Different Modes of Retrovirus Restriction by Human APOBEC3A and APOBEC3G In Vivo

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.United States. Public Health Service (Grant R01-AI-085015)United States. Public Health Service (Grant T32-CA115299 )United States. Public Health Service (Grant F32-AI100512

Characterization of Ovine A3Z1 Restriction Properties against Small Ruminant Lentiviruses (SRLVs)

Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motifWe sincerely acknowledge Sandra Hervás-Stubbs from CIMA for her fruitful help. We also acknowledge Greg Towers, University College London for plasmids and protocols. Funded by CICYT (AGL2010-22341-C04-01) and Navarra’s Government (IIQ010449.RI1, IIQ14064.RI1 and PI042-LENTIMOL). Ramsés Reina was supported by the Spanish Ministry of Science and Innovation “Ramón y Cajal” contract. We acknowledge support of the publication fee by the Public University of Navarra and CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance

APOBEC3 proteins are important for antiretroviral defense in mammals. The activity of these factors has been well characterized in vitro, identifying cytidine deamination as an active source of viral restriction leading to hypermutation of viral DNA synthesized during reverse transcription. These mutations can result in viral lethality via disruption of critical genes, but in some cases is insufficient to completely obstruct viral replication. This sublethal level of mutagenesis could aid in viral evolution. A cytidine deaminase-independent mechanism of restriction has also been identified, as catalytically inactive proteins are still able to inhibit infection in vitro. Murine retroviruses do not exhibit characteristics of hypermutation by mouse APOBEC3 in vivo. However, human APOBEC3G protein expressed in transgenic mice maintains antiviral restriction and actively deaminates viral genomes. The mechanism by which endogenous APOBEC3 proteins function is unclear. The mouse provides a system amenable to studying the interaction of APOBEC3 and retroviral targets in vivo. Virions packaging endogenous protein were isolated from mice for analysis of APOBEC3 without a need for protein overexpression. Biochemical and molecular studies are possible using endogenous protein and viral nucleic acids. Additionally, the effect of APOBEC3-mediated viral mutagenesis and subsequent drug resistance can be modeled in this system. Human APOBEC3G transgenic mice infected with murine retroviruses and treated with an antiretroviral drug allows examination of natural levels of viral replication, APOBEC3 induced hypermutation, and potential viral escape. Studies described herein explore mechanisms of APOBEC3-mediated restriction and drug resistance in vivo. We show that endogenous APOBEC3 protein is efficiently packaged into viral cores, and this protein maintains catalytic activity against artificial substrates. We recovered low levels of G-to-A mutations from natural reverse transcription products, although approximately five to ten fold lower than that thought to be necessary for efficient viral restriction. We show that inhibition of reverse transcription is the main mechanism of restriction in vivo, and can be targeted through virion-packaged or cell-associated protein. Transgenically-expressed human APOBEC3G is instead able to heavily deaminate viral DNA, although frequently to sublethal levels. We assessed the effect of both murine APOBEC3 and APOBEC3G on viral replication in the presence and absence of an antiretroviral drug, and examined viruses for drug resistance mutations. APOBEC3G has a clear effect on the rate of viral mutagenesis in vivo, with the potential to induce drug resistance mutations

Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA

deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast., we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance

Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development

INTERACTIONS BETWEEN THE GLYCOSYLATED GAG PROTEIN OF A MURINE LEUKEMIA VIRUS AND MURINE APOBEC3: NOVEL INSIGHTS INTO HOW A MURINE LEUKEMIA VIRUS COUNTERACTS A RESTRICTION FACTOR

APOBEC proteins have evolved in mice and humans as potent innate defences against retroviral infections. APOBEC3G (hA3G) in humans and mouse APOBEC3 (mA3) deaminate cytidine in single-stranded DNA which ultimately results in hypermutation of newly synthesized proviral DNA. Other deaminase-independent mechanisms of inhibition have been identified, such as directly inhibiting reverse transcription. Both HIV and murine leukemia viruses (MuLVs) have evolved mechanisms to evade the action of the APOBEC proteins. HIV encodes the Vif protein which binds to hA3G and facilitates its rapid degradation through the proteasome. The mechanism(s) by which exogenous MuLVs evade mA3 inhibitory activity is unknown. Exogenous MuLVs encode a glycosylated gag protein (gGag) originating from an alternate CUG start site upstream of the AUG start site of the Gag structural polyproteins. gGag is synthesized to similar amounts as the structural Gag polyprotein in MuLV infected cells but is glycosylated in the endoplasmic reticulum and undergoes distinct proteolytic processing. The function(s) of gGag remain unclear, but eliminating its synthesis through mutation markedly impedes in vivo replication of the virus with very little affect on in vitro replication. Endogenous retroviruses have not been found to express gGag and are tightly controlled by mA3. APOBEC3 proteins are expressed in many tissues in the mouse but are not expressed in most in vitro cell lines. These observations are consistent with a link between gGag expression and the evasion of mA3 by MuLVs. Studies described herein demonstrate that gGag is protective against both cellular and virion-associated mA3 in vitro and is protective against mA3 in vivo. While there was no direct interaction between mA3 and gGag in an infected cell, gGag and mA3 are localized in the same compartment in the virion and are able to be coprecipitated together from lysed virions. G-to-A hypermutation is not a mechanism used by mA3 to inhibit gGag-negative MuLV replication. Through an affect on reverse transcription, cellular and virion-associated mA3 reduce viral transcripts in MuLV infected cells in a gGag-dependent manner

