Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha

Mammalian cells express several factors that act in a cell-autonomous manner to inhibit retrovirus replication. Among these are the Friend virus susceptibility factor 1/lentivirus susceptibility factor 1/restriction factor 1 (Ref1) class of restriction factors, which block infection by targeting the capsids of diverse retroviruses. Here we show that lentivirus susceptibility factor 1 and Ref1 are species-specific variants of tripartite interaction motif 5alpha (TRIM5alpha), a cytoplasmic body component recently shown to block HIV-1 infection in rhesus macaque cells, and can indeed block infection by widely divergent retroviruses. Depletion of TRIM5alpha from human cells relieved restriction of N-tropic murine leukemia virus (N-MLV), and expression of human TRIM5alpha in otherwise nonrestricting cells conferred specific resistance to N-MLV infection, indicating that TRIM5alpha is Ref1 or an essential component of Ref1. TRIM5alpha variants from humans, rhesus monkeys, and African green monkeys displayed different but overlapping restriction specificities that were quite accurately predicted by the restriction properties of the cells from which they were derived. All TRIM5alpha variants could inhibit infection by at least two different retroviruses, and African green monkey TRIM5alpha was able to inhibit infection by no less than four divergent retroviruses of human, non-human primate, equine, and murine origin. However, each TRIM5alpha variant was unable to restrict retroviruses isolated from the same species. These data indicate that TRIM5alpha can confer broad innate immunity to retrovirus infection in primate cells and is likely to be an important natural barrier to cross-species retrovirus transmission.     

Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor

The host restriction factor TRIM5alpha mediates species-specific, early blocks to retrovirus infection; susceptibility to these blocks is determined by viral capsid sequences. Here we demonstrate that TRIM5alpha variants from Old World monkeys specifically associate with the HIV type 1 (HIV-1) capsid and that this interaction depends on the TRIM5alpha B30.2 domain. Human and New World monkey TRIM5alpha proteins associated less efficiently with the HIV-1 capsid, accounting for the lack of restriction in cells of these species. After infection, the expression of a restricting TRIM5alpha in the target cells correlated with a decrease in the amount of particulate capsid in the cytosol. In some cases, this loss of particulate capsid was accompanied by a detectable increase in soluble capsid protein. Inhibiting the proteasome did not abrogate restriction. Thus, TRIM5alpha restricts retroviral infection by specifically recognizing the capsid and promoting its rapid, premature disassembly.     

TRIM5 acts as more than a retroviral restriction factor

The retrovirus restriction factor TRIM5α blocks post-entry infection of retroviruses in a species-specific manner. As a cellular E3 ubiquitin ligase, TRIM5α binds to the retroviral capsid lattice in the cytoplasm of an infected cell and accelerates the uncoating process of retroviral capsid, thus providing a potent restriction to HIV-1 and other retrovirus infections. The precise mechanism by which this restriction is imposed remains under scrutiny, and evidence is lacking to link the E3 ubiquitin ligase activity of TRIM5α to its ability to restrict retrovirus infection. In a recent study, Pertel and colleagues have uncovered the link between the two, providing compelling evidence to suggest that following the interaction with the retroviral capsid, TRIM5 triggers an antiviral innate immune response by functioning as a pattern recognition receptor. This unique function of TRIM5 is dependent on its association with the E2 ubiquitin-conjugating enzyme complex UBC13-UEV1A and subsequent activation of the TAK1 kinase complex and downstream genes involved in innate immune responses. These findings have defined a novel function for TRIM5 as a pattern recognition receptor in innate immune recognition and provided valuable mechanistic insight into its role as a retroviral restriction factor. Here we discuss the significance of these new findings in understanding TRIM5-mediated HIV restriction.     

