Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

Abstract

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55^gag) is cleaved to produce a single VLP structural protein, p37^gag. Replacement of Asp⁴³⁶ within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55^gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195^gag-pol. The PR cleavage site within Pr55^gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55^gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55^gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37^gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

TED (transposable element D) is a 7.5-kb, middle-repetitive retrotransposon of the moth Trichoplusia ni. It was identified as a single-copy insertion (TED^FP-D) within the DNA genome of Autographa californica nucleopolyhedrovirus (AcMNPV), having transposed during infection of cultured T. ni cells with this baculovirus (33). Comprised of gag, pol, and env genes that are flanked by long terminal repeats (Fig. 1A), TED^FP-D was the first example of spontaneous, retroelement-mediated transfer of functional host genes to an animal virus (11, 30, 33). On the basis of sequence similarity within pol, TED is most closely related to the Drosophila retrotransposons 17.6, 297, tom, and gypsy (11, 18, 31, 37, 46) and thus is classified as a member of the gypsy family of retroelements (42). Recent studies have indicated that TED, tom, and gypsy possess env-like genes that encode membrane-associated glycoproteins with properties expected of vertebrate retroviral envelope proteins (35, 40, 45). These findings, combined with evidence that gypsy produces enveloped viruslike particles (VLP) that are infectious in Drosophila larvae (19, 40, 41), suggest that the gypsy family transposons may be facultative retroviruses of insects (9, 38).

FIG. 1.

(A) TED genetic organization. TED (7.5 kb) contains gag, pol, and env genes flanked by 270-bp long terminal repeats (solid boxes). PR, RT, and IN are conserved retroviruslike domains within pol. (B) TED sequences expressed by baculovirus vectors. TED gag and pol were fused to the polyhedrin (polh) promoter (striped box) and inserted into the AcMNPV genome, replacing the polh gene. Expression of TED pol requires a −1 translational frameshift for all virus vectors except vGAG/POL.fs⁻ and vGAG/POL.PR⁻fs⁻, which contain in-frame gag-pol fusions due to 4-bp insertions (▾). The apparent masses (in kilodaltons) of TED proteins are indicated to the right of each virus. Symbols: ×, D⁴³⁶V mutation; arrow, transcriptional start site; •, inserted stop codon. Restriction site abbreviations: B*, BamHI; R, EcoRI; H, HindIII; K, KpnI; N, NcoI; Nh, NheI; P, PstI; S, SalI; S3, Sau3AI, Sm, SmaI; Sp, SpeI. Only restriction sites used for cloning are indicated.

The assembly of VLP containing genomic RNA and the components necessary for reverse transcription is critical to the movement of retrotransposons and retroviruses (for reviews, see references 4, 17, and 38). In the case of retrotransposons Ty1 and Ty3 of Saccharomyces cerevisiae, synthesis of gag-encoded structural proteins is required for transposition, suggesting that VLP assembly is an important intermediate step in this process (2, 13). VLP may have multiple functions, including compartmentalization of genomic RNA, along with reverse transcriptase (RT) and integrase (IN), protection of the genome from intracellular nucleases, and genome transport to the nucleus, among others. In vertebrate retroviruses, distinct domains of the Gag precursor that are separated by proteolytic cleavage into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins accomplish these functions (reviewed in references 5, 7, 17, 49 and 50).

