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

Gene expression

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-?B pathway modulation [66]. Upon various pharmacological treatments of NF-?B pathway activation, transcription factors such as NF-?B 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-?B 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].