Positive selection of primate TRIM5alpha identifies a critical species-specific retroviral restriction domain

Primate genomes encode a variety of innate immune strategies to defend themselves against retroviruses. One of these, TRIM5alpha, can restrict diverse retroviruses in a species-specific manner. Thus, whereas rhesus TRIM5alpha can strongly restrict HIV-1, human TRIM5alpha only has weak HIV-1 restriction. The biology of TRIM5alpha restriction suggests that it is locked in an antagonistic conflict with the proteins encoding the viral capsid. Such antagonistic interactions frequently result in rapid amino acid replacements at the protein-protein interface, as each genetic entity vies for evolutionary dominance. By analyzing its evolutionary history, we find strong evidence for ancient positive selection in the primate TRIM5alpha gene. This selection is strikingly variable with some of the strongest selection occurring in the human lineage. This history suggests that TRIM5alpha evolution has been driven by antagonistic interactions with a wide variety of viruses and endogenous retroviruses that predate the origin of primate lentiviruses. A 13-aa "patch" in the SPRY protein domain bears a dense concentration of positively selected residues, potentially implicating it as an antiviral interface. By using functional studies of chimeric TRIM5alpha genes, we show that this patch is generally essential for retroviral restriction and is responsible for most of the species-specific antiretroviral restriction activity. Our study highlights the power of evolutionary analyses, in which positive selection identifies not only the age of genetic conflict but also the interaction interface where this conflict plays out.     

Balancing selection and the evolution of functional polymorphism in Old World monkey TRIM5alpha

Retroviral restriction factor TRIM5alpha exhibits a high degree of sequence variation among primate species. It has been proposed that this diversity is the cumulative result of ancient, lineage-specific episodes of positive selection. Here, we describe the contribution of within-species variation to the evolution of TRIM5alpha. Sampling within two geographically distinct Old World monkey species revealed extensive polymorphism, including individual polymorphisms that predate speciation (shared polymorphism). In some instances, alleles were more closely related to orthologues of other species than to one another. Both silent and nonsynonymous changes clustered in two domains. Functional assays revealed consequences of polymorphism, including differential restriction of a small panel of retroviruses by very similar alleles. Together, these features indicate that the primate TRIM5alpha locus has evolved under balancing selection. Except for the MHC there are few, if any, examples of long-term balancing selection in primates. Our results suggest a complex evolutionary scenario, in which fixation of lineage-specific adaptations is superimposed on a subset of critical polymorphisms that predate speciation events and have been maintained by balancing selection for millions of years.     

Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA.

The micrococcal nuclease cleavage of intact nuclear chromatin from African green monkey cells and of the completely deproteinized sequences was studied by using high-resolution analytical and DNA sequencing gels and secondary restriction enzyme analysis. When deproteinized component alpha DNA was used as substrate, not all phosphodiester bonds in the 172-base-pair repeat units were cleaved with equal frequency by the nuclease. A distinct preference for the cleavage of A-T rather than G-C bonds was observed; however, A + T-richness in itself did not confer susceptibility to cleavage by micrococcal nuclease. The results suggested that, in deproteinized DNA, nuclease cleavage at particular dinucleotides may be influenced more by the effect of adjacent sequences than by the composition of the dinucleotide. In contrast to complex cleavage patterns of the deproteinized component alpha DNA which arose because of multiple cleavage sites in the repeat unit, micrococcal nuclease cleaved component alpha nuclear chromatin at one site per nucleosome repeat, near position 126 in the nucleotide sequence. This simple chromatin cleavage pattern is consistent with the discrete nucleosomal structure of component alpha in chromatin and a direct phase relationship between the component alpha DNA sequence repeats and the nucleosome protein structural repeats.     

Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins

Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as alpha 3 chain of type IV collagen and alpha1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis with no effect on migration, whereas human endostatin prevents endothelial cell migration with no effect on proliferation. We demonstrate that human tumstatin binds to alpha v beta 3 integrin in a vitronectin/fibronectin/RGD cyclic peptide independent manner, whereas human endostatin competes with fibronectin/RGD cyclic peptide to bind alpha 5 beta 1 integrin. The activity of human tumstatin is mediated by alpha v beta 3 integrin, whereas the activity of human endostatin is mediated by alpha 5 beta 1 integrin. Additionally, although human tumstatin binding to alpha v beta 3 integrin leads to the inhibition of Cap-dependent translation (protein synthesis) mediated by focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway, human endostatin binding to alpha 5 beta 1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway, with no effect on phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 and Cap-dependent translation. Collectively, such distinct properties of human tumstatin and human endostatin provide the first insight into their diverse antiangiogenic actions and argue for combining them for targeting tumor angiogenesis.     

