Polyomavirus T Antigen InducesAPOBEC3BExpression Using an LXCXE-Dependent and TP53-Independent Mechanism

Abstract

APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B. Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B. Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5′-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.

Footprint of the host restriction factors APOBEC3 on the genome of human viruses

“…Indeed, high risk α-HPVs have been shown to trigger and stabilize A3A and A3B via their oncoproteins E6 and E7 [ 16 , 45 – 47 ]. Likewise, it has recently been shown that BK and JC β-PyV upregulate A3B through their large T antigen [ 17 , 48 ]. In both the α-HPVs and β-PyVs, the induced A3 proteins are enzymatically active and therefore capable of deamination [ 17 , 49 ].…”

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1

“…Although a role for A3B in cancer mutagenesis and evolvability has been welldocumented, universally accepted genomic substrates have yet to be described. Conflicting studies show A3B can target transcription-associated substrates as well as replication and recombination-associated substrates (18,54,74,95,96). Therefore, it is plausible that A3B-mediated mutagenesis can be exacerbated during G1/S stalling, which may create more exposed ssDNA substrates.…”

Characterization of the mechanism by which the RB/E2F pathway controls expression of the cancer genomic DNA deaminase APOBEC3B

“…Various DNA damaging agents also stimulate A3B expression including hydroxyurea, gemcitabine, aphidicolin, and camptothecin ( Kanu et al, 2016 ; Yamazaki et al, 2020 ). Interestingly, as alluded above, HPV infection induces A3B expression by mechanisms requiring the viral E6 and E7 oncoproteins ( Mori et al, 2015 ; Mori et al, 2017 ; Starrett et al, 2019 ; Verhalen et al, 2016 ; Vieira et al, 2014 ; Warren et al, 2015 ; Westrich et al, 2018 ). E6 appears to induce A3B in part by recruiting the transcription factor TEAD4 to promoter sequences ( Mori et al, 2015 ; Mori et al, 2017 ).…”

“…Fourth, A3B expression correlates positively with APOBEC signature mutation loads in breast cancer ( Burns et al, 2013a ), and its overexpression associates with branched evolution in breast and lung cancer ( de Bruin et al, 2014 ; Lee et al, 2019 ; Roper et al, 2019 ). Fifth, A3B expression is induced by human papillomavirus (HPV) and polyomavirus (PyV) infections, which relates to the fact that cervical, head/neck, and bladder cancers have high proportions of APOBEC signature mutations ( Gillison et al, 2019 ; Henderson et al, 2014 ; Starrett et al, 2019 ; Verhalen et al, 2016 ; Vieira et al, 2014 ). Last, A3B overexpression associates with poor clinical outcomes including drug resistance and metastasis ( Glaser et al, 2018 ; Law et al, 2016 ; Serebrenik et al, 2020 ; Sieuwerts et al, 2017 ; Sieuwerts et al, 2014 ; Walker et al, 2015 ; Xu et al, 2015 ; Yamazaki et al, 2019 ; Yan et al, 2016 ).…”

“…E6 appears to induce A3B in part by recruiting the transcription factor TEAD4 to promoter sequences ( Mori et al, 2015 ; Mori et al, 2017 ). JC and BK PyV upregulate A3B transcription by a mechanism requiring the LxCxE motif of the viral large T antigen (TAg; Starrett et al, 2019 ; Verhalen et al, 2016 ). HPV E7 also has a LxCxE motif suggesting a shared mechanism in which these viral oncoproteins may activate A3B transcription by antagonizing the canonical retinoblastoma tumor suppressor protein RB1 and the related pocket proteins RB-like 1 (RBL1) and RBL2 (reviewed by An et al, 2012 ; Bellacchio and Paggi, 2013 ; DeCaprio, 2014 ; DeCaprio and Garcea, 2013 ; Rashid et al, 2015 ).…”