A long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe expresses a Gag-like protein that assembles into virus-like particles which mediate reverse transcription

Abstract

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses.

FIG. 1.

Comparative analysis of the Gag proteins of Tf1 and Tf2. (A) Clustal alignment of Gag proteins from Tf1 and Tf2. The dark blue boxes with yellow letters highlight identical amino acids, while the light blue boxes with black letters highlight similar amino acids. The four open boxes (A, B, C, and D) contain the amino acids deleted for this study. The black and gray lines under the alignment indicate the two blocks used to measure the Ks/Ka ratios (see the text). (B and C) Kyte-Doolittle hydropathy profiles of the Tf1 (B) and Tf2 (C) Gag proteins. Arrows indicate the four conserved hydrophilic domains (A, B, C, and D) targeted by the mutagenesis.

FIG. 2.

Effects of Gag deletions on transposition and recombination activity. (A) Schematic of the genetic assay for measuring the levels of transposition and homologous recombination. The restriction sites (BstXI) used for the cDNA blot are shown. I indicates the artificial intron placed in neo, −thiamine indicates the absence of thiamine, and 5-FOA^R indicates resistance to 5-FOA. (B) Results of the transposition and recombination assays for the four transposons containing deletions in Gag (Tf1-ΔA, Tf1-ΔB, Tf1-ΔC, and Tf1-ΔD) as well as for the control strains (Tf1-WT, Tf1-PRfs, and Tf1-INfs). Patches of cells in which the expression of Tf1 was induced were replica printed to plates containing G418 to measure recombination activity (right panel). To measure transposition, cells were replica printed to plates with 5-FOA, then printed to plates with 5-FOA and G418.

FIG. 3.

Effects of mutations in Gag on the synthesis of cDNA. Total DNA was extracted from S. pombe strains in which Tf1 expression had been induced. The DNA was digested with BstXI (Fig. 2) and probed with a neo-specific sequence. The 9.5-kb band was produced by a plasmid sequence, and the 2.1-kb band was generated by Tf1 cDNA. The additional band is likely derived from single-LTR circles; however, this identification is not definite. The reduced level of the plasmid band from Tf1-ΔC was reproducible with independent transformants and may be due to degradation during extraction that is mediated by RT or IN.

FIG. 4.

Immunoblot of extracts made from S. pombe expressing different mutants of Tf1. (A) Schematic of the Tf1 polyprotein. (B to F) Immunoblots were performed by using polyclonal antibodies directed against Gag peptide amino acids (aa) 20 through 245 (bleed 660) (B and F) and 1 through 15 (bleed 63085-4+5) (C), against IN peptide (bleed 657) (D), and against RT peptide (bleed 1571) (E). The predicted sizes of the proteins are 27, 56, and 60 kDa, respectively, for Gag, IN, and RT. WT, wild type.

FIG. 5.

Electron micrographs of S. pombe cells expressing Tf1-WT (A, A', B, C, and D), Tf1-PRfs (G and G'), Tf1-ΔA (F), and Tf1-ΔD (E and E'). A', E', and G' are higher magnifications of the images shown in A, E, and G, respectively. a, aggregates of Tf1 material; P(r), particles with dense rings; P, particles without rings; m, membranes; n, heterochromatin; nm, nuclear membrane.

FIG. 5.

Electron micrographs of S. pombe cells expressing Tf1-WT (A, A', B, C, and D), Tf1-PRfs (G and G'), Tf1-ΔA (F), and Tf1-ΔD (E and E'). A', E', and G' are higher magnifications of the images shown in A, E, and G, respectively. a, aggregates of Tf1 material; P(r), particles with dense rings; P, particles without rings; m, membranes; n, heterochromatin; nm, nuclear membrane.

FIG. 6.

RNase protection assay to detect packaging of Tf1 RNA in VLPs. (A) Cellular extracts containing VLPs were treated with various amounts of Benzonase (0, 6, or 12 U, lanes 0, 6, and 12, respectively) for 6 min at room temperature. RNA was then transferred to membranes and subjected to RNA blot analysis with a probe specific for the Gag region of Tf1. (B) The total RNA was visualized by ethidium bromide staining of the agarose gel under UV light.