Stem cell factor modulates avidity of alpha 4 beta 1 and alpha 5 beta 1 integrins expressed on hematopoietic cell lines

Interactions between hematopoietic cells and bone marrow (BM) stroma, composed of extracellular matrix and stromal cells, are crucial for hematopoiesis. Integrins facilitate these interactions by mediating adherence of hematopoiesis. Integrins facilitate these interactions by mediating adherence of hematopoietic cells to both the extracellular matrix and stromal cells. Marrow stromal cells secrete a variety of growth factors, including stem cell factor (SCF). Because treatment with SCF in vivo mobilizes primitive hematopoietic cells from the BM, we investigated the effect of the growth factor SCF of hematopoietic cell adhesion. These studies show that SCF modulates adhesive function in a dose- and time-dependent manner, but does not modulate expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 in the SCF- responsive cell line MO7E. Treatment of MO7E cells with SCF (200 ng/mL) produced a transient increase in adherence to cytokine-activated human umbilical vein endothelial cells (HUVECs) or to vascular cell adhesion molecule 1 (VCAM-1)-transfected Chinese hamster ovary (CHO) cells with peak adhesion at 30 minutes and return to baseline by 60 to 90 minutes. This increase in adhesion was paralleled by increased binding of the beta 1 activation-dependent monoclonal antibody (MoAb) 15/7, as determined by flow cytometry. However, prolonged incubation of MO7E with SCF induced a marked decrease in integrin-mediated adherence, with maximal inhibition by 24 hours. No change in expression of integrins, as determined by flow cytometry, was observed with short- or long-term incubation with SCF. SCF-treated cells were still able to respond to phorbol esters and to the activating beta 1 MoAb 8A2 with increased adherence, but not to the level seen in control cells. This suggests that a subpopulation of expressed alpha 4 beta 1 and alpha 5 beta 1 integrins is disengaged by prolonged incubation with SCF.     

Polymorphisms in tumour necrosis factor alpha, interleukin-10 and transforming growth factor beta1 genes and end-stage liver disease

OBJECTIVE: To determine any relationship between polymorphisms in the genes encoding tumour necrosis factor alpha (TNFalpha), interleukin-10 (IL-10) and transforming growth factor beta1 (TGFbeta1) and end-stage liver disease. METHODS: Whole-blood samples were taken from patients attending the Scottish Liver Transplant Unit with end-stage liver disease (primary biliary cirrhosis, n = 61; alcoholic liver disease, n = 25; primary sclerosing cholangitis, n = 17; viral disease, n = 8; type 1 auto-immune hepatitis, n = 8; acute liver failure, n = 20). DNA was extracted and the polymorphisms at positions TNF -308, IL-10 -1082 and TGFbeta1 +869 and +915 were determined using sequence-specific oligonucleotide probes. Samples were also analysed from normal healthy controls. RESULTS: There was a significant difference between patients with primary sclerosing cholangitis and healthy controls, with 65% of patients (11/17) possessing at least one TNF2 allele (A at position -308) compared with 38% of controls (P = 0.02). Four of the eight patients with auto-immune hepatitis were homozygous for TNF2 while the other four were heterozygous (P = 0.001). No significant difference between controls and patients was seen in polymorphisms for IL-10 or TGFbeta1. No association between genotype and Child's class was found in primary biliary cirrhosis. CONCLUSION: Patients with primary sclerosing cholangitis and auto-immune hepatitis are more likely to possess TNF2 than normal controls. This allele has been associated with an increased production of TNFalpha in vitro and may indicate a predisposition to these inflammatory conditions.     

Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages.

We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation.     

BclAF1 restriction factor is neutralized by proteasomal degradation and microRNA repression during human cytomegalovirus infection

Cell proteins can restrict the replication of viruses. Here, we identify the cellular BclAF1 protein as a human cytomegalovirus restriction factor and describe two independent mechanisms the virus uses to decrease its steady-state levels. Immediately following infection, the viral pp71 and UL35 proteins, which are delivered to cells within virions, direct the proteasomal degradation of BclAF1. Although BclAF1 reaccumulates through the middle stages of infection, it is subsequently down-regulated at late times by miR-UL112-1, a virus-encoded microRNA. In the absence of BclAF1 neutralization, viral gene expression and replication are inhibited. These data identify two temporally and mechanistically distinct functions used by human cytomegalovirus to down-regulate a cellular antiviral protein.     

