Human Tribbles 3 Protects Nuclear DNA from Cytidine Deamination by APOBEC3A

The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.     

APOBEC3A通过脱氨基作用抑制HBV复制

目的:研究APOBEC3A抑制HBV复制的分子机制。方法:首先在肝癌细胞HuH7中过表达APOBEC3A,通过MTT法检测了APOBEC3A对细胞毒性的影响;通过免疫荧光检测了APOBEC3A在细胞中的定位,通过IP试验进一步证实了APOBEC3A与病毒颗粒的相互作用;并通过ELISA,特异性荧光定量PCR检测了HBV复制的参数包括上清中HBsAg,病毒核心颗粒中HBV DNA以及核内的cccDNA的表达水平;最后通过3D PCR方法分析了核心颗粒中HBV DNA脱氨基作用。结果:过表达APOBEC3A对HuH7细胞毒性没有显著性差异;APOBEC3A主要位于细胞核,但APOBEC3A可以与病毒颗粒结合,在逆转录环节对HBV复制发生抑制作用;共转染HBV复制质粒和APOBEC3A表达质粒后,细胞上清中HBsAg,核心颗粒中的HBV DNA以及核内的cccDNA均显著下降;最后通过3D PCR和克隆测序表明核心颗粒中的HBV DNA负链发生了大量的G-A突变,同时正链也发生了较多的C-T突变。结论:APOBEC3A可与病毒颗粒结合,在HBV复制的逆转录环节可作用于HBV单链,发生脱氨基作用,从而抑制HBV的复制。     

Methylcytosine and Normal Cytosine Deamination by the Foreign DNA Restriction Enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.     

116 Deamination of both methyl- and normal-cytosine by the foreign DNA restriction enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.     

Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.     

Efficient Deamination of 5-Methylcytidine and 5-Substituted Cytidine Residues in DNA by Human APOBEC3A Cytidine Deaminase

Deamination of 5-methylcytidine (5MeC) in DNA results in a G:T mismatch unlike cytidine (C) deamination which gives rise to a G:U pair. Deamination of C was generally considered to arise spontaneously. It is now clear that human APOBEC3A (A3A), a polynucleotide cytidine deaminase (PCD) with specificity for single stranded DNA, can extensively deaminate human nuclear DNA. It is shown here that A3A among all human PCDs can deaminate 5-methylcytidine in a variety of single stranded DNA substrates both in vitro and in transfected cells almost as efficiently as cytidine itself. This ability of A3A to accommodate 5-methyl moiety extends to other small and physiologically relevant substituted cytidine bases such as 5-hydroxy and 5-bromocytidine. As 5MeCpG deamination hotspots characterize many genes associated with cancer it is plausible that A3A is a major player in the onset of cancer.     

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3''→5'' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5''-end is deaminated more effectively than one less close to the 5''-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2''s activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3''→5'' polarity. Examination of the effects of different salt concentrations on the 3''→5'' polarity indicated that the higher the salt concentration, the less prominent the 3''→5'' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.     

The activity spectrum of Vif from multiple HIV-1 subtypes against APOBEC3G, APOBEC3F, and APOBEC3H

The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the proteasome. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3 proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and A3H haplotype II (hapII). We show that most Vif alleles neutralize A3G and A3F efficiently but display differences with respect to the inhibition of A3H hapII. The majority of non-subtype B Vif alleles tested presented some activity against A3H hapII, with two subtype F Vif variants being highly effective in counteracting A3H hapII. The residues required for activity were mapped to two residues in the amino-terminal region of Vif (positions 39F and 48H). Coimmunoprecipitations showed that these two amino acids were necessary for association of Vif with A3H hapII. These findings suggest that the A3H hapII binding site in Vif is distinct from the regions important for A3G and A3F recognition and that it requires specific amino acids at positions 39 and 48. The differential Vif activity spectra, especially against A3H hapII, suggest adaptation to APOBEC3 repertoires representative of different human ancestries. Phenotypic assessment of anti-APOBEC3 activity of Vif variants against several cytidine deaminases will help reveal the requirement for successful replication in vivo and ultimately point to interventions targeting the Vif-APOBEC3 interface.     

14-3-3 Protects against stress-induced apoptosis

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death''. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.     

Caspase-3 Protects Stressed Organs against Cell Death

The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response.     

