Excessive backfat of sows at 109 d of gestation induces lipotoxic placental environment and is associated with declining reproductive performance.

This study investigated the influence of sow backfat thickness at 109 d of gestation on sow and piglet performance. Data from 846 farrowing multiparous Yorkshire sows with parity from 3 to 5 were collected from a pig breeding farm. Sows were divided into six groups based on backfat thickness (≤16, 17-18, 19-20, 21-22, 23-24, and ≥25 mm) at 109 d of gestation. The evaluation of reproductive performance included the litter size, litter weight at birth and at weaning of 21 d, weight of placenta at parturition, placental efficiency, and sow daily feed intake of lactation. Parameters related to plasma lipids and the placental-lipid concentration were measured. Data were analyzed to determine the relationships among backfat thickness, placental lipids, and piglet performance. No differences were observed in the number of piglets born, born alive, after cross-foster, and at weaning among groups (P > 0.05). The litter weight at birth and weaning, piglet birth weight, weaning weight, placental efficiency, and the number and percentage of piglets born with weight of <800 g showed a significantly quadratic effect of the backfat thickness (P < 0.05). During lactation, sow daily feed intake linearly decreased with increased backfat thickness at 109 d of gestation (P < 0.05). Although triglycerides and low-density lipoprotein cholesterol (LDL-C) showed no significant difference, cholesterol and high-density lipoprotein cholesterol (HDL-C) and free fatty acid (FFA) concentrations significantly increased (P < 0.05) in both maternal and umbilical cord blood with increased backfat thickness of sow. Placental-lipid concentrations also significantly increased (P < 0.05) with increased backfat thickness. Moreover, backfat thickness and placental-lipid concentration were positively correlated with the number of piglets weighing <800 g (P < 0.01) but negatively correlated with birth weight, litter birth weight, and piglet weaned weight (P < 0.01). In conclusion, backfat thickness of sow at end of gestation correlates with birth and weaning weight of piglets. Placental ectopic lipid accumulation-induced lipotoxicity is likely responsible for such correlation.

Creative deaminases, self-inflicted damage, and genome evolution.

Organisms minimize genetic damage through complex pathways of DNA repair. Yet a gene family--the AID/APOBECs--has evolved in vertebrates with the sole purpose of producing targeted damage in DNA/RNA molecules through cytosine deamination. They likely originated from deaminases involved in A>I editing in tRNAs. AID, the archetypal AID/APOBEC, is the trigger of the somatic diversification processes of the antibody genes. Its homologs may have been associated with the immune system even before the evolution of the antibody genes. The APOBEC3s, arising from duplication of AID, are involved in the restriction of exogenous/endogenous threats such as retroviruses and mobile elements. Another family member, APOBEC1, has (re)acquired the ability to target RNA while maintaining its ability to act on DNA. The AID/APOBECs have shaped the evolution of vertebrate genomes, but their ability to mutate nucleic acids is a double-edged sword: AID is a key player in lymphoproliferative diseases by triggering mutations and chromosomal translocations in B cells, and there is increasing evidence suggesting that other AID/APOBECs could be involved in cancer development as well.

Induction of resistance to the lipophilic cytarabine prodrug elacytarabine (CP-4055) in CEM leukemic cells.

The deoxynucleoside analogs cytarabine (Ara-C) and gemcitabine (dFdC) are widely used in the treatment of cancer. Due to their hydrophilic nature they need the equilibrative (hENT) and concentrative (hCNT) nucleoside transporters to enter the cell. To bypass drug resistance due to decreased uptake, lipophilic 5'elaidic acid esters were synthesized, elacytarabine (CP-4055, from ara-C) and CP-4126 (from gemcitabine), which are currently in clinical development for solid and hematological tumors. We investigated whether resistance can be induced in vitro, and treated the CEM leukemic cell line with weekly increasing elacytarabine concentrations, up to 0.28 microM (10 times IC(50)). The IC(50) of the resistant CEM/CP-4055 was 35 microM, about 1,000 times that of the wildtype CEM, and comparable to that of CEM/dCK- (deoxycytidine kinase deficient) (22 microM). CEM/CP-4055 was also cross-resistant to Ara-C, gemcitabine and CP-4126 (28 and 33 microM, respectively). A low level of mRNA dCK was observed, and similar to CEM/dCK-, CEM/CP-4055 did not accumulate Ara-CTP after exposure to Ara-C or elacytarabine, which is consistent with a deficiency in dCK. In conclusion, elacytarabine induced resistance similar to Ara-C. This resistance was caused by downregulation of dCK.

Is the resistance of gemcitabine for pancreatic cancer settled only by overexpression of deoxycytidine kinase?

The prognosis of pancreatic cancer remains poor, and the standard first-line chemotherapy with gemcitabine (GEM) has a response rate of less than 20%. Since expression of deoxycytidine kinase (dCK) seems important for improvement of GEM sensitivity, overexpression of dCK was investigated using pancreatic cancer cell lines (Panc-1, MIAPaCa-2 and BxPC-3). dCK gene was introduced into the cell lines by retrovirus and changes in IC50 were examined. Sensitivity of two pancreatic cancer cell lines to GEM elevated dramatically in comparison with control cells, but change of sensitivity remained at 1.8 times in BxPC-3. Since addition of tetrahydro uridine (THU), an inhibitor of deoxycytidine deaminase (CDA), increased the sensitivity 54-fold, overexpression of CDA seems to be the mechanism for improvement of the sensitivity. In conclusion, dCK is a key enzyme of GEM, but resistance of GEM is not improved in all pancreatic cancer cells by overexpression of dCK. Combination treatment based on expression of GEM metabolism-related gene may become an effective therapy in the future.

Review of recent studies on resistance to cytotoxic deoxynucleoside analogues.

Cytotoxic deoxynucleoside analogues are widely used in the treatment of haematological malignancies and solid tumours. Their metabolism and mechanisms of action are relatively well known, but with ongoing technological development, a continuous flow of scientific data is constantly adding new knowledge to this field. Thus, what was already a well-developed area some years ago has continued its expansion and become a better understood part of medical sciences. In order to keep abreast of the latest advances on cellular and clinical resistance to deoxynucleoside analogues, we have reviewed the recent literature and provide here an update on the subject. We have particularly focused on changes in gene products involved in the metabolic pathway of these drugs, such as membrane transporters, kinases, deaminases and 5'-nucleotidases. We also gave an overview on the chemical and biological development of modified deoxynucleoside analogues such as conjugates and pronucleotides.

The intrinsic antiretroviral factor APOBEC3B contains two enzymatically active cytidine deaminase domains.

The mammalian APOBEC3 proteins are cytidine deaminases that function as inhibitors of retrovirus replication and retrotransposon mobility. An issue that has remained controversial is whether the editing of deoxycytidine residues to deoxyuridine is necessary and sufficient for this inhibition or whether APOBEC3 proteins also exert a second, distinct inhibitory mechanism. Here, we present an analysis of the ability of mutants of APOBEC3G and APOBEC3B, both of which contain two consensus cytidine deaminase active sites, to inhibit the replication of human immunodeficiency virus. Our data confirm that APOBEC3G only contains a single, carboxy-terminal active site but, surprisingly, reveal that both cytidine deaminase consensus sequences in APOBEC3B are enzymatically active. Enzymatically inactive mutant forms of APOBEC3G and APOBEC3B were found to retain the ability to inhibit the infectivity of HIV-1 virions produced in their presence by approximately 4-fold and approximately 8-fold, respectively. While this inhibition was significantly less than the level seen with wild-type forms of A3G or A3B, these data, nevertheless argue that the inhibition of HIV-1 by APOBEC3 proteins is at least partly independent of DNA editing.

Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study.

We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was far much higher than those of an APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. We finally monitored the incorporation of APOBEC3G into virions produced in A3G-C1. APOBEC3G was easily detected in progeny viral particles upon transfection of vif-minus proviral clone but not of wild type. These results indicated that our new A3G-C1 cell line is eminently useful for various studies on the interaction of human APOBEC3G and HIV-1 Vif.

Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H.

APOBEC3G (A3G) is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older "mature" A3G already residing in high-molecular-mass RNA-protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme.

Biochemical differentiation of APOBEC3F and APOBEC3G proteins associated with HIV-1 life cycle.

APOBEC3G and APOBEC3F are cytidine deaminase with duplicative cytidine deaminase motifs that restrict HIV-1 replication by catalyzing C-to-U transitions on nascent viral cDNA. Despite 60% protein sequence similarity, APOBEC3F and APOBEC3G have a different target consensus sequence for editing, and importantly, APOBEC3G has 10-fold higher anti-HIV activity than APOBEC3F. Thus, APOBEC3F and APOBEC3G may have distinctive characteristics that account for their functional differences. Here, we have biochemically characterized human APOBEC3F and APOBEC3G protein complexes as a function of the HIV-1 life cycle. APOBEC3G was previously shown to form RNase-sensitive, enzymatically inactive, high molecular mass complexes in immortalized cells, which are converted into enzymatically active, low molecular mass complexes by RNase digestion. We found that APOBEC3F also formed high molecular mass complexes in these cells, but these complexes were resistant to RNase treatment. Further, the N-terminal half determined RNase sensitivity and was necessary for the high molecular mass complex assembly of APOBEC3G but not APOBEC3F. Unlike APOBEC3F, APOBEC3G strongly interacted with cellular proteins via disulfide bonds. Inside virions, both APOBEC3F and APOBEC3G were found in viral cores, but APOBEC3G was associated with low molecular mass, whereas APOBEC3F was still retained in high molecular mass complexes. After cell entry, both APOBEC3F and APOBEC3G were localized in low molecular mass complexes associated with viral reverse transcriptional machinery. These results demonstrate that APOBEC3F and APOBEC3G complexes undergo dynamic conversion during HIV-1 infection and also reveal biochemical differences that likely determine their different anti-HIV-1 activity.

Distinct patterns of cytokine regulation of APOBEC3G expression and activity in primary lymphocytes, macrophages, and dendritic cells.

Human APOBEC3G (A3G), a deoxycytidine deaminase, is a broadly acting antiretroviral factor expressed in a variety of cells. Mitogen activation of CD4 T cells enhances A3G expression and leads to recruitment of low molecular mass (LMM) A3G, which functions as a post-entry human immunodeficiency virus (HIV) restriction factor, into enzymatically inactive, high molecular mass (HMM) RNA-protein complexes that include Staufen RNA-transporting granules. We now report that interleukin-2 (IL-2), IL-15 and, to a lesser extent, IL-7 enhance the expression of A3G in peripheral blood lymphocytes and that this effect is blocked by inhibitors of the JAK and MAPK signaling pathways. In mixed cultures of CD4+ T cells containing either HMM or LMM A3G, HIV preferentially infected cells containing HMM A3G. A3G shifted into a HMM complex when IL-2, -7, or -15 was added to resting T cells, likely explaining how cytokine treatment renders resting CD4+ T cells permissive to HIV infection. Similarly, poly(I:C)/tumor necrosis factor-alpha-induced maturation of dendritic cells was associated with a sharp increase in A3G expression; however, this induction led to the accumulation of LMM A3G. Together, these results highlight the distinct inductive effects of select cytokines on A3G gene expression and A3G complex assembly that occur in natural cellular targets of HIV infection.

Cellular interactions of virion infectivity factor (Vif) as potential therapeutic targets: APOBEC3G and more?

Vif is an HIV accessory protein whose primary function is to negate the action of APOBEC3G, a naturally occurring cellular inhibitor of HIV replication. Vif acts by binding to APOBEC3G, inducing its protein degradation within infected cells and reducing its levels in progeny virions. Interventions that interfere with the Vif-APOBEC3G interaction, raise intracellular or virion associated levels of APOBEC3G, or reduce intracellular levels of Vif, all could hold promise as potential therapeutic approaches aimed at enhancing the cells innate antiviral activity. Levels of APOBEC3G might be increased or Vif levels decreased, by strategies targeting protein synthesis, protein degradation or cellular localisation and function, and properties of APOBEC3G and Vif relevant to these strategies are discussed. Recent data have suggested that Vif may have other mechanisms of action apart from the above activities against APOBEC3G, including effects against other anti-viral mechanisms independent of APOBEC3G cytidine deaminase activity. In addition to interaction with APOBEC3G, Vif may have other accessory functions, which are discussed in relation to potential therapies that may affect multiple stages of the HIV life cycle. Future development of strategies that combine enhancement of APBOEC3G functional with inhibition of multiple Vif functions may become useful tools for HIV therapy.

APOBEC3G/3F mediates intrinsic resistance of monocyte-derived dendritic cells to HIV-1 infection.

HIV-1 infects immature dendritic cells (iDCs), but infection is inefficient compared with activated CD4+ T cells and only involves a small subset of iDCs. We analyzed whether this could be attributed to specific cellular restrictions during the viral life cycle. To study env-independent restriction to HIV-1 infection, we used a single-round infection assay with HIV-1 pseudotyped with vesicular stomatitis virus G protein (HIV-VSVG). Small interfering RNA-mediated depletion of APOBEC3G/3F (A3G/3F), but not TRIM5alpha, enhanced HIV-1 infection of iDCs, indicating that A3G/3F controls the sensitivity of iDCs to HIV-1 infection. Furthermore, sequences of HIV reverse transcripts revealed G-to-A hypermutation of HIV genomes during iDC infection, demonstrating A3G/3F cytidine deaminase activity in iDCs. When we separated the fraction of iDCs that was susceptible to HIV, we found the cells to be deficient in A3G messenger RNA and protein. We also noted that during DC maturation, which further reduces susceptibility to infection, A3G levels increased. These findings highlight a role for A3G/3F in explaining the resistance of most DCs to HIV-1 infection, as well as the susceptibility of a fraction of iDCs. An increase in the A3G/3F-mediated intrinsic resistance of iDCs could result in a block of HIV infection at its mucosal point of entry.

The anti-HIV-1 editing enzyme APOBEC3G binds HIV-1 RNA and messenger RNAs that shuttle between polysomes and stress granules.

Deoxycytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family) have RNA-binding motifs, invade assembling human immunodeficiency virus (HIV-1), and hypermutate reverse transcripts. Antagonistically, HIV-1 viral infectivity factor degrades these enzymes. A3G is enzymatically inhibited by binding RNA within an unidentified large cytosolic ribonucleoprotein, implying that RNA degradation during reverse transcription may activate intravirion A3G at the necessary moment. We purified a biologically active tandem affinity-tagged A3G from human HEK293T cells. Mass spectrometry and coimmunoprecipitation from HEK293T and T lymphocyte extracts identified many RNA-binding proteins specifically associated with A3G and A3F, including poly(A)-binding proteins (PABPs), YB-1, Ro-La, RNA helicases, ribosomal proteins, and Staufen1. Most strikingly, nearly all A3G-associated proteins were known to bind exclusively or intermittently to translating and/or dormant mRNAs. Accordingly, A3G in HEK293T and T lymphocyte extracts was almost completely in A3G-mRNA-PABP complexes that shifted reversibly between polysomes and dormant pools in response to translational inhibitors. For example arsenite, which inhibits 5'-cap-dependent translational initiation, shifted mRNA-A3G-PABP from polysomes into stress granules in a manner that was blocked and reversed by the elongation inhibitor cycloheximide. Immunofluorescence microscopy showed A3G-mRNA-PABP stress granules only partially overlapping with Staufen1. A3G coimmunoprecipitated HIV-1 RNA and many mRNAs. Ribonuclease released nearly all A3G-associated proteins, including A3G homo-oligomers and A3G-A3F hetero-oligomers, but the viral infectivity factor remained bound. Many proteins and RNAs associated with A3G are excluded from A3G-containing virions, implying that A3G competitively partitions into virions based on affinity for HIV-1 RNA.