Regulation of activation induced deaminase

Activation Induced Deaminase (AID) belongs to the protein family of DNA deaminases, which catalyse the deamination of the cytosine residues in single stranded DNA, resulting in the formation of deoxy-uracils. The enzymatic activity of AID is required for the immunoglobulin gene modifications by class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (iGC). While being essential for antibody diversification, the activity of AID can be harmful for the organism due to its direct mutagenic activities and induction of genomic instability. This thesis investigates AID regulation both, on the level of gene expression and its interaction partners, and the DNA repair pathways triggered by AIDmediated DNA deamination. Firstly, I have identified estrogen and progesterone as regulators of AID expression. This is achieved via direct binding of estrogen and progesterone receptors to AID promoter. Estrogen leads to an induction of AID expression and increase in AID-mediated downstream pathways – SHM, CSR as well as oncogenic translocations between Ig and c-myc loci. In contrast, progesterone results in a decrease in AID expression and an attenuation of its downstream pathways. Secondly, by generating DT40 cell lines with endogenously tagged AID, we used co-immunoprecipitation and subsequent mass spectrometry for identifying proteins that form a complex with AID in the cytoplasm, nucleoplasm and chromatin. The results of this approach gave us possible insight into the mechanistic process of AID-mediated DNA deamination in vivo, suggesting that chromatin bound AID resides in a complex with elongating RNA polymerase II. Thirdly, by expressing AID in meiotic recombination deficient fission yeast and nematode, we have established that a meiotic cell can process a base mismatch, using the base excision repair machinery, to give rise to meiotic recombination. This suggests that meiotic cells can process lesions other than Spo-11 induced DSBs for recombination

CHARACTERIZATION OF APOBEC3 REPERTOIRES AND EFFECTS ON RETROVIRAL REPLICATION

APOBEC3 (A3) enzymes are a family of intrinsic retroviral restriction factors that are coordinately expressed in CD4+ T-cells and function to restrict retroviral replication. In the case of HIV-1, this primarily occurs in the absence of the HIV-1 protein, viral infectivity factor (Vif). Vif induces the polyubiquitination and degradation of A3 enzymes using the host proteasome pathway. The A3 enzyme family are single stranded DNA deaminases, capable of deaminating cytosine to form promutagenic uracil on single-stranded DNA substrates. In humans there are seven paralogs that comprise the A3 family, including: A3A, A3B, A3C, A3D, A3F, A3G, and A3H. However, only four paralogs have been associated with inhibition of retroviral replication in a large majority of the human populations, including: A3D, A3F, A3G and A3H. These four key enzymes have well-characterized repertoires of single nucleotide polymorphisms (SNPs) that affect both cellular stability and deaminase activity. Each repertoire of polymorphisms is variable in human populations and is influenced by human ancestry. Here we have undertaken a Saskatchewan-based mixed population study examining human genotypes for common APOBEC3H (A3H), APOBEC3F (A3F) and APOBEC3G (A3G) SNPs and how combinatory SNP variations affect HIV-1 replication. Using Sanger-sequencing based genotyping we were able to isolate dominant polymorphisms in our population. We found that the dominant genotypes in our population were heterozygous for A3F 231V and 231I SNPs, homozygous for the A3G 186H SNP, and predominantly inactive A3H SNP profiles. We tested the dominant A3F and A3G polymorphisms for cellular stability, viral packaging and restriction capacity, and found that the A3F 231V SNP provided the most robust restriction response when co-expressed with both A3G and A3F 231I. We also show that A3F 231V can hetero-oligomerize with both A3G and A3F 231I polymorphic variants, resulting in greater cellular stabilization and steady-state cellular expression in the presence and absence of HIV-1 Vif. The observed rise in cellular stability and virion packaging when A3F 231V was co-expressed with A3F 231 and A3G had a positive correlation with HIV-1 replication restriction efficiency. The various states of oligomerization between co-expressed A3 enzymes demonstrates that whole genotype analysis of A3 repertoires is essential in accurately understanding host-pathogen interactions on a population level

Biochemical And Cellular Studies Of Apobec3 Family Dna-Cytosine Deaminases

The AID/APOBEC family of enzymes deaminate cytosines in single-stranded DNA to uracils leading to base substitutions and strand breaks. Members of APOBEC3 family in humans are induced by cytokines produced during the body\u27s inflammatory response to infections and provide innate immunity against viruses. However, there is emerging consensus that these enzymes can cause mutations in the cellular genome depending on the physiological state of the cell and the phase of the cell cycle they are expressed. Since aberrant expression of APOBEC3B was recently identified as a possible source of cancer, we initiated a study to determine the maximally active catalytic domain of this enzyme. The results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations. Next we determined the effects of inflammatory stimuli on APOBEC3 expression and its consequences of genomic DNA by using a phorbol ester to induce APOBEC3A expression in a keratinocyte cell line. The inability of APOBEC3A to cause uracil accumulation in the genome was due to the reversible cessation of DNA replication and absence of single-stranded DNA substrates. We identify the reversible arrest in cell growth as an attractive mechanism to protect the human genome against APOBEC enzymes