Mannitol Protects against Oxidation by Hydroxyl Radicals

Hydroxyl radicals may be responsible for oxidative damage during drought or chilling stress. We have shown that the presence of mannitol in chloroplasts can protect plants against oxidative damage by hydroxyl radicals (B. Shen, R.G. Jensen, H.J. Bohnert [1997] Plant Physiol 113: 1177-1183). Here we identify one of the target enzymes that may be protected by mannitol. Isolated thylakoids in the presence of physiological concentrations of Fe2+ generated hydroxyl radicals that were detected by the conversion of phenylalanine into tyrosine. The activity of phosphoribulokinase (PRK), a thiol-regulated enzyme of the Calvin cycle, was reduced by 65% in illuminated thylakoids producing hydroxyl radicals. Mannitol (125 mM) and sodium formate (15 mM), both hydroxyl radical scavengers, and catalase (3000 units mL-1) prevented loss of PRK activity. In contrast, superoxide dismutase (300 units mL-1) and glycine betaine (125 mM) were not effective in protecting PRK against oxidative inactivation. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity was not affected by hydroxyl radicals. We suggest that the stress-protective role of mannitol may be to shield susceptible thiol-regulated enzymes like PRK plus thioredoxin, ferredoxin, and glutathione from inactivation by hydroxyl radicals in plants.     

Remarkable Lethal G-to-A Mutations in vif-Proficient HIV-1 Provirus by Individual APOBEC3 Proteins in Humanized Mice

Genomic hypermutation of RNA viruses, including human immunodeficiency virus type 1 (HIV-1), can be provoked by intrinsic and extrinsic pressures, which lead to the inhibition of viral replication and/or the progression of viral diversity. Human APOBEC3G was identified as an HIV-1 restriction factor, which edits nascent HIV-1 DNA by inducing G-to-A hypermutations and debilitates the infectivity of vif-deficient HIV-1. On the other hand, HIV-1 Vif protein has the robust potential to degrade APOBEC3G protein. Although subsequent investigations have revealed that lines of APOBEC3 family proteins have the capacity to mutate HIV-1 DNA, it remains unclear whether these endogenous APOBEC3s, including APOBEC3G, contribute to mutations of vif-proficient HIV-1 provirus in vivo and, if so, what is the significance of these mutations. In this study, we use a human hematopoietic stem cell-transplanted humanized mouse (NOG-hCD34 mouse) model and demonstrate the predominant accumulation of G-to-A mutations in vif-proficient HIV-1 provirus displaying characteristics of APOBEC3-mediated mutagenesis. Notably, the APOBEC3-associated G-to-A mutation of HIV-1 DNA that leads to the termination of translation was significantly observed. We further provide a novel insight suggesting that HIV-1 G-to-A hypermutation is independently induced by individual APOBEC3 proteins. In contrast to the prominent mutation in intracellular proviral DNA, viral RNA in plasma possessed fewer G-to-A mutations. Taken together, these results provide the evidence indicating that endogenous APOBEC3s are associated with G-to-A mutation of HIV-1 provirus in vivo, which can result in the abrogation of HIV-1 infection.Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 [A3]) family proteins are potent mutators of a broad spectrum of retroviruses, including human immunodeficiency virus type 1 (HIV-1) (4, 5, 13, 16, 29, 61). A3s are cellular cytidine deaminases that convert C in the viral minus-strand cDNA to U, resulting in the alteration of G to A in the nascent proviral DNA. Several A3 proteins are incorporated into progeny virions and mutate viral cDNA in the invaded cells, which is thought to result in the inhibition of viral replication (4, 5, 13, 16, 29, 46, 61). On the other hand, an HIV-1 accessory protein, viral infectivity factor (Vif), has the ability to counteract the incorporation of certain A3 proteins such as A3G and A3F into progeny virions by degrading these proteins through the proteasome-dependent pathway (31, 45, 47, 50). Lines of in vitro investigations have elucidated the mechanisms of G-to-A hypermutation of HIV-1 DNA mediated by A3s and the counteracting ability of Vif against A3s, which have shed light on the relevance of host-retrovirus interaction (4, 5, 21, 59, 60). Nevertheless, the physiological balance between intrinsic A3s and Vif in vivo is poorly understood, and the significance of A3-mediated mutagenesis for HIV-1 replication in vivo remains unresolved.In order to investigate the dynamics of human-specific pathogens in vivo, we have recently constructed a humanized mouse (NOG-hCD34 mouse) model by xenotransplanting human CD34⁺ hematopoietic stem cells into an immunodeficient NOD/SCID/IL-2R-γ^null (NOG) mouse (15, 34). In the humanized mice, human leukocytes, including human CD4⁺ T cells, are successfully differentiated de novo and are stably and longitudinally maintained for more than 1 year (15, 34). By utilizing the humanized mice, we have established a novel animal model for HIV-1 infection (34). Our humanized mice are capable of supporting persistent replication of CCR5-tropic HIV-1 for more than 7 months and mirror the characteristics of HIV-1 pathogenesis, such as the depletion of memory CD4⁺ T cells in the periphery and the preferential infection of effector memory T cells (34).Recently, Ince et al. reported the significance of HIV-1 mutation and its influence on HIV-1 expansion by using a humanized mouse model system (14). In that paper, however, the authors particularly focused on the diversity of the HIV-1 env gene, and therefore, the involvement and the significance of A3-associated mutagenesis in HIV-1 expansion in vivo remain unclear.In this study, by using the humanized mouse (NOG-hCD34 mouse) model, we show that G-to-A mutation of vif-proficient HIV-1 provirus exhibiting the characteristics of A3-mediated mutagenesis occurs in vivo. We also provide a novel insight indicating that intrinsic A3-mediated G-to-A mutation is independently caused by endogenous A3 protein. Furthermore, in contrast to the prominent accumulation of G-to-A mutation in provirus, we observed few mutations in virion-associated RNA in plasma. Based on our findings, we discuss the possibility that endogenous A3s have a significant influence on HIV-1 infection in vivo.     

Redirecting Lentiviral Vectors Pseudotyped with Sindbis Virus-Derived Envelope Proteins to DC-SIGN by Modification of N-Linked Glycans of Envelope Proteins

Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.The ultimate goal of gene therapy is cell- and tissue-specific targeted delivery of therapeutic genes. A targeted system increases the therapeutic effects of transgenes at the site of action while reducing adverse effects in surrounding cells and tissues that commonly occur through nonspecific modes of gene delivery (5-8). Gene therapy vectors that can home to specific cells and tissues after intravenous administration, also known as targeting vectors, are ideal for targeted delivery (62). In the past, many attempts have been made to develop targeting viral vectors by using adenovirus, adeno-associated virus, oncoretrovirus, lentivirus, measles virus, and alphavirus (70, 89).To create targeting viral vectors, the natural tropisms of the viruses must first be eliminated and new binding specificities conferred (89). The binding of envelope viruses, such as oncoretrovirus, lentivirus, measles virus, and alphavirus, is mediated by envelope proteins. To redirect the tropisms of these viruses, the original receptor-binding regions of their envelope proteins must be eliminated. We have developed targeting oncoretroviral and lentiviral vectors by pseudotyping them with modified Sindbis virus envelope proteins and by mutating the receptor-binding regions of the envelope proteins, thereby reducing the nonspecific transduction of untargeted cells (61, 63-66). The mutated regions of the envelope protein originally interact directly with other receptors, including heparan sulfate, laminin receptor, and/or unknown molecules (10, 46, 67, 90). These mutations reduced the nonspecific transduction of the liver and spleen when the vectors were administered intravenously (66). By conjugating the virus with targeting ligands, including antibodies and peptides, the virus can transduce specific cells and tissues both in vitro and in vivo (53, 61, 63-66, 71, 72). These results demonstrated that we can eliminate the natural tropism of the Sindbis virus envelope protein while maintaining its fusion activity.However, the N-glycans of the envelope proteins are still intact and possibly interact with cell surface lectins. DC-SIGN is the best-known cell surface lectin expressed on dendritic cells, certain macrophages, and activated B cells (27, 29, 30).Structural and biochemical studies show flexible modes of DC-SIGN binding to cognate saccharides. The trimannose core unit of high-mannose N-glycans is the primary binding site for DC-SIGN (23), while nonreducing alpha1-2-linked terminal mannose moieties contribute to the high avidity seen when DC-SIGN binds the Man8 or Man9 structures common to many viral envelope glycoproteins (22). DC-SIGN traps a wide variety of viruses and viral vectors (HIV [29, 30], simian immunodeficiency virus [50], human T-cell leukemia virus type 1 [12], measles virus [17, 18], dengue virus [86], feline corona virus [77], herpes simplex virus type 1 [16], human cytomegalovirus [36], human herpesvirus type 8 [76], Ebola virus [1], West Nile virus [15], influenza virus [91], Marburg virus [57], and severe acute respiratory syndrome virus [93]) by binding to the N-glycans of the viruses and viral vectors. Binding of DC-SIGN with virus and viral vectors results in enhanced infection and/or transduction of DC-SIGN-positive cells (cis infection/transduction) and/or neighboring cells (trans infection/transduction).If any targeting vector can be trapped by DC-SIGN, it is necessary to eliminate its binding to DC-SIGN to increase the targeting specificity of the virus in vivo (28, 49, 73). In addition to enhanced infection/transduction, binding to DC-SIGN causes signaling that can activate DC-SIGN-expressing antigen-presenting cells (32, 38). Activation of antigen-presenting cells can lead to adverse effects, including systemic inflammation and immune reactions to viral vectors and their transgene products (7, 8, 32, 59, 88). Therefore, investigation of the interactions between viral vectors and DC-SIGN, identification of N-glycans that mediate binding to DC-SIGN, and elimination of interactions with DC-SIGN are important aspects of reducing adverse effects of vector administration and prolonging transgene expression.The envelope protein of our targeting lentiviral vectors, the Sindbis virus envelope protein, contains four N-linked glycans (9, 48). Sindbis virus can replicate in insect and mammalian cells, which have different types of enzymes to process N-glycans (3). Therefore, the structures of N-glycans differ between the virus produced in insect cells and that produced in mammalian cells (40, 58). The N-glycans of the virus produced in insect cells have either the high-mannose or the paucimannosidic structure. Paucimannosidic structure N-glycans, as well as high-mannose structure N-glycans, have terminal mannose residues, and all N-glycans produced in insect cells are predicted to be able to bind DC-SIGN (Fig. ​(Fig.11 a) (39, 47). On the other hand, two N-glycans of the virus produced in mammalian cells have the high-mannose structure, while two others have the complex structure (40, 58). The two complex structure N-glycans have been shown to be exposed on the surface of the envelope protein, while the two high-mannose structure N-glycans are buried within the center of the trimer of the envelope proteins (74, 94). Therefore, the virus produced in insect cells can access DC-SIGN as its receptor while the virus produced in mammalian cells cannot (47). Because our targeting vectors are produced in mammalian cells, they should not bind DC-SIGN efficiently. However, one group demonstrated that lentiviral vectors pseudotyped with a modified Sindbis virus envelope protein bind to DC-SIGN and target DC-SIGN-positive cells (92), in contrast to the results seen with replication-competent Sindbis virus. Both Sindbis virus and the pseudotyped lentiviral vectors were produced in mammalian cells; Sindbis virus was produced in baby hamster kidney (BHK) cells, chicken embryonic fibroblasts, and hamster fibroblast cells; and the pseudotyped vector was produced in human embryonic kidney fibroblast (293T) cells (69). Because it is known that the N-glycans of the HIV envelope protein produced in lymphocytes have structures different from those produced in macrophages, the different producer cells may account for the differences between the N-glycan structures of the virus and Sindbis virus envelope-pseudotyped lentivectors (54, 55). It is also known that the N-glycan structure of dengue virus can be altered by the presence of viral capsid (35). Thus, the capsid of Sindbis virus and HIV could also affect the structures of the N-glycans of envelope proteins differently.Open in a separate windowFIG. 1.(a) N-glycan structures and processing pathway. All N-glycans are first produced as the high-mannose structure in both mammalian cells and insect cells. In mammalian cells, certain N-glycans are further processed to the complex structure. In insect cells, certain N-glycans are further processed to the paucimannosidic structure. DMNJ inhibits mannosidase I, which is necessary for the formation of the complex structure; thus, all N-glycans have the high-mannose structure when generated in the presence of DMNJ. One representative structure of each N-glycan is shown. Man, mannose; GlcNAc, N-acetylglucosamine; SA, sialic acid; Gal, galactose. (b) Schematic representation of chimeric Sindbis virus envelope proteins. The Sindbis virus envelope protein is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate the E3, E2, 6K, and E1 proteins. E1 and E2 are incorporated into the viral envelope, and E3 and 6K are leader sequences for E2 and E1, respectively. The N-linked glycosylation sites of the envelope proteins are shown. 2.2 is a modified Sindbis virus envelope protein in which the IgG-binding domain of protein A (ZZ) was inserted into the E2 region at aa 70. 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) at aa 70 of the E2 protein. 2.2 ΔE2-196N does not have the N-glycan at E2 aa 196, 2.2 ΔE1-139N does not have the N-glycan at E1 aa 139, and 2.2 ΔE2-196N E1-139N does not have the N-glycans at either E2 aa 196 or E1 aa 139.In this study, we investigated whether our targeting vector binds DC-SIGN. We found that DC-SIGN does not mediate the transduction of our targeting vectors efficiently. The vectors can be redirected to DC-SIGN by modifying the structures of the N-glycans of the envelope proteins by using the mannosidase I inhibitor deoxymannojirimycin (DMNJ) (25, 47, 51).     

Antiviral defense by APOBEC3 family proteins

APOBEC3G is a potent antiretroviral factor, which belongs to the APOBEC superfamily of cytidine deaminases. It deaminates cytidine to uridine in nascent minus-strand viral DNA, inducing G-to-A hypermutation in the plus-strand viral DNA. HIV-1 Vif protein overcomes the antiviral activity of APOBEC3G by targeting it for ubiquitin-dependent degradation. Recent accumulating evidences that other members of APOBEC proteins also show antiviral activity on a wide variety of viruses suggest that APOBEC family proteins play a crucial role in an antiviral defense as an innate immunity. Here, we review recent progress in research on APOBEC3 proteins.