HTLV-1 Replication/Reactivation-Induced DNA Damage: Mechanisms and Pathogenesis

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The human T-cell leukemia virus type 1 (HTLV-1) is a complex human delta retrovirus that infects 10-20 million people worldwide. Approximately 3-5% of infected individuals develop an intractable T cell malignancy known as adult T cell leukemia/lymphoma (ATL) decades later. How HTLV-1 infection progresses to ATL is not fully understood. Two viral regulatory proteins, Tax and HBZ, encoded by the sense and antisense viral mRNA transcripts, respectively, are crucial for causing ATL. Tax, the viral trans-activator/oncoprotein, is a robust activator of viral replication and IKK/NF-κB signaling, and a potent clastogen that induces DNA double-strand breaks (DSBs). It is thought to be the cause for the extensive genomic instability of ATL, which, on average, contains 59.5 structural variations in its genome. We recently discovered that NF-κB hyperactivation by Tax results in the co-transcriptional accumulation of a nucleic acid structure known as an R-loop. And R-loop excision by Xeroderma pigmentosum F (XPF) and XPG, two endonucleases of the transcription-coupled nucleotide excision repair (TC-NER) pathway, leads to DNA DSBs. This finding provides a mechanistic explanation for the genomic instability of ATL and the senescence-inducing activities of Tax. Indeed, NF-κB blockade prevents Tax- induced R-loop accumulation, DNA damage, and senescence. And the silencing of XPF and XPG mitigates Tax senescence, while deficiency in either or both frequently occurs in ATL cells of all types. Importantly, NF-κB is constitutively active in ATL, and ATL cells are resistant to Tax-senescence, suggesting that they have evolved adaptive changes to benefit from the survival and proliferation advantages conferred by Tax and NF-κB. Considering our discoveries outlined above and recent evidence showing that Tax is expressed intermittently in intense bursts in ATL cell lines and HTLV-1-infected T lymphocytes cultured ex vivo, we posit that most de novo HTLV-1 infections result in active viral replication, during which potent NF-κB activation by Tax drives the accumulation of R-loops that promote DNA DSBs, leading to senescence. In a fraction of infected T cells, HTLV- 1 proviruses likely integrate into transcriptionally quiescent chromosomal regions, establishing latent viral infection. During latency, HBZ, expressed from a distinct promoter in the 3’ LTR, dampens Tax activities to inhibit viral replication and, at the same time, stimulates the clonal expansion of the latently infected cells. We further posit that Tax, expressed intermittently during viral reactivation, targets R-loops and DNA DSBs to specific NF- kB-regulated genes, causing their inactivation and facilitating ATL development. Finally, we hypothesize that during ATL evolution, gain-of-function mutations and copy number variants of interferon regulatory factor 4 (IRF4) gene are selected to enable cellular adaptation to the DNA damage and senescence response induced by Tax and NF-κB. We will test these hypotheses in two specific aims: Aim 1 To determine how Tax/NF-κB - induced R-loops impact host cell genome and cell fate. Aim 2 To elucidate how ATL cells accommodate chronic NF-κB activation.