Acquir. A3-induced mutations from reporter cells as well as the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 is accompanied by limited viremia (8). The human cytidine deaminase family of apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3, A3) consists of seven proteins (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) that can inhibit retroviruses, endogenous retroelements, and DNA viruses to different degrees (10, 55, 76, 80, 86). The human A3 locus on chromosome 22 is polymorphic on a genome level with a large structural variation deleting the entire A3B coding region as well Tenapanor as several single-nucleotide polymorphisms in introns and exons of each cytidine deaminase (2, 32, 70). Haplotypes with distinct antiviral phenotypes have been reported only for A3H: A3H haplotype 2 (A3H hapII) confers strong anti-HIV-1 activity, while most other A3H variants are unstable proteins that lack inhibitory activity (12, 22, 58). The enzymatic activity of virion-packaged A3 molecules results in deamination of cytosine bases in the single-stranded viral DNA during reverse transcription, which leads to guanosine (G)-to-adenosine (A) mutations in the provirus (4, 23, 36, 43, 45). Of note, A3 proteins have also been shown to restrict through deaminase-independent mechanisms by reducing reverse transcription products and integration (3, 25, 28, 49, 50, 57). The accessory HIV-1 protein Vif counteracts the restriction of several A3 proteins (e.g., A3F and A3G) by mediating its proteasomal degradation in the producer cell (11, 46, 51, 77, 93) or by preventing their packaging into virions (21, 31). HTLV-1 encounters A3 proteins as it replicates in the same CD4+ T cells as HIV-1, but it lacks a functional Vif to counteract them (42). Yet, several groups reported that wild-type HTLV-1 is largely resistant to A3G restriction in cell culture assays (41, 56, 59). Only one group reported that HTLV-1 is mildly sensitive to A3G and that its catalytic deaminase activity was dispensable for restriction (74). Moreover, proviruses with multiple G-to-A mutations suggestive of A3 action are frequently detected in HIV-1-infected individuals (30, 33), but HTLV-1 proviruses carrying footprints of past deamination are rarely found in cell culture or HTLV-1-infected patients. In Rabbit Polyclonal to PDGFR alpha the few cases in which G-to-A mutations have been found, they have been attributed to A3G activity based on the dinucleotide context in which they occur (17, 41, 44). Derse and colleagues reported that HTLV-1 evolved a strategy different from HIV to counteract APOBEC3 proteins (14). In contrast to HIV-1 Vif-mediated proteosomal degradation, a portion of the HTLV-1 nucleocapsid results in A3G exclusion from the virion. Tenapanor Wild-type HTLV-1 particles failed to encapsidate A3G, whereas a deletion of a HTLV-1-specific 20-amino-acid (20-aa) region near the C terminus of NC resulted in enhanced A3G virion encapsidation and restriction, suggesting that this domain excludes A3G from packaging (14). We hypothesized that one or more A3 cytidine deaminases other than A3G may escape this NC peptide-mediated exclusion from virions and reduce HTLV-1 infectivity. In this study, we tested Tenapanor all seven human A3 proteins for their ability to restrict HTLV-1. We found that A3A, A3B, and A3H hapII potently decrease HTLV-1 infectivity. A3A and A3B required catalytic deaminase activity for restriction, whereas A3H hapII restricted HTLV-1 in a deaminase-independent manner. Analysis of HTLV-1 sequences in cell lines derived from ATLL and HAM/TSP patients revealed multiple independently mutated proviruses, suggesting that HTLV-1 is targeted by several A3 proteins, such as A3A, A3B, and A3G. MATERIALS AND METHODS Cell culture. TZM-bl cells were provided by J. C. Kappes and X. Wu through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, NIH Reagent program (90). The human cell lines HEK-293T and TZM-bl were maintained at 37C in a humidified atmosphere of 5% CO2 in Dulbecco’s Tenapanor high-glucose modified Eagle’s medium, supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 50 units/ml penicillin, and 50 g/ml streptomycin. Jurkat cells were cultured at 37C in a humidified atmosphere of 5% CO2 in Roswell Park Memorial Institute medium 1640 (RPMI 1640), supplemented with 10% FBS, 2 mM glutamine, 50 units/ml penicillin, and 50 g/ml streptomycin. Patient-derived HTLV-1-infected cell lines. Cell lines derived from HTLV-1-infected.