Cerebellum-specific and age-dependent expression of an endogenous retrovirus with intact coding potential

Background

Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear.

Results

Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.

Conclusions

Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.     

Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts human immunodeficiency virus (HIV)-1 infection in a vif (the virion infectivity factor from HIV)-dependent manner. hA3G from HIV-permissive activated CD4+ T-cells exists as an inactive, high molecular mass (HMM) complex that can be transformed in vitro into an active, low molecular mass (LMM) variant comparable with that of HIV-non-permissive CD4+ T-cells. Here we present low resolution structures of hA3G in HMM and LMM forms determined by small angle x-ray scattering and advanced shape reconstruction methods. The results show that LMM particles have an extended shape, dissimilar to known cytidine deaminases, featuring novel tail-to-tail dimerization. Shape analysis of LMM and HMM structures revealed how symmetric association of dimers could lead to minimal HMM variants. These observations imply that the disruption of cellular HMM particles may require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.     

Apobec 3G Efficiently Reduces Infectivity of the Human Exogenous Gammaretrovirus XMRV

Background

The human exogenous gammaretrovirus XMRV is thought to be implicated in prostate cancer and chronic fatigue syndrome. Besides pressing epidemiologic questions, the elucidation of the tissue and cell tropism of the virus, as well as its sensitivity to retroviral restriction factors is of fundamental importance. The Apobec3 (A3) proteins, a family of cytidine deaminases, are one important group of host proteins that control primary infection and efficient viral spread.

Methodology/Principal Findings

Here we demonstrate that XMRV is resistant to human Apobec 3B, 3C and 3F, while being highly susceptible to the human A3G protein, a factor which is known to confer antiviral activity against most retroviruses. We show that XMRV as well as MoMLV virions package Apobec proteins independent of their specific restriction activity. hA3G was found to be a potent inhibitor of XMRV as well as of MoMLV infectivity. In contrast to MoMLV, XMRV infection can also be partially reduced by low concentrations of mA3. Interestingly, established prostate cancer cell lines, which are highly susceptible to XMRV infection, do not or only weakly express hA3G.

Conclusions

Our findings confirm and extend recently published data that show restriction of XMRV infection by hA3G. The results will be of value to explore which cells are infected with XMRV and efficiently support viral spread in vivo. Furthermore, the observation that XMRV infection can be reduced by mA3 is of interest with regard to the current natural reservoir of XMRV infection.     

Mov10 and APOBEC3G Localization to Processing Bodies Is Not Required for Virion Incorporation and Antiviral Activity

Mov10 and APOBEC3G (A3G) localize to cytoplasmic granules called processing bodies (P bodies), incorporate into human immunodeficiency virus type 1 (HIV-1) virions, and inhibit viral replication. The functional relevance of Mov10/A3G P-body localization to virion incorporation and antiviral activity has not been fully explored. We found that a helicase V mutant of Mov10 exhibits significantly reduced localization to P bodies but still efficiently inhibits viral infectivity via virion incorporation. Disruption of the P bodies by DDX6 knockdown also confirmed Mov10 antiviral activity without P-body localization. In addition, overexpression of SRP19, which binds to 7SL RNA, depleted A3G from P bodies but did not affect its virion incorporation. Sucrose gradient sedimentation assays revealed that the majority of Mov10, A3G, HIV-1 RNA, and Gag formed high-molecular-mass (HMM) complexes that are converted to low-molecular-mass (LMM) complexes after RNase A treatment. In contrast, the P-body markers DCP2, LSM1, eIF4e, DDX6, and AGO1 were in LMM complexes, whereas AGO2, an effector protein of the RNA-induced silencing complex that localizes to P bodies, was present in both LMM and HMM complexes. Depletion of AGO2 indicated that RNA-induced silencing function is required for Mov10''s ability to reduce Gag expression upon overexpression, but not its virion incorporation or effect on virus infectivity. We conclude that the majority of Mov10 and A3G are in HMM complexes, whereas most of the P-body markers are in LMM complexes, and that virion incorporation and the antiviral activities of Mov10 and A3G do not require their localization to P bodies.     

Expression of APOBEC3G/3F and G-to-A hypermutation levels in HIV-1-infected children with different profiles of disease progression

Objective

Increasing evidence has accumulated showing the role of APOBEC3G (A3G) and 3F (A3F) in the control of HIV-1 replication and disease progression in humans. However, very few studies have been conducted in HIV-infected children. Here, we analyzed the levels of A3G and A3F expression and induced G-to-A hypermutation in a group of children with distinct profiles of disease progression.

Methodology/Principal Findings

Perinatally HIV-infected children were classified as progressors or long-term non-progressors according to criteria based on HIV viral load and CD4 T-cell counts over time. A group of uninfected control children were also enrolled in the study. PBMC proviral DNA was assessed for G-to-A hypermutation, whereas A3G and A3F mRNA were isolated and quantified through TaqMan® real-time PCR. No correlation was observed between disease progression and A3G/A3F expression or hypermutation levels. Although all children analyzed showed higher expression levels of A3G compared to A3F (an average fold of 5 times), a surprisingly high A3F-related hypermutation rate was evidenced in the cohort, irrespective of the child''s disease progression profile.

Conclusion

Our results contribute to the current controversy as to whether HIV disease progression is related to A3G/A3F enzymatic activity. To our knowledge, this is the first study analyzing A3G/F expression in HIV-infected children, and it may pave the way to a better understanding of the host factors governing HIV disease in the pediatric setting.     

