Distribution and fate of HIV-1 unintegrated DNA species: a comprehensive update

Integration is required for successful retroviral replication. Cellular proteins RAD50, MRE11, and NBS1 are nuclease components that reportedly participate in 1-LTR circle formation [3, 13]. DNA-dependent protein kinase (DNA-PK), a nuclear serine-threonine protein kinase that can phosphorylate downstream proteins after sensing DNA breaks, is essential in the repair of dsDNA breaks by NHEJ. It is composed of a DNA-binding Ku70/Ku86 heterodimer and a 469 kDa catalytic subunit, DNA-PK_cs [48]. It has a significant role in V(D)J recombination as well. The RAG1/RAG2 proteins generate breaks in the strands of dsDNA. At the break-sites, the Ku70/Ku80 heterodimer binds to the free DNA termini, keeping them localized. DNA-PK_cs binds the DNA-bound Ku to form the DNA-PK complex, which stimulates DNA-PK_cs activity through phosphorylation [49]. It is known that 2-LTR circles are products of DNA repair mechanisms in the nucleus as a host reaction to foreign dsDNA [50]. Viral cDNA replication intermediates have been found to interact with host Ku components of the NHEJ pathway [51, 52]. Silencing of the NHEJ components Ku, ligase 4, or XRCC4 decreases the number of 2-LTR circles. Interestingly, the absence of DNA-PKcs, a component of the NHEJ machinery, showed the opposite effect [13, 52].

Circular DNAs are considered as replicative dead end. Linear DNA, the substrate of integration, is degraded in dividing T cells within hours [53], but persists for several days in resting T cells; it may even persist up to one month in macrophages [8, 54, 55] (Fig. 1). In the pre-integration state, viruses express their genes and produce half of the rescuable virions in resting CD4+ T cells [55]. The shortage of the virus production may be due to the virus-induced programmed cell death (apoptosis) or blockage of reverse transcription. In addition to these fates, in the high-titer infections of cultured cells, the majority of viral DNA undergoes intracellular degradation after synthesis [41, 46, 56, 57]. Several further proposals have also been made for DNA repair factors acting on retroviral DNA [58, 59].

Highly active antiretroviral therapy (HAART) causes a dramatic reduction in HIV-1 replication during the first 3 years and lowers the level of 2-LTR circles as well [60]. Furthermore, after 7–8 years of uninterrupted therapy, 2-LTR circles were almost undetectable in peripheral blood mononuclear cells (PBMCs) of most of the patients. These observations indicate the labile nature of 2-LTR circles, which may be subject to continuous HAART therapy. However, HIV-1 DNA was still detectable in the infected patients [60], probably due to the ongoing viral replication in newly infected dividing cells. Previously, it was demonstrated that the gradual decrease of the amount of circular DNA in proliferating cells occurs due to the absence of an origin of replication and is a function of dilution resulting from cell division [53]. IN mutant-infected macrophages produce DNA that persists at low levels even after 2 weeks, most of which is presumably unintegrated DNA. It was confirmed that the stability of 2-LTR was not due to viral replication [61].

Latent unintegrated genomes present in viremic HIV-1 patients after HAART treatment act as reservoirs and are an obstacle to effective treatment. 2-LTR circles persist in HIV-1 infected, growth-arrested T cell lines beyond their estimated half-life as previously determined in dividing cell populations [45, 56]. The drop of 2-LTR circle levels in dividing cells might be due to cell division, not due to degradation [54, 62]. In non-dividing macrophages, the 2-LTR circle levels remained the same for up to 21 days post infection (dpi) (Fig. 1). The unvarying 2-LTR circle level indicates their high stability in macrophages [45]. Moreover, macrophages are resistant to cytopathic effects caused by HIV-1, which makes macrophages a suitable reservoir for further infection [8, 33, 45, 62]. This stability in non-replicating cells may be due to their association with host proteins, e.g. histones, which protects them from decay [62]. E-DNA would be diluted out with successive rounds of cell division. In contrast to highly-active antiretroviral therapy (HAART)-treated HIV-1 patients, elite controllers, a group of HIV-1 infected persons who can control HIV-1 replication naturally in the absence of antiretroviral therapy and maintain untraceable viral loads, had higher 2-LTR circles than integrated proviruses in Ex Vivo experiments [63, 64].