Despite the importance of gag in transposition, little is known about Gag protein processing and VLP assembly by the env-containing retrotransposons. Due to their striking resemblance to the vertebrate retroproviruses, these simple elements may provide useful models for retrovirus CA assembly, morphogenesis, and evolution. An effective means for investigation of TED Gag structure and function has been the overexpression of TED by using baculovirus vectors (10). Infection of cultured lepidopteran cells with TED-containing AcMNPV recombinants demonstrated that the primary translation product of TED gag is Pr55^gag (30). Consistent with Gag function, Pr55^gag assembles to produce 60-nm-diameter VLP. Expression of TED gag-pol, which requires a −1 ribosomal frameshift (Fig. 1A), yields VLP composed of the major CA protein p37^gag, traces of Pr55^gag, and RT activity. The appearance of p37^gag correlated with expression of the 5′ portion of TED pol with sequence similarity to retroviral proteases (PR). Thus, it was proposed that p37^gag is derived by PR-mediated processing of Pr55^gag (30). Moreover, PR cleavage of the TED Gag-Pol polyprotein Pr195^gag-pol was predicted to yield RT and other functions necessary for transposition.

To examine the processing and assembly of TED Gag proteins, we have extended the use of baculovirus vectors to overexpress gag and pol in cultured Spodoptera frugiperda (SF21) cells that lack endogenous copies of TED. By using polyclonal antisera raised against TED Gag proteins, we confirmed that Pr55^gag is cleaved to produce the major VLP protein p37^gag. Moreover, we report here that cleavage is mediated by the TED pol-encoded PR, which is generated from the frameshifted polyprotein Pr195^gag-pol. The Gag cleavage site was mapped to the junction of a central NC-like domain and a C-terminal acidic domain within Pr55^gag. Analogous acidic domains were identified at the C termini of Drosophila retroelements 17.6, 297, and tom, suggesting that the function of the acidic domain is conserved. Nonetheless, the acidic domain was dispensable for VLP assembly, whereas the adjacent NC-like domain was required. These findings suggested that p37^gag provides both CA and NC functions and that TED incorporates multiple functions into a single Gag protein. This feature distinguishes TED from those vertebrate retroviruses in which the gag products are proteolytically separated.

MATERIALS AND METHODS

Cells and AcMNPV recombinants.

S. frugiperda IPLB-SF21 cells (48) were cultured in TC100 growth medium (GIBCO Laboratories) supplemented with 2.6 mg of tryptose broth per ml and 10% heat-inactivated fetal bovine serum. Viruses vGAG and vGAG/POL (Fig. 1B) have been described previously (30). TED-containing recombinants derived from the wild-type L1 strain of AcMNPV (28) were constructed and propagated by standard methods (21, 34). For each virus, the polyhedrin gene (polh) was replaced with TED^FP-D sequences under the control of the polh promoter. In brief, SF21 cells (2 × 10⁶ per plate) were transfected with Lipofectin (Bethesda Research Laboratories), 2 μg of NdeI-linearized transplacement plasmid, and 0.2 μg of Bsu36I-digested vΔp35/lacZ viral DNA (15). Extracellular budded virus was collected 2 to 4 days later and plaque purified by using the non-apoptotic plaque phenotype conferred by acquisition of the apoptotic suppressor gene p35 (29). Proper insertion of TED sequences was confirmed by PCR amplification and restriction analyses of viral DNA.

Recombinant plasmids. (i) Transplacement vector pEV/35K.

AcMNPV p35 was inserted in the opposite orientation adjacent to polh of a modified form of plasmid pEVocc⁺/PA (8). An XhoI-KpnI fragment containing polh was replaced by the polh promoter fused to a polylinker. The resulting transplacement vector, pEV/35K, contained a modified polylinker for insertion of foreign genes downstream of a polh 5′ noncoding leader identical to that of pEV55 (34).

(ii) TED pol mutations.

