1174.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Pace Matthew JEHG, Agosto Luis M, Mexas Angela M, Male Frances, Brady Troy, Bushman Frederic D, O'Doherty Una. Directly Infected Resting CD4+T Cells Can Produce HIV Gag Without Spreading Infection in a Model of HIV Latency. PLoS Pathog in press. doi: 10.1371/journal.ppat.1002818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Hosmalin A, Lebon P. Type I interferon production in HIV-infected patients. J Leukoc Biol. 2006;80:984–993. doi: 10.1189/jlb.0306154. [DOI] [PubMed] [Google Scholar]
  • 63.Portales P, Reynes J, Rouzier-Panis R, Baillat V, Clot J, Corbeau P. Perforin expression in T cells and virological response to PEG-interferon alpha2b in HIV-1 infection. AIDS. 2003;17:505–511. doi: 10.1097/00002030-200303070-00005. [DOI] [PubMed] [Google Scholar]
  • 64.Garcia-Sastre A, Biron CA. Type 1 interferons and the virus-host relationship: a lesson in detente. Science. 2006;312:879–882. doi: 10.1126/science.1125676. [DOI] [PubMed] [Google Scholar]
  • 65.Herbeuval JP, Shearer GM. HIV-1 immunopathogenesis: how good interferon turns bad. Clin Immunol. 2007;123:121–128. doi: 10.1016/j.clim.2006.09.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Pitha PM. Multiple effects of interferon on the replication of human immunodeficiency virus type 1. Antiviral Research. 1994;24:205–219. doi: 10.1016/0166-3542(94)90068-x. [DOI] [PubMed] [Google Scholar]
  • 67.Goujon C, Malim MH. Characterization of the alpha interferon-induced postentry block to HIV-1 infection in primary human macrophages and T cells. J Virol. 2010;84:9254–9266. doi: 10.1128/JVI.00854-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Chen K, Huang J, Zhang C, Huang S, Nunnari G, Wang FX, et al. Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells. J Virol. 2006;80:7645–7657. doi: 10.1128/JVI.00206-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES