APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1 - PubMed (original) (raw)

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1

Marcel Ooms et al. J Virol. 2012 Jun.

Abstract

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.

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Figures

Fig 1

Fig 1

APOBEC3 restriction of HTLV-1 and HIV-1. (A) The indicated A3 expression plasmids were cotransfected with HTLV-1 helper (GagPol and nonstructural proteins), luciferase reporter, and VSV-G plasmids in HEK-293T cells. The cells were overlaid with Jurkat cells, and luciferase (relative light units [RLU]) was measured after 3 days. The GFP control is set at 100% infectivity. Values are means plus standard deviations (error bars) for three independent experiments. (B) Similar to panel A but with increasing amounts of A3 expression plasmids. (C) HIV-1 WT and HIV-1 ΔVif expression plasmids were cotransfected with the indicated A3 plasmids in HEK-293T cells. Two days posttransfection, supernatants were used to infect TZM-bl reporter cells, and β-galactosidase activity (RLU) was measured 2 days postinfection. Infectivity without A3 is set at 100%. Values are means plus standard deviations (error bars) of a representative experiment performed in triplicate. Unpaired t tests were computed to determine whether differences between GFP and each A3 protein reach the level of statistical significance (P < 0.05 [*] and P < 0.01[**], using GraphPad Prism 5 software).

Fig 2

Fig 2

Packaging of APOBEC3 into HTLV-1 virions. (A) The indicated A3 expression plasmids were cotransfected with an irrelevant plasmid (−) or with an HTLV-1 helper plasmid (+ HTLV) (all HTLV proteins except Env) in HEK-293T cells, and the cells were lysed 2 days after transfection. The lysates were analyzed by immunoblotting and probed for A3 expression (HA), and tubulin served as a loading control. α-HA, anti-HA antibody. (B) Filtered supernatants from panel A were concentrated through a 20% sucrose cushion and were either mock treated or treated with subtilisin A to remove exosomes/microvesicles and reconcentrated through a 20% sucrose cushion. Virion lysates were analyzed by immunoblotting. HTLV capsid (CA), matrix (MA), and nucleocapsid (NC) were detected by anti-HTLV-1 antibodies (α-HTLV).

Fig 3

Fig 3

Comparison between human and macaque A3A restriction of HTLV-1. (A) Protein sequence alignment of human A3A and Macaca mulatta A3A. Human A3A contains a specific deletion of 27S, 28V, and 29R. (B) The indicated A3A WT and mutant expression plasmids were cotransfected with HTLV-1 helper (GagPol and nonstructural proteins), luciferase reporter, and VSV-G plasmids in HEK-293T cells. The cells were overlaid with Jurkat cells, and luciferase (RLU) was measured after 3 days. GFP is set at 100%. Values are means plus standard deviations (error bars) for three independent experiments. P values were computed to determine whether differences between GFP and each A3 protein or between A3A mutants reach significance (P < 0.05 [*] and P < 0.01[**] by unpaired t test using GraphPad Prism 5 software). no luc, no luciferase. (C) The indicated A3A WT and mutant expression plasmids were cotransfected with HTLV-1 helper (GagPol and nonstructural proteins), luciferase reporter, and VSV-G plasmids in HEK-293T cells, and the cells were lysed 2 days after transfection. Supernatants were cleared and concentrated through a 20% sucrose cushion. Cell and virion lysates were analyzed by Western blotting. Of note, human A3A is 3× HA tagged and is therefore larger than the 1× HA tagged macaque A3A. Tubulin serves as a loading control.

