Upregulation of the APOBEC3 Family Is Associated with a Poor Prognosis and Influences Treatment Response to Raf Inhibitors in Low Grade Glioma

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) has been identified as a group of enzymes that catalyze cytosine deamination in single-stranded (ss) DNA to form uracil, causing somatic mutations in some cancers. We analyzed the APOBEC3 family in 33 TCGA cancer types and the results indicated that APOBEC3s are upregulated in multiple cancers and strongly correlate with prognosis, particularly in low grade glioma (LGG). Then we constructed a prognostic model based on family expression in LGG where the APOBEC3 family signature is an accurate predictive model (AUC of 0.85). Gene mutation, copy number variation (CNV), and a differential gene expression (DEG) analysis were performed in different risk groups, and the weighted gene co-expression network analysis (WGCNA) was employed to clarify the role of various members in LGG; CIBERSORT algorithm was deployed to evaluate the landscape of LGG immune infiltration. We found that upregulation of the APOBEC3 family expression can strengthen Ras/MAPK signaling pathway, promote tumor progression, and ultimately reduce the treatment benefits of Raf inhibitors. Moreover, the APOBEC3 family was shown to enhance the immune response mediated by myeloid cells and interferon gamma, as well as PD-L1 and PD-L2 expression, implying that they have immunotherapy potential. Therefore, the APOBEC3 signature enables an efficient assessment of LGG patient survival outcomes and expansion of clinical benefits by selecting appropriate individualized treatment strategies.

Genomic Instability and DNA Damage Repair Pathways Induced by Human Papillomaviruses

Human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as those of the oropharynx. HPV proteins activate host DNA damage repair factors to promote their viral life cycle in stratified epithelia. Activation of both the ATR pathway and the ATM pathway are essential for viral replication and differentiation-dependent genome amplification. These pathways are also important for maintaining host genomic integrity and their dysregulation or mutation is often seen in human cancers. The APOBEC3 family of cytidine deaminases are innate immune factors that are increased in HPV positive cells leading to the accumulation of TpC mutations in cellular DNAs that contribute to malignant progression. The activation of DNA damage repair factors may corelate with expression of APOBEC3 in HPV positive cells. These pathways may actively drive tumor development implicating/suggesting DNA damage repair factors and APOBEC3 as possible therapeutic targets.

The Battle between Retroviruses and APOBEC3 Genes: Its Past and Present

The APOBEC3 family of proteins in mammals consists of cellular cytosine deaminases and well-known restriction factors against retroviruses, including lentiviruses. APOBEC3 genes are highly amplified and diversified in mammals, suggesting that their evolution and diversification have been driven by conflicts with ancient viruses. At present, lentiviruses, including HIV, the causative agent of AIDS, are known to encode a viral protein called Vif to overcome the antiviral effects of the APOBEC3 proteins of their hosts. Recent studies have revealed that the acquisition of an anti-APOBEC3 ability by lentiviruses is a key step in achieving successful cross-species transmission. Here, we summarize the current knowledge of the interplay between mammalian APOBEC3 proteins and viral infections and introduce a scenario of the coevolution of mammalian APOBEC3 genes and viruses.

APOBEC3A catalyzes mutation and drives carcinogenesis in vivo

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members, including APOBEC3B, fail to promote liver tumor formation. Tumor DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor datasets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

Polymorphisms in Human APOBEC3H Differentially Regulate Ubiquitination and Antiviral Activity

The APOBEC3 family of cytidine deaminases are an important part of the host innate immune defense against endogenous retroelements and retroviruses like Human Immunodeficiency Virus (HIV). APOBEC3H (A3H) is the most polymorphic of the human APOBEC3 genes, with four major haplotypes circulating in the population. Haplotype II is the only antivirally-active variant of A3H, while the majority of the population possess independently destabilizing polymorphisms present in haplotype I (R105G) and haplotypes III and IV (N15del). In this paper, we show that instability introduced by either polymorphism is positively correlated with degradative ubiquitination, while haplotype II is protected from this modification. Inhibiting ubiquitination by mutating all of the A3H lysines increased the expression of haplotypes III and IV, but these stabilized forms of haplotype III and IV had a strict nuclear localization, and did not incorporate into virions, nor exhibit antiviral activity. Fusion chimeras with haplotype II allowed for stabilization, cytoplasmic retention, and packaging of the N15del-containing haplotype III, but the haplotype III component of these chimeras was unable to restrict HIV-1 on its own. Thus, the evolutionary loss of A3H activity in many humans involves functional deficiencies independent of protein stability.

