Human immunodeficiency virus type 1 (HIV-1) replication involves a series of highly orchestrated steps that are regulated by both viral and cellular factors. Like other retroviruses, HIV-1’s genome is reverse transcribed before the proviral DNA integrates into host cell chromatin. HIV-1 proviral DNA preferentially integrates into open transcriptionally active sites in the host cell chromatin where robust viral gene transcription can occur. Integration of HIV-1 DNA, mediated by viral integrase, allows the viral genome to persist throughout the lifespan of the cell. However, despite the critical role of integration in the lifecycle of all retroviruses, during the course of HIV-1 infection the majority of viral DNA remains unintegrated .
Unintegrated HIV-1 DNA (uDNA) is generated during virus replication and serves as DNA templates for transcription. The majority of viral uDNA genomes remain linear. Linear HIV-1 DNA, the substrate for integration, is found in both the cytoplasm and nucleus and can be degraded or modified by host factors [1–3]. uDNA can circularize by homologous recombination  or ligation of interrupted reverse transcription intermediates  to form 1-LTR circles. uDNA can also circularize by non-homologous end joining DNA repair events to form 2-LTR circles [6, 7]. In addition, formation of 1-LTR and 2-LTR circles can result from activity of host factors involved in DNA repair. Circular forms of the viral genome are found exclusively in the nucleus. Interestingly, the host restriction factor APOBEC3G has been shown to cause a 2-fold decrease in 2-LTR circle formation in cells infected with Δvif HIV-1 .
In HIV-1-infected patients, high levels of uDNA can be found in the blood, lymphoid tissues, and brain [1, 6, 9–12]. uDNA also accumulates in non-dividing cells such as macrophages and resting T cells but is lost in dividing cells through dilution during cell division [13–15]. Clinically, detection of high levels of uDNA in the brain is associated with development of AIDS dementia and correlates with a decline in CD4+ T cells. In addition, uDNA templates are increased in HIV-infected cells of patients given ART regimens with the integrase inhibitor, Raltegravir. The accumulation of uDNA in patients appears to be independent of viral load [16, 17].
Several studies have characterized important roles for uDNA with respect to HIV-1 infection [18–24]. Linear uDNA is the form used by HIV-1 integrase to incorporate the viral genome into host cell chromatin. uDNA can contribute to virus genetic diversity by providing templates for viral genome complementation of the integrated HIV-1 genome during virus assembly and production [25, 26]. Transcriptional activity in uDNA is lower than that of integrated genomes. However, early gene products of HIV-1 infection, Tat and Nef, are transcribed and translated in cells containing only uDNA [20, 24, 27, 28]. Nef protein production from uDNA is sufficient to promote T cell activation, down regulate CD4 and enhance HIV-1 infection . Tat, an important viral protein in HIV replication, is initially produced from uDNA. Tat production from uDNA transactivates the HIV-1 LTR of both uDNA and integrated HIV-1 DNA .
Transcription of uDNA is enhanced by viral protein R (Vpr) a late gene product of HIV-1 infection. Interestingly both virion-associated Vpr and newly synthesized Vpr preferentially increase transcription of nef from uDNA templates . Another late gene product of HIV-1 infection is viral protein U (Vpu). Vpu promotes the efficient release of virus particles from the cell surface, induces the degradation of CD4 in the ER and suppresses the activation of NF-κβ [31–33]. Vpu also restricts the function of several host proteins including tetherin and Twik-related Acid Sensitive K+ channel-1 (TASK-1) [34, 35]. TASK proteins are a part of the family of two-pore domain background potassium (K(2P)) channels.
TASK channels are widely expressed by many cell types and establish resting membrane potential and cell excitability. They are functional as dimers and have been shown to form homo- and heterodimers in vivo. Hsu et al. demonstrated that HIV-1 Vpu shows a high degree of sequence similarity to the first transmembrane domain of TASK-1 (Ttm1) . In addition, Vpu can self-assemble into homo-oligomeric complexes and form ion channels in lipid bilayers. Interestingly Vpu’s ion channel function is not required to antagonize CD317-mediated restriction of HIV release [37, 38]. In their study characterizing the homology between Vpu and the first transmembrane domain of TASK-1, Hsu et al. demonstrated that Vpu and Ttm1 were each capable of functional inhibition of the other. Furthermore it was found that cells infected with Vpu-deleted HIV-1, NL4-3 Udel, produced low levels of virus and this was reversed when Ttm1 was expressed in trans.
These observations suggest that some form of molecular piracy may have occurred during the evolution of HIV-1 infection. Here we demonstrate for the first time that TASK proteins and Vpu preferentially suppress transcription of uDNA. Our results have important implications for further understanding host-pathogen interactions that regulate HIV-1 replication and pathogenesis.