Fig 4

Fig 4

Deaminase activity requirements for restriction of HTLV-1 and HIV-1. (A) Schematic representations of the deaminase domains present in A3A, A3B, and A3H. A3A and A3H hapII contain a single deaminase domain, whereas A3B contains two domains, which are indicated by a black box. The letter “A” denotes the alanine mutations of the essential glutamic acid in the catalytic active site. N-DD and C-DD denote the N-terminal and C-terminal deaminase domains, respectively. (B) The indicated A3 WT and deaminase mutants were cotransfected with HTLV-1 helper (GagPol and nonstructural proteins), luciferase reporter, and VSV-G plasmids in HEK-293T cells. The cells were overlaid with Jurkat cells, and luciferase (RLU) was measured after 3 days. GFP is set at 100%. Values are means plus standard deviations (error bars) for three independent experiments. Unpaired t tests were computed to determine whether differences between GFP and each A3 protein are statistically different (P < 0.05 [*] and P < 0.01[**], using GraphPad Prism 5 software). (C) HIV-1 ΔVif expression plasmids were cotransfected with the indicated A3 WT and deaminase mutants in HEK-293T cells. Two days posttransfection, supernatants were used to infect TZM-bl reporter cells, and β-galactosidase (RLU) was measured 2 days postinfection. Infectivity with GFP is set at 100%. Values are means plus standard deviations (error bars) of a representative experiment performed in triplicate. The values were compared for statistical significance (P < 0.05 [*] and P < 0.01[**] by unpaired t test, using GraphPad Prism 5 software). (D) Immunoblot analysis of A3A, A3B, and A3H hapII and their corresponding deaminase mutants in HEK-293T cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control.

Fig 5

Fig 5

A3-induced editing in HTLV-1 genomes. (A) A region overlapping with the intron in the luciferase gene of the HTLV-1 reporter sequence was PCR amplified from DNA obtained from HTLV-1 infectivity assays (Fig. 1A). PCR was performed using a range of denaturing temperatures (84 to 88°C) and analyzed on agarose gels. The denaturing temperature (Td) (84, 85.3, 86.3, 87, 87.5, and 88°C) is indicated by the height of the black triangle above the gel. (B) PCR products amplified at the lowest denaturing temperature were cloned, and eight individual clones were sequenced (501 bp). The mutations are indicated by color as follows: GA-to-AA mutations (blue lines), GG-to-AG mutations (red lines), GT-to-AT mutations (pink lines), and GC-to-AC mutations (green lines). (C) Pie chart representation of the relative dinucleotide preferences of A3A and A3B.

Fig 6

Fig 6

A3G editing of HIV-1 ΔVif and HTLV-1. (A) Supernatants of cells cotransfected with HIV-1 ΔVif and GFP or A3G was used to infect TZM-bl cells. Proviral DNA was extracted, and a 1,905-bp fragment in HIV-1 pol was PCR amplified and purified from gel. PCR fragments were subsequently used as a template in the 3DPCR with a range of denaturing temperatures (80.5 to 83.2°C) and analyzed on agarose gels. The denaturing temperature (Td) (80.5, 80.9, 81.2, 81.5, 81.2, and 83.2°C) is indicated by the height of the black triangle above the gel. HTLV-1 editing was performed as described in the legend to Fig. 5. (B) PCR products amplified at the lowest denaturing temperature were cloned, and eight individual clones were sequenced. Red lines indicate GG-to-AG mutations, blue lines indicate GA-to-AA mutations, pink lines indicate GT-to-AT mutations, green lines indicate GC-to-AC mutations, and black lines indicate non-G-to-A mutations. (C) Pie chart representation of the relative dinucleotide preferences of A3G for HIV-1 and HTLV-1.

Fig 7

Fig 7

A3 editing of HTLV-1 in vivo. (A) Cellular DNA was extracted from several cell lines obtained from HTLV-1-infected individuals diagnosed with ATLL or HAM/TSP. Nested 3DPCR was performed with HTLV-1 _tax_-specific primers. DNA extracted from HTLV-1 plasmid-transfected HEK-293T cells served as a negative control for editing (plasmid). (B) Nested 3DPCR was performed on DNA extracted from the different HTLV-1 cell lines using specific primers to amplify a fragment of the cellular myc gene. DNA extracted from a healthy donor and from HTLV-transfected HEK-293T cells were used as controls (PBMC and plasmid). (C) PCR fragments from the 3DPCR were cloned and sequenced. Only unique sequences (419 bp) are shown. A3-specific dinucleotide contexts are shown in the indicated colors. All non-G-to-A mutations are represented in black. + strand, plus strand.

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