Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism.

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.

Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9.

Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV). Currently, there is no anti-viral therapeutic available against JCPyV infection. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) is a genome editing tool capable of introducing sequence specific breaks in double stranded DNA. Here we show that the CRISPR/Cas9 system can restrict the JCPyV life cycle in cultured cells. We utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. We found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.

Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

Understanding restriction factors and intrinsic immunity: insights and lessons from the primate lentiviruses.

Primate lentiviruses include the HIVs, HIV-1 and HIV-2; the SIVs, which are endemic to more than 40 species of nonhuman primates in Africa; and SIVmac, an AIDS-causing pathogen that emerged in US macaque colonies in the 1970s. Because of the worldwide spread of HIV and AIDS, primate lentiviruses have been intensively investigated for more than 30 years. Research on these viruses has played a leading role in the discovery and characterization of intrinsic immunity, and in particular the identification of several antiviral effectors (also known as restriction factors) including APOBEC3G, TRIM5α, BST-2/tetherin and SAMHD1. Comparative studies of the primate lentiviruses and their hosts have proven critical for understanding both the evolutionary significance and biological relevance of intrinsic immunity, and the role intrinsic immunity plays in governing viral host range and interspecies transmission of viruses in nature.

APOBEC3G polymorphism as a selective barrier to cross-species transmission and emergence of pathogenic SIV and AIDS in a primate host.

Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary "arms-race" between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary "arms-race" hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.