A Sau3AI fragment encoding TED PR (nucleotides 1943 to 2490) was inserted into the BamHI site of pBluescript KS(+) (Stratagene) to generate plasmid pTEDpr⁺. Asp⁴³⁶ in the putative PR active site was replaced with Val by using the oligonucleotide 5′-ATTCTTGATTGTCACTGCCAA-3′ (A→T at nucleotide 1969 is underlined) (26) to generate pTEDpr⁻. The D⁴³⁶V mutant sequence was inserted into TED pol by successive steps. First, TED nucleotides 1611 to 1951 were amplified by PCR (39) using primers 5′-CGTAAATCCGGGAATCCGCC-3′ and 5′-GGTGGATCCGAAAATTCTATATAT-3′ (T→G at nucleotide 1942 introduced a BamHI site). After EcoRI and BamHI digestion, the amplified fragment (TED nucleotides 1672 to 1943) was inserted at the corresponding sites of pGAG_tr (30) to generate pGag.2. A BamHI fragment of pTEDpr⁻ containing D⁴³⁶V was inserted at the BamHI site of pGag.2 to generate pGagpr⁻. An XbaI/amber stop linker (New England Biolabs) was inserted at the XbaI site downstream of TED sequences to generate pGagpr⁻.X. After treatment with T4 DNA polymerase, an XhoI-SstI fragment from pGagpr⁻.X was inserted into XhoI and SmaI sites of pEV/35K to generate pEV/35K/gagpr⁻. An NcoI-SstII fragment (TED nucleotides 2418 to 6070) from pGAG/POL (30) was inserted into the corresponding sites of pEV/35K/gagpr⁻ to generate pEV/35K/gagpol.pr⁻. The BamHI fragment of pTEDpr⁺ was used to replace the homologous fragment in pEV/35K/gagpr⁻ to generate pEV/35K/gagpr⁺. The sequence of nucleotides 1672 to 2490 was determined in order to verify all mutations. The corresponding viruses vGAG/PR⁻ and vGAG/PR⁺ encoded 8 heterologous residues (PLVLASLD) at the 3′ end of pol.

A TED gag-pol in-frame fusion was created by inserting 4 bp (underlined) immediately after the −1 frameshift by using oligonucleotide 5′-GTTTGGTCGATTGCTAGCAAATCCTGACTTTC-3′ (TED nucleotides 1875 to 1848) to generate plasmid pTEDfs⁻. A 271-bp EcoRI-BamHI fragment from pTEDfs⁻ was inserted into the corresponding sites of pGagpr⁺.B2⁻ (see below) to generate pGagpr⁺fs⁻. The XhoI-NcoI fragment of pGagpr⁺fs⁻ was replaced with the analogous fragment of pEV/35K/gagpol.pr⁻.

(iii) TED gag mutations.

TED gag was truncated at either the EcoRI (nucleotide 1672) or the SalI (nucleotide 1705) site by EcoRI or SalI digestion of pGAG_tr (30), followed by digestion with XbaI. After end repair with Klenow fragment, an XbaI/amber linker was inserted to generate pGag338 and pGag349, respectively. The gag sequences were inserted as XhoI-XbaI fragments into the transplacement vector pEV/35K to generate pEV/35K/gag338 and pEV/35K/gag349. pEV/35K/gag338 was digested with KpnI and XbaI, end repaired with Klenow fragment, and ligated to an XbaI/amber linker to generate pEV/35K/gag229. Virus vGAG²²⁹ encoded two heterologous residues (LV) at the 3′ end of gag. The 1,024-bp EcoRI fragment from pPRM⁻35K-ORF (16) with p35 was inserted into the EcoRI site of pEV/35K/gag338 to generate pEV/35K/gag-P35. The gag-P35 hybrid encoded 6 heterologous residues (DLYHSK) between gag and p35.

Sequences encoding the influenza hemagglutinin (HA) epitope YPYDVPDYA were inserted at TED nucleotide 1728 (Gag residue 355) by introducing an NheI site (underlined) into pGagpr⁺.X with oligonucleotide 5′-GTATAATAGTCGCAGCTAGCCCCTGGTTCGG-3′. Complementary oligonucleotides encoding the HA epitope (5′-CTAGCATGTACCCATACGACGTCCCAGACTACGCTG-3′) were inserted to generate pGagpr⁺.HA. pGagpr⁺.X was partially digested with BamHI, Klenow repaired, and ligated to a BglII linker, 5′-GAAGATCTTC-3′, to generate pGagpr⁺.B2⁻, in which a BglII site replaced the BamHI site at TED nucleotide 2490. An EcoRI-BamHI fragment (nucleotides 1672 to 1943) from pGagpr⁺.HA was used to replace the analogous fragment of pGagpr⁺.B2⁻ to generate pGagpr⁺.HAB⁻. An XhoI-NcoI fragment (TED nucleotides 559 to 2418) from pGagpr⁺.HAB⁻ was used to replace the analogous fragment of pEV/35K/gagpol to generate pEV/35K/gagpol.HA.

