Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

APOBEC3G, a member of an RNA/DNA cytidine deaminase superfamily, has been identified as a cellular inhibitor of HIV-1 infectivity, possibly through the dC to dU deamination of the first minus strand cDNA synthesized during reverse transcription. Virions incorporate APOBEC3G during viral assembly in non-permissive cells, and this incorporation is inhibited by the viral protein Vif. The mechanism of APOBEC3G incorporation into HIV-1 is examined in this report. In the absence of Vif, cytoplasmic APOBEC3G becomes membrane-bound in cells expressing HIV-1 Gag, and its incorporation into Gag viral-like particles (VLPs) is proportional to the amount of APOBEC3G expressed in the cell. The expression of Vif, or mutant Gag unable to bind to membrane, prevents the APOBEC3G association with membrane. HIV-1 Gag alone among viral proteins is sufficient for packaging of APOBEC3G into Gag VLPs, and this incorporation requires the presence of Gag nucleocapsid. The presence of amino acids 104-156 in APOBEC3G, located in the linker region between two zinc coordination motifs, is also required for its incorporation into Gag VLPs. Evidence against an RNA bridge facilitating the Gag/APOBEC3G interaction includes data indicating that 1) the incorporation of APOBEC3G occurs independently of viral genomic RNA, 2) a Gag/APOBEC3G complex is immunoprecipitated from cell lysate after RNase treatment, and 3) the zinc coordination motif, rather than the regions flanking this motif, have been implicated in RNA binding in another family member, APOBEC1.
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PMID:The interaction between HIV-1 Gag and APOBEC3G. 1515 5

In contrast to activated CD4+ T cells, resting human CD4+ T cells circulating in blood are highly resistant to infection with human immunodeficiency virus (HIV). Whether the inability of HIV to infect these resting CD4+ T cells is due to the lack of a key factor, or alternatively reflects the presence of an efficient mechanism for defence against HIV, is not clear. Here we show that the anti-retroviral deoxycytidine deaminase APOBEC3G strongly protects unstimulated peripheral blood CD4+ T cells against HIV-1 infection. In activated CD4+ T cells, cytoplasmic APOBEC3G resides in an enzymatically inactive, high-molecular-mass (HMM) ribonucleoprotein complex that converts to an enzymatically active low-molecular-mass (LMM) form after treatment with RNase. In contrast, LMM APOBEC3G predominates in unstimulated CD4+ T cells, where HIV-1 replication is blocked and reverse transcription is impaired. Mitogen activation induces the recruitment of LMM APOBEC3G into the HMM complex, and this correlates with a sharp increase in permissivity for HIV infection in these stimulated cells. Notably, when APOBEC3G-specific small interfering RNAs are introduced into unstimulated CD4+ T cells, the early replication block encountered by HIV-1 is greatly relieved. Thus, LMM APOBEC3G functions as a potent post-entry restriction factor for HIV-1 in unstimulated CD4+ T cells. Surprisingly, sequencing of the reverse transcripts slowly formed in unstimulated CD4+ T cells reveals only low levels of dG dA hypermutation, raising the possibility that the APOBEC3G-restricting activity may not be strictly dependent on deoxycytidine deamination
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PMID:Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. 2061 46

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.
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PMID:Monomeric APOBEC3G is catalytically active and has antiviral activity. 1664 Dec 60

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.
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PMID:Biochemical differentiation of APOBEC3F and APOBEC3G proteins associated with HIV-1 life cycle. 1714 55

Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function.
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PMID:Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules. 1716 10

Human immunodeficiency virus type 1 (HIV-1) Vif counteracts the antiviral activity of the human cytidine deaminase APOBEC3G (APO3G) by inhibiting its incorporation into virions. This has been attributed to the Vif-induced degradation of APO3G by cytoplasmic proteasomes. We recently demonstrated that although APO3G has a natural tendency to form RNA-dependent homo-multimers, multimerization was not essential for encapsidation into HIV-1 virions or antiviral activity. We now demonstrate that a multimerization-defective APO3G variant (APO3G C97A) is able to assemble into RNase-sensitive high-molecular-mass (HMM) complexes, suggesting that homo-multimerization of APO3G and assembly into HMM complexes are unrelated RNA-dependent processes. Interestingly, APO3G C97A was highly resistant to Vif-induced degradation even though the two proteins were found to interact in coimmunoprecipitation experiments and exhibited partial colocalization in transfected HeLa cells. Surprisingly, encapsidation and antiviral activity of APO3G C97A were both inhibited by Vif despite resistance to degradation. These results demonstrate that targeting of APO3G to proteasome degradation and interference with viral encapsidation are distinct functional properties of Vif.
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PMID:Human immunodeficiency virus type 1 Vif inhibits packaging and antiviral activity of a degradation-resistant APOBEC3G variant. 1752 11

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells.
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PMID:T cells contain an RNase-insensitive inhibitor of APOBEC3G deaminase activity. 1789 23

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.
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PMID:Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation. 1845 46

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or APOBEC3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. However, the cellular function of APOBEC3G remains to be further clarified. It has been reported that APOBEC3s can restrict the mobility of endogenous retroviruses and LTR-retrotransposons, suggesting that they can maintain stability in host genomes. However, APOBEC3G is normally cytoplasmic. Further studies have demonstrated that it is associated with an RNase-sensitive high molecular mass (HMM) and located in processing bodies (P-bodies) of replicating T-cells, indicating that the major cellular function of APOBEC3G seems to be related to P-body-related RNA processing and metabolism. As the function of P-body is closely related to miRNA activity, APOBEC3G could affect the miRNA function. Recent studies have demonstrated that APOBEC3G and its family members counteract miRNA-mediated repression of protein translation. Further, APOBEC3G enhances the association of miRNA-targeted mRNA with polysomes, and facilitates the dissociation of miRNA-targeted mRNA from P-bodies. As such, APOBEC3G regulate the activity of cellular miRNAs. Whether this function is related to its potent antiviral activity remains to be further determined.
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PMID:The inhibitory effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members on the activity of cellular microRNAs. 1984 82

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription.
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PMID:APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element. 2485 35


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