APOBEC3G subunits self-associate via the C-terminal deaminase domain

Human APOBEC3G (hA3G) is a cytidine deaminase active on HIV single-stranded DNA. Small angle x-ray scattering and molecular envelope restorations predicted a C-terminal dimeric model for RNA-depleted hA3G in solution. Each subunit was elongated, suggesting that individual domains of hA3G are solvent-exposed and therefore may interact with other macromolecules even as isolated substructures. In this study, co-immunoprecipitation and in-cell quenched fluorescence resonance energy transfer assays reveal that hA3G forms RNA-independent oligomers through interactions within its C terminus. Residues 209-336 were necessary and sufficient for homoligomerization. N-terminal domains of hA3G were unable to multimerize but remained functional for Gag and viral infectivity factor (Vif) interactions when expressed apart from the C terminus. These findings corroborate the small angle x-ray scattering structural model and are instructive for development of high throughput screens that target specific domains and their functions to identify HIV/AIDS therapeutics.     

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.     

APOBEC3G/CEM15 (hA3G) mRNA levels associate inversely with human immunodeficiency virus viremia

APOBEC3G/CEM15 (hA3G) is a novel host factor that confers resistance to lentiviral infection under experimental conditions. Human immunodeficiency virus (HIV) type 1, however, produces viral infectivity factor (Vif) that targets hA3G for proteolysis, thereby escaping this defense system. To examine hA3G's contribution to the protection against HIV disease progression in humans, we quantified hA3G mRNA levels in peripheral blood mononuclear cells from 6 HIV-uninfected and 25 HIV-infected subjects; the latter group included 8 long-term nonprogressors (LTNPs) and 17 progressors. None of the HIV-infected subjects were receiving antiretroviral therapy. We found a striking inverse correlation between hA3G mRNA levels and HIV viral loads (P ≤ 0.00009) and a highly significant positive correlation between hA3G mRNA levels and CD4 cell counts (P ≤ 0.00012) in these patients. Furthermore, we discovered that the order of hA3G mRNA levels is LTNPs > HIV-uninfected subjects > progressors.     

Expression analysis of LEDGF/p75, APOBEC3G, TRIM5alpha, and tetherin in a Senegalese cohort of HIV-1-exposed seronegative individuals

Background

HIV-1 replication depends on a delicate balance between cellular co-factors and antiviral restriction factors. Lens epithelium-derived growth factor (LEDGF/p75) benefits HIV, whereas apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and tetherin exert anti-HIV activity. Expression levels of these proteins possibly contribute to HIV-1 resistance in HIV-1-exposed populations.

Methodology/Principal Findings

We used real-time PCR and flow cytometry to study mRNA and protein levels respectively in PBMC and PBMC subsets. We observed significantly reduced LEDGF/p75 protein levels in CD4+ lymphocytes of HIV-1-exposed seronegative subjects relative to healthy controls, whereas we found no differences in APOBEC3G, TRIM5α, or tetherin expression. Untreated HIV-1-infected patients generally expressed higher mRNA and protein levels than healthy controls. Increased tetherin levels, in particular, correlated with markers of disease progression: directly with the viral load and T cell activation and inversely with the CD4 count.

Conclusions/Significance

Our data suggest that reduced LEDGF/p75 levels may play a role in resistance to HIV-1 infection, while increased tetherin levels could be a marker of advanced HIV disease. Host factors that influence HIV-1 infection and disease could be important targets for new antiviral therapies.     

Proteomic identification of ADAM12 as a regulator for TGF-β1-induced differentiation of human mesenchymal stem cells to smooth muscle cells

Background

Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.

Methods and Results

Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.

Conclusions

These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.     

APOBEC3F and APOBEC3G mRNA levels do not correlate with human immunodeficiency virus type 1 plasma viremia or CD4+ T-cell count

APOBEC3F and APOBEC3G (hA3F and hA3G) are part of an innate mechanism of antiretroviral defense. The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif targets both proteins for proteasomal degradation. Using mRNA from peripheral blood mononuclear cells of 92 HIV-infected subjects not taking antiretroviral therapy and 19 HIV-uninfected controls, we found that hA3F (P < 0.001) and hA3G (P = 0.016) mRNA levels were lower in HIV-infected subjects and were positively correlated with one another (P = 0.003). However, we found no correlation in the abundance of either hA3F or hA3G mRNA with either viral load or CD4 counts in HIV-infected subjects.     

Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

Background

Dengue virus is endemic in peninsular Malaysia. The clinical manifestations vary depending on the incubation period of the virus as well as the immunity of the patients. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in Malaysia where the incidence is 3.2%. It has been noted that some G6PD-deficient individuals suffer from more severe clinical presentation of dengue infection. In this study, we aim to investigate the oxidative responses of DENV2-infected monocytes from G6PD-deficient individuals.

Methodology

Monocytes from G6PD-deficient individuals were infected with DENV2 and infection rate, levels of oxidative species, nitric oxide (NO), superoxide anions (O₂ ⁻), and oxidative stress were determined and compared with normal controls.

Principal Findings

Monocytes from G6PD-deficient individuals exhibited significantly higher infection rates compared to normal controls. In an effort to explain the reason for this enhanced susceptibility, we investigated the production of NO and O₂ ⁻ in the monocytes of individuals with G6PD deficiency compared with normal controls. We found that levels of NO and O₂ ⁻ were significantly lower in the DENV-infected monocytes from G6PD-deficient individuals compared with normal controls. Furthermore, the overall oxidative stress in DENV-infected monocytes from G6PD-deficient individuals was significantly higher when compared to normal controls. Correlation studies between DENV-infected cells and oxidative state of monocytes further confirmed these findings.

Conclusions/Significance

Altered redox state of DENV-infected monocytes from G6PD-deficient individuals appears to augment viral replication in these cells. DENV-infected G6PD-deficient individuals may contain higher viral titers, which may be significant in enhanced virus transmission. Furthermore, granulocyte dysfunction and higher viral loads in G6PD-deificient individuals may result in severe form of dengue infection.