APOBEC3B potently restricts HIV-2 but not HIV-1 in a Vif-dependent manner

Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2 but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partner’s in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif -encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

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.

Hepatitis B Virus (HBV) - Induced Hepatocarcinogenesis, a Founding Framework of Cancer Evolution & Development (Cancer Evo-Dev)

In this chapter, we present the founding framework of a novel theory termed as Cancer Evolution-Development (Cancer Evo-Dev), based on the current understanding of hepatitis B virus (HBV) induced hepatocarcinogenesis. The interactions of genetic predispositions and HBV infection is responsible for the maintenance of chronic non-resolving inflammation. Under the inflammatory microenvironment, pro-inflammatory factors trans-activate the expression of cytidine deaminases and suppress the expression of uracil DNA glycosylase. The imbalance between the mutagenic forces and mutation-correcting forces facilitates the generations of somatic mutations, viral mutations, and viral integrations into the host genomes. The majority of cells with genomic mutations and mutated viruses are eliminated in survival competition. Only a small percentage of the mutated cells adapted to the hostile environment can survive, retro-differentiate, and function as cancer-initiating cells, representing a process of “mutation-selection-adaptation”. Cancer Evo-Dev lays the theoretical foundation for understanding the mechanisms by which chronic infection of HBV promotes hepatocarcinogenesis. This theory also plays an important role in specific prophylaxis, prediction, early diagnosis, and targeted treatment of cancers.

Divergence in dimerization and activity of primate APOBEC3C

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and the Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Multiple integrated analyses determined why rhA3C was not active against HIV-1 and how to increase this activity. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity also promoted dimerization. Although rhA3C shares a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C, establish the amino acid network for dimerization and increased activity, and track the loss and gain of A3C antiviral activity in primates. The coevolutionary analysis of the A3C dimerization interface provides a basis from which to analyze dimerization interfaces of other A3 family members.

Examination of the APOBEC3 Barrier to Cross Species Transmission of Primate Lentiviruses

The transmission of viruses from animal hosts into humans have led to the emergence of several diseases. Usually these cross-species transmissions are blocked by host restriction factors, which are proteins that can block virus replication at a specific step. In the natural virus host, the restriction factor activity is usually suppressed by a viral antagonist protein, but this is not the case for restriction factors from an unnatural host. However, due to ongoing viral evolution, sometimes the viral antagonist can evolve to suppress restriction factors in a new host, enabling cross-species transmission. Here we examine the classical case of this paradigm by reviewing research on APOBEC3 restriction factors and how they can suppress human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). APOBEC3 enzymes are single-stranded DNA cytidine deaminases that can induce mutagenesis of proviral DNA by catalyzing the conversion of cytidine to promutagenic uridine on single-stranded viral (−)DNA if they escape the HIV/SIV antagonist protein, Vif. APOBEC3 degradation is induced by Vif through the proteasome pathway. SIV has been transmitted between Old World Monkeys and to hominids. Here we examine the adaptations that enabled such events and the ongoing impact of the APOBEC3-Vif interface on HIV in humans.

Protocol for examining and eliminating base editor-induced genome-wide and transcriptome-wide off-target mutations

Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.

Mutational pressure by host APOBEC3s more strongly affects genes expressed early in the lytic phase of herpes simplex virus-1 (HSV-1) and human polyomavirus (HPyV) infection

Herpes-Simplex Virus 1 (HSV-1) infects most humans when they are young, sometimes with fatal consequences. Gene expression occurs in a temporal order upon lytic HSV-1 infection: immediate early (IE) genes are expressed, then early (E) genes, followed by late (L) genes. During this infection cycle, the HSV-1 genome has the potential for exposure to APOBEC3 (A3) proteins, a family of cytidine deaminases that cause C>U mutations on single-stranded DNA (ssDNA), often resulting in a C>T transition. We developed a computational model for the mutational pressure of A3 on the lytic cycle of HSV-1 to determine which viral kinetic gene class is most vulnerable to A3 mutations. Using in silico stochastic methods, we simulated the infectious cycle under varying intensities of A3 mutational pressure. We found that the IE and E genes are more vulnerable to A3 than L genes. We validated this model by analyzing the A3 evolutionary footprints in 25 HSV-1 isolates. We find that IE and E genes have evolved to underrepresent A3 hotspot motifs more so than L genes, consistent with greater selection pressure on IE and E genes. We extend this model to two-step infections, such as those of polyomavirus, and find that the same pattern holds for over 25 human Polyomavirus (HPyVs) genomes. Genes expressed earlier during infection are more vulnerable to mutations than those expressed later.

mmsig: a fitting approach to accurately identify somatic mutational signatures in hematological malignancies

Mutational signatures have emerged as powerful biomarkers in cancer patients, with prognostic and therapeutic implications. Wider clinical utility requires access to reproducible algorithms, which allow characterization of mutational signatures in a given tumor sample. Here, we show how mutational signature fitting can be applied to hematological cancer genomes to identify biologically and clinically important mutational processes, highlighting the importance of careful interpretation in light of biological knowledge. Our newly released R package mmsig comes with a dynamic error-suppression procedure that improves specificity in important clinical and biological settings. In particular, mmsig allows accurate detection of mutational signatures with low abundance, such as those introduced by APOBEC cytidine deaminases. This is particularly important in the most recent mutational signature reference (COSMIC v3.1) where each signature is more clearly defined. Our mutational signature fitting algorithm mmsig is a robust tool that can be implemented immediately in the clinic.