HIV pathogenesis: knowledge gained after two decades of research.

Great progress has been made in our understanding of HIV since its initial discovery about 20 years ago. The ability of HIV to infect CD4+ lymphocytes and a wide variety of other cells in the body is appreciated, as is its role in immunologic, gastrointestinal, and brain disorders. HIV enters cells via the CD4 molecule, chemokine co-receptors (CXCR4, CCR5), and other cell-surface proteins. Several accessory virus-associated genes (e.g., Rev, Tat, Nef) have uncovered unique pathways that can also be observed in normal cells. Recently, the discovery of natural cellular resistant factors (APOBEC3G and TRIM5a) has provided avenues for novel antiviral therapies. Studies of long-term survivors have given insight into immune responses that control HIV and can prevent infection. Neutralizing antibodies and CD8+ cell cytotoxic responses, as well as plasmacytoid dendritic cells and CD8+ cell non-cytotoxic antiviral responses, are adaptive and innate immune activities mediating this anti-HIV effect. HIV vaccine studies have indicated that conventional approaches do not work against this integrated intracellular parasite. While much has been learned about HIV, more details are needed about its infection cycle and its pathologic effects in the body. The past 20 years have yielded important information on HIV/AIDS that should lead to effective anti-HIV therapies and a vaccine.

Monomeric APOBEC3G is catalytically active and has antiviral activity.

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.

Comparative analysis of the antiretroviral activity of APOBEC3G and APOBEC3F from primates.

APOBEC3G and APOBEC3F exhibit antiretroviral activity primarily as a consequence of their ability to deaminate cytidines in retroviral DNA. Here, we compare the properties of APOBEC3F and APOBEC3G from human, macaque, and African green monkey (AGM). While all APOBEC proteins tested exhibited anti-HIV-1 activity, human APOBEC3F was, surprisingly, 10- to 50-fold less potent than human APOBEC3G. However, similar discrepancies in antiviral potency were not found when pairs of proteins from macaque and AGM were compared. Intrinsic differences in the ability of each APOBEC protein to induce hypermutation, rather than differences in packaging efficiency, partially accounted for variable antiretroviral activity. Each of four primate lentivirus Vif proteins reduced human and AGM APOBEC3F expression and antiviral activity, but all were only partially effective and species-specific effects were relatively minor. Overall, highly efficient and species-specific neutralization of APOBEC3G, and less efficient neutralization of APOBEC3F, appears to be a general property of Vif proteins.

Anti-viral protein APOBEC3G is induced by interferon-alpha stimulation in human hepatocytes.

Apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) is a potent inhibitor of infection by a wide range of retroviruses. Although recent reports have suggested that human APOBEC3G exerts antiviral activity against hepatitis B virus, APOBEC3G expression is normally low in the human liver. To clarify the role of APOBEC3G in cellular defenses against hepatitis viruses, the regulation of the APOBEC3G expression was investigated in human hepatocytes. Endogenous transcripts of nine APOBEC family members were barely detectable in quiescent liver cells. However, APOBEC3G was significantly up-regulated in response to interferon-alpha (IFN-alpha) stimulation in HepG2, Huh-7, and primary human hepatocytes. IFN regulatory factor elements that are important for IFN-inducible promoter activity were identified 5' upstream from the human APOBEC3G gene. Our findings provided the first evidence showing that APOBEC3G is induced by IFN stimulation in human hepatocytes and thus could be involved in host defense mechanisms directed against hepatitis viruses.

Uracil DNA glycosylase is dispensable for human immunodeficiency virus type 1 replication and does not contribute to the antiviral effects of the cytidine deaminase Apobec3G.

It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous uracils in the viral DNA during virus replication. We used a natural human UNG-/- cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G.