Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1

Abstract

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.

HIV-1 infection renders brain vascular pericytes susceptible to the extracellular glutamate

“…We first selected primary naïve CD4 + T cells from peripheral blood mononuclear cells (PBMC) and primed them towards differentiation into nonpolarized (NP) cells, which resemble the phenotype of the T CM cells (Fig. 3e) (Bosque and Planelles, 2009; Piekna-Przybylska et al , 2017). Infections with HIV-1 were performed when cells were still in an activated stage, and latency was established by progression of activated cells to a quiescent, memory-like stage.…”

“…We recently reported that HIV-1 latency in T CM cells affects cellular DDR, and results in increased susceptibility to DNA damage (Piekna-Przybylska et al , 2017). Pericytes are permissive to HIV-1 infection, although the rate of virus replication appears to be even lower than in astrocytes, where HIV-1 replication is also restricted (Nakagawa et al , 2012).…”

“…The cells were cultured in complete medium provided by the supplier, containing 2% fetal bovine serum (FBS), antibiotics (penicillin and streptomycin) and pericytes or astrocytes growth supplement. Primary central memory T (T CM ) cells were generated from naïve CD4 + T cells isolated from peripheral blood mononuclear cells (PBMC) selected from healthy blood donors, as previously described (Bosque and Planelles, 2009; Piekna-Przybylska et al , 2017). ATM Kinase Inhibitor (KU55933) and PARP inhibitor I (3-ABA, 3-Aminobenzamide) were obtained from Calbiochem.…”

“…When the cultures reached a confluence of 80–90%, they were seeded again at 40–50% confluence. Generation of latently infected primary memory T cells were done as previously described (Bosque and Planelles, 2009; Piekna-Przybylska et al , 2017). …”

“…Permissiveness of pericytes to non-productive HIV-1 infection suggests that the cells might support viral latency. We and others showed that HIV-1 affects DNA damage response (DDR) in cells during virus expression, as well as during latency (Belzile et al , 2010; Piekna-Przybylska et al , 2017; Roshal et al , 2003; Sun et al , 2006; Zhu et al , 2001; Zimmerman et al , 2004). We hypothesize that the loss of pericyte coverage in brains of patients with HAND is also related to an increased death rate of infected pericytes due to higher susceptibility to oxidative stress induced by chronic neuroinflammation, particularly glutamate and TNFα.…”