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Excerpt
Many studies have characterized the forms of retroviral complementary DNA (cDNA) present in cells after infection [2]. The immediate product of reverse transcription is a linear cDNA molecule which is the substrate for integration to form the provirus which supports subsequent viral replication. The viral cDNA can also undergo other transformations which yield dead-end products (see [3] and references therein). The viral cDNA can be circularized by: ligation of the ends of the viral cDNA by the host non-homologous DNA end-joining pathway [4] to yield 2-LTRs circles; homologous recombination between LTR to yield 1-LTR circles [3]; autointegration, in which the integration reaction uses the viral cDNA itself as an integration target yielding a rearranged circle. Other more complex pathways can also yield aberrant forms, and much of the viral cDNA is in fact degraded after synthesis without doing anything (at least in high titer infections of cultured cells [5–7]).
The idea that 2-LTR circle abundance might be useful as an in vivo marker came primarily from Sharkey et al. [1], who began by studying experimental infections of cultured cells. They infected cells with HIV-1, added a reverse transcriptase inhibitor to prevent viral spread, then monitored the abundance of viral cDNA forms produced by a PCR assay. In their cultures the numbers of 2-LTR circles per cell declined with time, suggesting to them that the 2-LTR circles within cells were being degraded at a high rate—within 48 h the abundance of 2-LTR circles apparently declined by 90%.
Two-LTR circles can be readily measured in clinical samples. If they are quite short lived, then detection of 2-LTR circles would indicate new infection, providing a convenient assay. Such analysis was applied to samples from patients who had undergone long term successful HAART, and 2-LTR circles were detected in the peripheral blood mononuclear cells (PBMC) of 76% of the patients. The inference was that this indicated ongoing covert replication despite successful HAART, as the detection of 2-LTR circles appeared to require de novo infection in patients with good viral control. Following this report, measuring the presence of 2-LTR circles has come into use as a marker for ongoing covert replication in clinical studies (see, for example [8,9]).
The idea began to unravel when the stability of 2-LTR circles was looked at more closely. It seemed odd from the start that 2-LTR circles should be unstable, as other extrachromosomal circular DNAs were known to be quite stable, such as the TREC circles generated during DNA rearrangements at the T-cell receptor locus [10,11].
In 2002, Pierson et al.