Antiserum preparation.

For anti-Pr55^gag (α-p55) antiserum, Triton X-100 lysate (see below) was prepared from SF21 cells 48 h after infection with vGAG. Particulate material was pelleted through a 30% (wt/vol) sucrose cushion by centrifugation (at 250,000 × g for 2.5 h) and sedimented on a 20 to 70% (wt/vol) linear sucrose gradient (at 150,000 × g for 16 h). Pr55^gag-containing gradient fractions were pooled for antigen preparation. For anti-TrpE-Gag (α-Tg) antiserum, an SpeI-PstI fragment containing TED gag nucleotides 765 to 1238 was inserted into the XbaI and PstI sites of vector pATH22 (22) to generate pATH22/TrpE-gag. Insoluble TrpE-Gag fusion protein was isolated from Escherichia coli JM83 that contained pATH22/TrpE-Gag. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (27), gel slices containing either Pr55^gag or TrpE-Gag protein were crushed and emulsified with adjuvant. New Zealand White rabbits were immunized with 200 μg of antigen and boosted 2 weeks later with 100 μg of antigen. Immune and preimmune sera were collected and treated with an acetone powder from wild-type AcMNPV-infected SF21 cells by standard methods (6, 14).

Analysis of virus-infected cell lysates and VLP.

Except as noted, SF21 cells were inoculated with a multiplicity of infection (MOI) of 10 PFU per cell. Infected cells were washed with phosphate-buffered saline 42 or 48 h later, suspended in lysis buffer (1% SDS–2.5% β-mercaptoethanol), and boiled for 5 min. For VLP isolation, cells were collected 42 h after infection, washed with TNE (10 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA), and suspended in TNE lysis buffer (TNE plus 250 mM sucrose–0.1% Triton X-100) for 15 min at 0°C to produce Triton X-100 lysates. After clarification by centrifugation (at 800 × g for 15 min), particulate material was collected by centrifugation through a 30% (wt/vol) sucrose cushion (at 250,000 × g for 2.5 h). Pellets were either suspended in lysis buffer and boiled for 5 min or sedimented on a 20 to 70% (wt/vol) sucrose gradient (at 150,000 × g for 16 h). TED Gag-containing gradient fractions were pooled.

Immunoblot analysis.

Protein samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (47). The membranes were incubated in 5% nonfat dry milk or 3% bovine serum albumin in TBST (50 mM Tris [pH 7.4], 150 mM NaCl, 0.05% Tween 20), followed by a 5 × 10⁻⁴ dilution of α-Tg, a 10⁻³ dilution of α-p55, or a 10⁻³ dilution of anti-HA 12CA5 monoclonal serum (BAbCO) in 5% nonfat milk- or 3% bovine serum albumin-containing TBST by standard methods (6). Immune complexes were detected with horseradish peroxidase-conjugated goat or mouse anti-rabbit immunoglobulin G (Jackson Laboratories) and developed by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham).

Pulse-chase analysis.

