Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases

Abstract

The mammalian APOBEC3 family of cytidine deaminases includes several members that possess potent antiretroviral activity. Human APOBEC3F and APOBEC3G are specifically incorporated into human immunodeficiency virus type 1 (HIV-1) progeny virions in the absence of virion infectivity factor (Vif), where they deaminate deoxycytidine to deoxyuridine on the minus strand of nascent reverse transcripts. Editing of the HIV-1 cDNA leads to its degradation or to G to A hypermutation of the integrated provirus. Here, we show that APOBEC3 proteins also restrict the activity of a distantly related long terminal repeat (LTR) retrotransposon. When expressed in the yeast Saccharomyces cerevisiae, human APOBEC3C, APOBEC3F, or APOBEC3G or mouse APOBEC3 potently inhibit replication of the Ty1 LTR retrotransposon. APOBEC3G interacts with Ty1 Gag and is packaged into Ty1 virus-like particles (VLPs) by a mechanism that closely resembles the one it uses to enter HIV-1 virions. Expression of APOBEC3G results in a reduced level of Ty1 cDNA integration and G to A editing of integrated Ty1 cDNA. Our findings indicate that APOBEC3G restricts Ty1 and HIV-1 by similar mechanisms and suggest that the APOBEC3 proteins target a substantially broader spectrum of retroelements than previously appreciated.

Figure 1. Human and Mouse APOBEC3 Proteins Specifically Inhibit Ty1 Retrotransposition

(A) Western blot analysis of whole cell lysates from strain JC3212 induced for expression of the HA-tagged APOBEC3 protein indicated, or no APOBEC3, with anti-HA antibodies. (B) Relative frequency of Ty1his3AI transposition in three transformants harboring pYES2 (no APOBEC) or pYES2 with the APOBEC3 allele indicated in strain JC3212. The percentage of Ty1his3AI transposition is the average frequency of transposition in APOBEC3 plasmid transformants divided by the average frequency of Ty1his3AI transposition in pYES2 transformants. (C) Similar to (B) except that strain JC297 containing Ty1his3AI, or strain JC560 harboring Ty2his3AI, was used in the analysis. Error bars, ± standard error.

Figure 2. Human APOBEC3G Cosediments with Ty1 VLPs

(A) Western blot analysis of sucrose gradient fractions of cell lysates from strain JC3212 expressing APOBEC3G or no APOBEC with rabbit antiserum specific for Ty1 Gag (top) or anti-HA antibodies (bottom). For each strain, 10 ug of whole cell lysate (IN), 7 μl (1×) and 0.7 μl (0.1×) of the 45% fraction of the first sucrose gradient and 11.5 μl of the first and then every third fraction of the second 20% to 60% gradient (20%–60%) were analyzed. (B) Similar to (A) except an spt3 strain expressing APOBEC3G was analyzed with anti-HA antibodies.

Figure 3. Ty1 Gag Binds APOBEC3G Specifically

(A) Western blot analysis of whole cell lysates from human 293T cells cotransfected with pK-GST (lanes 1 and 2), pK/HIVGAG-GST (lanes 3 and 4), or pK/TYA-GST (lanes 5 and 6) and with pK-h3G-HA (lanes 1, 3, and 5) or pK-βARR-HA (lanes 2, 4, and 6) by using anti-HA antibodies (top, input) or anti-GST antibodies (bottom). The fraction of each lysate that bound to glutathione-Sepharose beads was subjected to Western analysis with anti-HA antibodies (middle, bound). (B) 293T cells were cotransfected with phA3G-HA together with pK/TY1GAG-GST (lanes 1 and 2) or pK/MS2-GST and pDM128/4XMS2 (lanes 3 and 4). Binding assays were performed as described in (A) except that the cell lysate was incubated at 37°C for 10 min in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 50 μg of RNase A, prior to addition of the glutathione-Sepharose beads.

Figure 4. Human APOBEC3G Edits C Residues in Ty1 cDNA and Inhibits Integration of Total Ty1 cDNA Upstream of tRNA^gly Genes

(A) Sequence context for eleven independent dC deaminations on the minus strand of Ty1 cDNA. The fraction of events (expressed as a percentage) in which a particular base (A, C, G, or T) was found at the −2, −1, or +1 position relative to the deaminated C residue is indicated. (B) Top, PCR analysis of genomic DNA from a wild-type (WT) strain expressing no APOBEC protein (−) or the APOBEC3 protein indicated, or from a tec1Δ strain lacking APOBEC, with one primer that hybridizes in the Ty1 pol domain (T) and one that hybridizes to 16 tRNA^gly genes (S). PCR reactions were terminated after 29, 31, 33, or 35 cycles (black triangles). Control PCR reactions contained one primer (T or S). Bottom, PCR reactions of the TEL1 gene terminated after 17, 19, 21, or 23 cycles, as a control for equivalent template concentrations.