The story of CGP 40 215: studies on its efficacy and pharmacokinetics in African green monkey infected with Trypanosoma brucei rhodesiense

CGP 40 215 is an inhibitor of S-adenosylmethionine decarboxylase, a key enzyme in trypanosomal polyamine biosynthesis. It is highly active against Trypanosoma brucei rhodesiense and T. b. gambiense in vitro and in the corresponding rodent models, and therefore was a promising candidate for further development as a new drug against human African trypanosomiasis. We conducted initial pharmacokinetic and efficacy studies in African green monkeys: based on two dose-finding studies, an infection-treatment and a pharmacokinetic study in eight monkeys infected with T. b. rhodesiense in the 1st stage of infection. PK analysis revealed curative drug levels in the serum but complete absence of the drug in the cerebrospinal fluid. No adverse effects of the drug were observed, although in rats CGP 40 215 had caused hypotension. The following PK parameters were calculated using a two-compartment model: t1/2=1.8 h, VSS/f=0.4 l/kg, CL/f=3.0 ml/min x kg and AUC=21 900 ng x h/ml. Six of the eight monkeys were cured, one animal relapsed on day 222 and one animal died of unknown reasons, but was aparasitaemic. The study confirmed the curative potential of CGP 40 215 for 1st stage T. b. rhodesiense infection. Unfortunately, it was also found that the compound did not pass the blood-brain barrier, a pre-requisite for cure of 2nd stage (CNS) infection. As the majority of sleeping sickness patients seeking treatment are in the 2nd stage of the disease, further development of the compound was stopped.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor alpha, tumor necrosis factor beta (lymphotoxin), interleukin-1 alpha, and interleukin-1 beta

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor alpha, tumor necrosis factor beta (lymphotoxin), interleukin-1 alpha, and interleukin-1 beta stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.     

The effect of age and calorie restriction on HIF-1-responsive genes in aged liver

Hypoxia inducible factor-1 (HIF-1) regulates transactivation of several genes in response to hypoxia condition. We explore hepatic HIF-1 responsive gene regulation during aging and the age-related changes of the HIF-1 related gene activation in young and old rats. Results indicate that the aging process induces the activation of HIF-1, which is accompanied by increased HIF-1 DNA binding. This increased binding activity is accompanied by the increase of HIF-1-dependent genes, heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), erythropoietin (EPO), and inducible nitric oxide synthase (iNOS), which all showed remarkable up-regulation during aging process. In contrast, the increased HIF-1 related gene expression was effectively blunted by the anti-oxidative action of calorie restriction in aged rat liver. We propose that age-related HIF-1 binding activity may well be influenced by the increased pro-oxidative conditions of aged animals, which up-regulate HIF-1-dependent gene expression.Min Ju Kang and Hyon Jeen Kim Contributed equally to this work     

Rapid selective effects by a growth inhibitor and epidermal growth factor on the incorporation of [35S]methionine into proteins secreted by African green monkey (BSC-1) cells.

Confluent African green monkey kidney (BSC-1) cells secrete a protein (Mr approximately equal to 24,000) that inhibits DNA synthesis and growth of the same cells. Using [35S]methionine to metabolically label proteins, we have found that this growth inhibitor selectively induces the BSC-1 cells to synthesize and secrete another protein with a relative Mr of 48,000 on NaDodSO4/polyacrylamide gels. We have called this protein "inhibitor-inducible protein" (IIP48). The maximal increase in rate of labeling of IIP48 due to treatment with the growth inhibitor averages 12-fold over the control. IIP48 is an N-glycosidically linked glycoprotein, and it is not a major intracellular protein. This protein is maximally induced within 4 to 6 hr of adding the growth inhibitor to the cells. This is an early response of these cells to the growth inhibitor and may represent a primary response to the growth inhibitor. Epidermal growth factor (EGF) increases the rate of labeling of three other secreted proteins (MrS 28,000, 59,000, and 61,000), which we have called "mitogen-inducible proteins" (MIP28, MIP59, and MIP61). The specific effects of both EGF and the growth inhibitor on the secreted levels of these proteins are inhibited if actinomycin D is added with the growth effectors. Thus, RNA synthesis appears necessary for the inductions. EGF and the growth inhibitor induce these secreted proteins by independent and noninteracting pathways.