The human cytidine deaminase APOBEC3G (A3G) and other APOBEC3 proteins exhibit differential inhibitory activities against diverse endogenous retroelements and retroviruses, including Vif-deficient human immunodeficiency virus type 1. The potential inhibitory activity of human APOBEC proteins against long interspersed element 1 (LINE-1) has not been fully evaluated. Here, we demonstrate inhibition of LINE-1 by multiple human APOBEC3 cytidine deaminases, including previously unreported activity for A3DE and A3G. More ancient members of APOBEC, cytidine deaminases AID and APOBEC2, had no detectable activity against LINE-1. A3A, which did not form high-molecular-mass (HMM) complexes and interacted poorly with P bodies, was the most potent inhibitor of LINE-1. A3A specifically recognizes LINE-1 RNA but not the other cellular RNAs tested. However, in the presence of LINE-1, A3A became associated with HMM complexes containing LINE-1 RNA. The ability of A3A to recognize LINE-1 RNA required its catalytic domain and was important for its LINE-1 suppression. Although the mechanism of LINE-1 restriction did not seem to involve DNA editing, A3A inhibited the accumulation of nascent LINE-1 DNA, suggesting interference with LINE-1 reverse transcription and/or integration or intracellular movement of LINE-1 ribonucleoprotein. Thus, association with P bodies or cellular HMM complexes could not predict the potency of APOBEC3 anti-LINE-1 activities. The catalytic domain of APOBEC3 proteins may be important for proper folding and target factors such as RNA or protein interaction in addition to cytidine deamination.A3G and other APOBEC3 proteins (22) belong to a family of proteins that also includes activation-induced cytidine deaminase (AID), apolipoprotein B-editing catalytic subunit 1 (APOBEC1), and APOBEC2 (1,16,19,35,39,45). These proteins have cytidine deaminase activities that can modify RNA or DNA. A3G was the first APOBEC3 protein to be identified as a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) in the absence of Vif (41). Subsequently, several other human APOBEC3 proteins, including APOBE C3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOB EC3DE (A3DE), and APOBEC3F (A3F), were identified as broad antiviral factors against HIV-1, simian immunodeficiency virus, murine leukemia virus, and various endogenous retroelements (3, 4, 11-14, 24, 26, 37, 40, 48, 49, 53) as well as hepatitis B virus (44).Like AID, which edits single-stranded immunoglobulin gene DNA, APOBEC3 proteins prefer minus-strand retroviral DNAs as targets (1,10,16,19,29,35,39,45,51). In the absence of Vif-induced A3G degradation in virus-producing cells, virion-packaged A3G induces C-to-U mutations in minus-strand viral DNA during reverse transcription (18,25,30,31,43,50,52). Both cytidine deamination-dependent and -independent antiviral functions of APOBEC3 proteins have been reported (2,8,17,20,28,33,36,44).The potential inhibitory activity of certain human APOBEC3 proteins against LINE-1 has been reported. A3A and A3B are potent inhibitors...
Vpr-mediated induction of G 2 cell cycle arrest has been postulated to be important for human immunodeficiency virus type 1 (HIV-1) replication, but the precise role of Vpr in this cell cycle arrest is unclear. In the present study, we have shown that HIV-1 Vpr interacts with damaged DNA binding protein 1 (DDB1) but not its partner DDB2. The interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by short interfering RNA (siRNA) treatment. The Vpr mutant (Q65R) that was defective for DCAF1 interaction also had a defect in DDB1 binding. However, Vpr binding to DDB1 was not sufficient to induce G 2 arrest. A reduction in DDB1 or DDB2 expression in the absence of Vpr also did not induce G 2 arrest. On the other hand, Vpr-induced G 2 arrest was impaired when the intracellular level of DDB1 or Cullin 4A was reduced by siRNA treatment. Furthermore, Vpr-induced G 2 arrest was largely abolished by a proteasome inhibitor. These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G 2 arrest.
A method has been developed to use the polymerase chain reaction to amplify and sequence the chain determining region 3 (CDR 3) of the human immunoglobulin heavy-chain gene, and to use the sequence as a marker for rare neoplastic B lymphocytes. Consensus primers for the Variable and Joining regions of the gene were constructed and shown to enable efficient amplification, directed cloning, and sequencing of CDR 3. Using leukaemic cell line PFMC as a test system, CDR 3 was sequenced, specific primers synthesized, and PFMC DNA was detected down to a dilution of 1:1300 in DNA from normal lymphocytes. This strategy should be useful for monitoring therapy and detecting early disease relapse in B lymphoproliferative disease.
Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins have been classified as either Z1- or Z2-type cytidine deaminases on the basis of phylogenetic analysis of their catalytic domains. Despite the identification of a number of Z1-type domain-containing cytidine deaminases, only one copy of Z2-type cytidine deaminase has been detected in each of the mammalian species evaluated thus far. Z1-type human APOBEC3 proteins are known to exhibit broad activities against diverse retroelements. However, the potential role of the only human Z2-type cytidine deaminase, APOBEC3H (A3H), in the restriction of retroelements has not yet been fully characterized. Here, we demonstrate that human A3H is a potent inhibitor of non-LTR LINE-1 transposition. Interestingly, it was also as efficient as A3G in inhibiting Alu retrotransposition, despite its poor association with Alu RNA. We have further demonstrated, for the first time, that human APOBEC3DE is also a potent inhibitor of Alu retrotransposition. Variants of A3H have divergent antiviral activities against HIV-1-Vif-deficient viruses. Unlike the anti-HIV-1 cytidine deaminases A3G and A3F, A3H is moderately regulated by interferons. These observations suggest that human Z2-type cytidine deaminase A3H variants have varying intrinsic abilities to restrict retroelements and that various APOBEC3 proteins may have evolved distinct inhibitory mechanisms against retroelements.
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