Dorota Piekna-Przybylska

Altered telomere maintenance mechanism (TMM) is linked to increased DNA damage at telomeres and telomere uncapping. We previously showed that HIV-1 latent cells have altered TMM and are susceptible to ligands that target G-quadruplexes (G4) at telomeres. Susceptibility of latent cells to telomere targeting could potentially be used to support approaches to eradicate HIV reservoirs. However, G4 ligands also target G-quadruplexes in promoters blocking gene transcription. Since HIV promoter sequence can form G-quadruplexes, we investigated whether G4 ligands interfere with HIV-1 promoter activity and virus reactivation from latency, and whether telomere targeting could be combined with latency reversing agents (LRAs) to promote elimination of HIV reservoirs. Our results indicate that Sp1 binding region in HIV-1 promoter can adopt G4 structures in duplex DNA, and that in vitro binding of Sp1 to G-quadruplex is blocked by G4 ligand, suggesting that agents targeting telomeres interfere with virus reactivation. However, our studies show that G4 agents do not affect HIV-1 promoter activity in cell culture, and do not interfere with latency reversal. Importantly, primary memory CD4 + T cells infected with latent HIV-1 are more susceptible to combined treatment with LRAs and G4 ligands, indicating that drugs targeting TMM may enhance killing of HIV reservoirs. Using a cell-based DNA repair assay, we also found that HIV-1 infected cells have reduced efficiency of DNA mismatch repair (MMR), and base excision repair (BER), suggesting that altered TMM in latently infected cells could be associated with accumulation of DNA damage at telomeres and changes in telomeric caps.

Base excision repair (BER) is one of the most active DNA repair pathways in cells correcting DNA damage from oxidation, deamination, alkylation, and damages induced by free radicals and ionizing radiation. Deregulation or deficiencies in BER mechanisms increase the level of mutations leading to carcinogenesis, and single-strand DNA break formation, which may be converted to double-strand breaks and induce apoptosis. BER deficiency is associated with development of diseases causing neurodegenerative disorders, such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). In addition, BER mechanisms can be affected by viral infections, such as HPV, HTLV-1, and HIV-1. Deficiencies in DNA repair in cells can be analyzed using a very convenient and effective approach, where mammalian cells are transfected with plasmids carrying a reporter gene of fluorescent protein that contain inactivating damages. The repair of DNA damages depends on the cellular machinery and is reflected by expression of the reporter gene measured by flow cytometry. In this chapter, we describe this plasmid-based reporter gene system to investigate in cell the repairs of DNA damages involving BER mechanisms.

Reduced pericytes’ coverage of endothelium in the brain is one of the structural changes leading to breach of the blood-brain barrier during HIV infection. We previously showed in central memory T (T_CM) cells that HIV latency increases cellular susceptibility to DNA damage. In this study, we investigated susceptibility of primary brain pericytes infected with HIV-1 to DNA damage in response to glutamate and TNF-α, both known to induce neuronal death during chronic inflammatory conditions. To infect pericytes, we used a single-cycle HIV-1 pseudotyped with VSV-G envelope glycoprotein and maintained the cultures until latency was established. Our data indicate that pericytes silence HIV-1 expression at similar rate compared to primary T_CM cells. TNF-α and IL-1β caused partial reactivation of the virus suggesting that progression of disease and neuroinflammation might facilitate virus reactivation from latency. Significant increases in the level of γH2AX, which reflect DNA damage, were observed in infected cultures exposed to TNF-α and glutamate at day 2 post-infection. Glutamate, an excitatory neurologic stimuli, also caused increases in the γH2AX level in latently infected pericytes, whereas PARP and DNA-PK inhibitors caused reductions in cell population suggesting that HIV-1 latency affects repairs of single- and double-strand DNA breaks. For comparison, we also analyzed latently infected astrocytes and determined that DNA damage response in astrocytes is less affected by HIV-1. In conclusion, our results indicate that productive infection and HIV-1 latency in pericytes interfere with DNA damage response, rendering them vulnerable to the agents that are characteristic of chronic neuroinflammatory disease conditions.

The population of HIV reservoir in infected person is very small, but extremely long-lived and is a major obstacle for an HIV cure. We previously showed that cells with established HIV latency have deficiencies in DNA damage response (DDR). Here, we investigated ability of HIV-1 to interfere with telomere maintenance, and the effects of targeting telomeres on latently infected cells. Our results show that telomeres are elongated in cultured primary memory CD4 + T cells (T_CM) after HIV-1 infection and when virus latency is established. Similarly, much longer telomeres were found in several Jurkat-derived latently infected cell lines, indicating that virus stimulates telomere elongation. Exposing primary CD4+ T_CM cells to BRACO19, an agent targeting telomeres, resulted in a higher rate of apoptosis for infected cultures at day 3 post-infection, during HIV-1 latency and for PMA-stimulated cultures with low level of HIV-1 reactivation. Importantly, BRACO19 induced apoptosis in infected cells with potency similar to etoposide and camptothecin, whereas uninfected cells were less affected by BRACO19. We also determined that apoptosis induced by BRACO19 is not caused by telomeres shortening, but is related to formation of gamma-H2AX, implicating DNA damage or uncapping of telomeres, which triggers genome instability. In conclusion, our results indicate that HIV-1 stimulates telomere elongation during latency, suggesting that HIV reservoir has greater capacity for clonal expansion and extended lifespan. Higher rates of apoptosis in response to BRACO19 treatment suggest that HIV reservoirs are more susceptible to targeting telomere maintenance and to inhibitors targeting DDR, which is also involved in stabilizing telomeres.

Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.