The integrated HIV-1 provirus is transcribed into new genomic RNA, also serving as mRNA, which is in turn translated into viral proteins [2‐6, 65] (Fig. 1). Although it is speculated that the integrated copies of viral DNA are the sole template for viral gene expression, there is also evidence of preintegration transcription from unintegrated DNA [5, 65] (Fig. 1). Most recently, it has been shown that the transcriptional interplay is regulated oppositely between integrated and unintegrated DNA following NF-κB pathway modulation [66]. Upon various pharmacological treatments of NF-κB pathway activation, transcription factors such as NF-κB p65 and AP-1 (cFos/cJun) binds to integrated DNA and increases its expression, though the uDNA expression is declined. On the other hand, inhibition of the NF-κB pathway supports the expression of circular uDNA, and Bcl-3 and AP-1 is associated with its LTR region [66]. However, the persistent expression of HIV-1 proteins has already been reported not only in vitro in non-dividing cells, such as primary rat neurons and growth arrested fibroblasts, and dividing cells, such as SV40 T-antigen expressing cells [67‐70], but also in vivo in rodent ocular and brain tissues [69]. Even IN-mutant HIV-1 or integration-arrested viruses produce transcriptionally active circular DNA [4, 5, 67, 71]. It is already established that HIV-1 circular unintegrated DNA can promote viral replication itself, but several groups disapproved the notion [40, 47]. Most recently, Shimura et al. [72] reported that the expression of circular DNA genes is possible even after IN strand-transfer inhibitor treatment, probably due to cell-to-cell infection. Particularly, 2-LTR circles can express not only early viral genes, such as Nef [5, 65, 72], Tat [40, 73], and Rev [5, 40, 72], but also late, non-spliced, singly- or multiply-spliced transcripts prior to integration. Among them, the Nef and Tat proteins are translated only from the fully spliced mRNA [4]. It has been reported that the expression of circular DNA genes, especially Nef, is augmented by Vpr in HIV-1 infected cells [74]. Interestingly, Nef can stimulate T cell activation and decrease the expression of co-receptors CD4, CXCR4, and CCR5, thus increasing HIV-1 infectivity [75]. In addition, Tat, a transactivator protein of HIV-1, can activate the transcription from LTRs of both unintegrated and integrated viral DNA [76]. Particularly, viral transcripts are expressed from the upstream promoter, probably from the beginning of the R region located within the tandem LTR repeats of a 2-LTR circle [72]. In non-dividing cells, such as macrophages, gene expression is induced by Vpr only when it is driven by the HIV-1 LTR promoter, but not by the cytomegalovirus promoter [45]. However, after integration, Rev activity increases in order to support the production of late genes, such as Vpu, both from spliced and unspliced genes [5, 75]. Recently, Emeagwali and his colleagues (2012) showed that Vpu and the antagonistic host protein TWIK-related acid sensitive K+ channel 1 (TASK1) can preferentially downregulate the transcription of episomal DNA [75]. However, a greater part of nonintegrated viral DNA might be inactive templates for the transcription machinery. When it was observed experimentally, the Rev transcripts were synthesized from nonintegrated DNA, although the expression level was not high (approximately 0.03–5% of total viral DNA) and was transient. Inadequate Rev expression was likely the result of the activity of the late structural gene gag in nonintegrated HIV-1 DNA, which was similar to IN defective virus-infected, inhibitor-treated, or quiescent cells [5]. Upon latency reversing agents including PKC activators, histone deacetylase inhibitors and P-TEFb agonists) treatment, latent uDNA was reported to initiate lately virus production as well as the latent integrated proviruses [14].

Surprisingly, 2-LTR circles can be used as a substrate for integration by IN and contribute to the spumaviral lifecycle, even after integration [77]. Previously, spleen necrosis virus (SNV), Rous sarcoma virus (RSV), avian sarcoma virus (ASV), and avian leukosis virus (ALV) were assumed to generate the LTR–LTR circles that are used as templates for integration [19, 77]. Generally, IN can cleave the viral double stranded cDNA ends in a staggered manner, which then undergoes covalent transesterification to the 5′ phosphates of the host dsDNA [2, 21]. However, IN also shows the novel pleiotropic action in the mechanism underlying the requirement in the circular DNA integration: it can directly cleave the conserved palindromic sequence found at LTR–LTR junctions and produce linear DNA from 2-LTR circles [77]. Most recently, it has been reported that HIV-1 PIC, specifically IN, can also cleave 2-LTR circles in a similar way [78]. HIV-1 IN required for linearization of 2-LTR circles was found at the palindromic junction, recognized as integration site, and subsequently executed a de novo integration process. It can explain the decrease in the amount of 2-LTR circles and surge of linear DNA, as well as proviral DNA, after the withdrawal of raltegravir in vitro [78] or HAART administration in vivo [39].

Despite the expression level of circular DNA that was reported previously, improved efficiency was observed in the long U3 deletion mutants, both in vivo and in vitro [32]. The U3-region truncation does not alter the diversity of the four types of episomal DNA. Interestingly, the U3 deletion causes high transgene expression from episomes in different levels in non-dividing brain cells and slowly dividing liver cells of rats. The effect of the large U3 deletion on episomal expression indicates that the cis-acting elements of the retroviral genome can regulate the extrachromosomal transcriptional activity and that the cell-specific trans-acting factors are presumably implicated in negatively regulated transgene expression from lentiviral episomes [32]. Moreover, the addition of HDAC inhibitors in the form of short-chain fatty acids can also induce gene expression, as well as replication, from episomal DNA. To crosstalk genetically and functionally between integrated and unintegrated DNA, it has been found that HIV-1 gene expression, such as of Vpr, from unintegrated DNA can be complemented by co-infection with the integrated viral genome [47, 79].

The cytocidal effect of retroviruses is thought to exist due to the rapid accumulation of unintegrated viral DNA in the cells. The reinfection property might be the reason of cytopathogenesis. Most acutely and chronically infected cells show the cytocidal effect [35]. A lower level of unintegrated DNA indicates that the cells are resistant to superinfection and maintain a persistent infection [35].

The durable nature of HIV-1 reservoir in all CD4+ T-cell subsets, including naïve and memory cells is a key problem to eradication. The establishment of the pool starts from the very initial time of primary infection associated with cytopathic effect and rounds of new infection. CD4+ T cells are reported to maintain high levels of HIV DNA even after HAART and it can resume high levels of new replication following ART withdrawal [64, 80]. However, the samples, the analytes, and the assays must be taken into consideration during the measurement of the reservoir. To date, several assays have been used to measure the different analytes which are considered as the HIV reservoirs (Table 2), but shortage of standardization and validation have made the assessment difficult. The problem of all PCR-based assays is that they can not discriminate between the infectious virion producing cells and latent viral reservoir containg cells (Table 2) [80]. So different real time PCRs have been used to measure the analytes such as viral RNA, 2-LTR and proviruses more accurately. But they are still lack of fidelity of quality and accuracy. Some of the current techniques used to measure the analytes that reflect the HIV-1 reservior have been discussed in Table 2.