Polymorphisms in human APOBEC3H differentially regulate ubiquitination and antiviral activity

The APOBEC3 family of cytidine deaminases are an important part of the host innate immune defense against endogenous retroelements and retroviruses like human immunodeficiency virus (HIV). APOBEC3H (A3H) is the most polymorphic of the human APOBEC3 genes, with four major haplotypes circulating in the population. Haplotype II is the only antivirally-active variant of A3H, while the majority of the population possess independently destabilizing polymorphisms present in haplotype I (R105G) and haplotypes III and IV (N15del). Here, we show that instability introduced by either polymorphism is positively correlated with degradative ubiquitination, while haplotype II is protected from this modification. Inhibiting ubiquitination by mutating all of the A3H lysines increased expression of haplotypes III and IV, but these stabilized forms of haplotype III and IV had a strict nuclear localization, and did not incorporate into virions, nor exhibit antiviral activity, thus separating stabilization from function. On the other hand, the instability and functional deficiencies of haplotype III could be rescued by fusion to haplotype II, supporting a model by which antiviral A3H is actively stabilized through a cytoplasmic retention mechanism. Thus, the evolutionary loss of A3H activity in many humans involves functional deficiencies independent of protein stability.

APOBEC3A Catalyzes Mutation and Drives Carcinogenesis In Vivo

SUMMARYThe APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members including APOBEC3B did not as strongly promote liver tumor formation. DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor data sets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

An Alu Element Insertion in Intron 1 Results in Aberrant Alternative Splicing of APOBEC3G Pre-mRNA in Northern Pig-Tailed Macaques (Macaca leonina) That May Reduce APOBEC3G-Mediated Hypermutation Pressure on HIV-1

ABSTRACT APOBEC3 family members, particularly APOBEC3F and APOBEC3G, inhibit the replication and spread of various retroviruses by inducing hypermutation in newly synthesized viral DNA. Viral hypermutation by APOBEC3 is associated with viral evolution, viral transmission, and disease progression. In recent years, increasing attention has been paid to targeting APOBEC3G for AIDS therapy. Thus, a controllable model system using species such as macaques, which provide a relatively ideal in vivo system, is needed for the study of APOBEC3-related issues. To appropriately utilize this animal model for biomedical research, important differences between human and macaque APOBEC3s must be considered. In this study, we found that the ratio of APOBEC3G-mediated/APOBEC3-mediated HIV-1 hypermutation footprints was much lower in peripheral blood mononuclear cells (PBMCs) from northern pig-tailed macaques than in PBMCs from humans. Next, we identified a novel and conserved APOBEC3G pre-mRNA alternative splicing pattern in macaques, which differed from that in humans and resulted from an Alu element insertion into macaque APOBEC3G gene intron 1. This alternative splicing pattern generating an aberrant APOBEC3G mRNA isoform may significantly dilute full-length APOBEC3G and reduce APOBEC3G-mediated hypermutation pressure on HIV-1 in northern pig-tailed macaques, which was supported by the elimination of other possibilities accounting for this hypermutation difference between the two hosts. IMPORTANCE APOBEC3 family members, particularly APOBEC3F and APOBEC3G, are important cellular antiviral factors. Recently, more attention has been paid to targeting APOBEC3G for AIDS therapy. To appropriately utilize macaque animal models for the study of APOBEC3-related issues, it is important that the differences between human and macaque APOBEC3s are clarified. In this study, we identified a novel and conserved APOBEC3G pre-mRNA alternative splicing pattern in macaques, which differed from that in humans and which may reduce the APOBEC3G-mediated hypermutation pressure on HIV-1 in northern pig-tailed macaques (NPMs). Our work provides important information for the proper application of macaque animal models for APOBEC3-related issues in AIDS research and a better understanding of the biological functions of APOBEC3 proteins.

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits

ABSTRACT An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes. IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.