Functional analysis of N-terminal residues of ty1 integrase

Abstract

The Ty1 retrotransposon of Saccharomyces cerevisiae is comprised of structural and enzymatic proteins that are functionally similar to those of retroviruses. Despite overall sequence divergence, certain motifs are highly conserved. We have examined the Ty1 integrase (IN) zinc binding domain by mutating the definitive histidine and cysteine residues and thirteen residues in the intervening (X(32)) sequence between IN-H22 and IN-C55. Mutation of the zinc-coordinating histidine or cysteine residues reduced transposition by more than 4,000-fold and led to IN and reverse transcriptase (RT) instability as well as inefficient proteolytic processing. Alanine substitution of the hydrophobic residues I28, L32, I37 and V45 in the X(32) region reduced transposition 85- to 688-fold. Three of these residues, L32, I37, and V45, are highly conserved among retroviruses, although their effects on integration or viral infectivity have not been characterized. In contrast to the HHCC mutants, all the X(32) mutants exhibited stable IN and RT, and protein processing and cDNA production were unaffected. However, glutathione S-transferase pulldowns and intragenic complementation analysis of selected transposition-defective X(32) mutants revealed decreased IN-IN interactions. Furthermore, virus-like particles with in-L32A and in-V45A mutations did not exhibit substantial levels of concerted integration products in vitro. Our results suggest that the histidine/cysteine residues are important for steps in transposition prior to integration, while the hydrophobic residues function in IN multimerization.

FIG. 1.

Comparison of the Ty1-H3 IN N-terminal sequence with other Ty and selected retroviral sequences. Alignment of the N-terminal regions of INs of Ty retrotransposons and selected retroviruses. Arrows at the top indicate Ty1 residues mutated in this study; numbering is relative to the Ty1 IN sequence. Minus signs indicate reduction of transposition when the residue is mutated; plus marks indicate WT levels of transposition with mutants. The highly conserved histidine/cysteine residues are represented by black filled circles. Other similar residues include hydrophobic L, V, M, and I residues (boxed in green); acidic D, E, Q, and N residues (boxed in red); and nucleophilic S, T, Y, A, and G residues (boxed in blue). For the HIV-1 N-terminal IN sequence and solution structure determined by nuclear magnetic resonance, see Fig. 5c in reference .

FIG. 2.

Immunoblot of Ty1 proteins from partially purified VLPs. Ty1 mutations are indicated above each lane. (A) Immunoblot of IN. Upper portions of the gels are included to demonstrate that IN and RT deficiencies were not simply due to accumulation of processing intermediates. (B) Immunoblot of Ty1 RT. (C) Immunoblot of Ty1 Gag. The 49-kDa (p49) precursor and 45-kDa mature Gag (p45) are indicated at the right. Retrotransposition (rTn) competence (+) or deficiency (−) is indicated below each panel.

FIG. 3.

Comparison of Ty1 cDNAs from the WT and ZBR and X₃₂ mutants. (A) Diagram of cDNA analysis by restriction and Southern blotting. Restriction of Ty1-his3-AI cDNA with the enzyme DraI results in a 650-bp fragment. (B) Four independent colonies were chosen from the WT and each mutant as indicated. The single-copy CPR7 gene, represented by two fragments of 2,190 and 2,313 bp, was used as a loading control and for normalization of cDNA.

FIG. 4.

IN-IN interactions of representative transposition-competent and transposition-defective X₃₂ mutants visualized by GST pulldown experiments. Top row, GST-WT, GST-in-K33A, GST-in-T38A and GST-inY39A coexpressed with cognate mutants in IN carrying a C-terminal c-myc epitope. Lowercase (in) represents input proteins; pd indicates pulldown proteins. Efficiency of pulldown (pd eff.) was calculated as [c-myc IN_pd × (GST-IN_in/GST-IN_pd)]/c-myc-IN_in. Middle row, GST-in-L32A, GST-in-I28A, GST-in-I37A, and GST-inV45A, each coexpressed with c-myc-WT IN. Bottom row, GST-in-L32A, GST-in-I28A, GST-in-I37A, and GST-in-V45A coexpressed with c-myc-tagged cognate mutant pulldown partners. In all panels, INs were visualized with polyclonal antiserum B2 against Ty1 IN (28).

FIG. 5.

Intragenic complementation analysis of X₃₂ mutants with IN catalytic and NLS mutants. The black bar represents the transposition efficiency of two elements coexpressed from different plasmids, each carrying a WT IN, normalized to 100%. Dark gray bars indicate the percentage of transposition of in-I28A coexpressed with another plasmid carrying either the identical mutation, the catalytic in-2600 (45) mutation or the NLS in-K596,597G (48) mutation. Light gray bars show the same coexpression for in-V45A. The white bar represents the percentage of transposition of in-I28A coexpressed with in-V45A. Complementation data for in-2600 plus in2600 and in-K596,597G plus in-K596,597G (last two bars, intermediate gray) are taken from a previous publication (48). Error bars indicate standard deviations.

FIG. 6.

In vitro integration assay using WT and mutant VLPs. (A) Diagram of integration assay showing reactants and expected products. This assay monitors the insertion of a linear donor molecule into a supercoiled plasmid target. (B) Southern blotting using a ³²P-labeled probe with the donor as a template. Asterisks next to the mutant labels indicate those mutations that were defective for transposition. Volume analysis as determined by ImageQuant software of combined bands in each lane, after subtraction of the control lane, was as follows: WT, 1.6 × 10⁶; in-L32A, 4.0 × 10⁴; in-K33A, 7.9 × 10⁵; in-V45A, <0.