The medium from SF21 cells infected 42 h previously was replaced with phosphate-buffered saline containing 200 μCi of Tran³⁵S-label (>1,000 Ci/mmol; ICN Biomedicals, Inc.)/ml. After 20 min at 27°C, the radiolabel was replaced with growth medium containing a 50-fold excess of unlabeled methionine and cysteine. SDS cell lysates were prepared at the indicated times. For immunoprecipitations, the lysates were clarified by centrifugation (at 6,000 × g for 2 min), diluted in Nonidet P-40 (NP-40) buffer (50 mM Tris [pH 8.0], 1% NP-40, 150 mM NaCl, 5 mM EDTA), and incubated with α-p55 for 30 min at 0°C. Protein A-Sepharose beads were added, and the mixture was incubated for 1 h at 7°C. Immune complexes were washed three times with NP-40 buffer, boiled in lysis buffer, and subjected to SDS-polyacrylamide gel electrophoresis and fluorography (En³Hance; DuPont).

Image processing.

Films were scanned at a resolution of 300 dpi by using a Microtek Scanmaker III equipped with a transparency adapter. The resulting files were printed from Adobe Photoshop 3.0 by using a Kodak 8650 PS dye sublimation printer.

RESULTS

Identity of TED gag proteins.

To establish and verify the precursor-product relationships of TED Gag and Gag-Pol polyproteins, we first generated polyclonal antisera to TED gag-specific proteins. α-Tg serum was raised against an E. coli-generated TrpE protein fused to TED gag amino acid residues 35 to 192. Immunoblot analysis with α-Tg detected Pr55^gag (Fig. 2, lanes 2 and 8), the primary translation product of TED gag that is synthesized in cultured SF21 cells infected with AcMNPV recombinant vGAG (Fig. 1B). The TED-related protein p77, of unknown function, was also recognized (30). In addition, p37^gag was detected (Fig. 2, lanes 6 and 12) upon expression of TED gag and pol by recombinant vGAG/POL (Fig. 1B). As demonstrated by the failure to recognize proteins from wild-type AcMNPV-infected SF21 cells (Fig. 2, lanes 1 and 7), α-Tg was specific for TED proteins. A different antiserum, α-p55 (see below), which was raised against full-length Pr55^gag isolated from purified TED VLP, exhibited similar specificity.

FIG. 2.

Effect of D⁴³⁶V mutant pol on proteolytic processing of TED Gag proteins. Triton X-100 (TX-100) extracts were prepared from SF21 cells 42 h after infection with the indicated AcMNPV recombinants (MOI of 10). Samples (10⁴ cell equivalents) of Triton X-100 extract (lanes 1 to 6) or the VLP collected from 10⁴ cells (lanes 7 to 12) were subjected to immunoblot analysis by using α-Tg serum. Positions of molecular mass standards (in kilodaltons), TED proteins, and degradation products (arrows) are indicated. WT, wild type.

Requirement of Asp⁴³⁶ for PR activity and Pr55^gag cleavage.

The active-site hexapeptide (hydrophobic residue)₂-D-(T/S)-G-(A/S) is conserved among pol-encoded aspartyl PR of retroelements (25). To assess the role of TED PR in Gag processing, we substituted a valine for aspartate residue 436 within the predicted active site (LID⁴³⁶TGA) of TED PR and generated AcMNPV recombinants vGAG/POL.PR⁻ and vGAG/PR⁻, containing gag and all or part of the D⁴³⁶V-mutated pol gene (Fig. 1B). Immunoblot analysis of infected cells revealed that vGAG/PR⁻ and vGAG/POL.PR⁻ produced little or no p37^gag (Fig. 2, lanes 3 and 5). Moreover, the steady-state level of Pr55^gag was higher than that in cells infected with the wild-type PR-containing virus vGAG/PR⁺ or vGAG/POL (lanes 4 and 6). The active-site D⁴³⁶V substitution also caused an accumulation of the larger gag-related proteins p80 and Pr195^gag-pol in cells infected with vGAG/PR⁻ (lane 3) and vGAG/POL.PR⁻ (lane 5), respectively.

The requirement for Asp⁴³⁶ in Gag processing was confirmed by an analysis of TED VLP. As measured by recovery of particulate material by centrifugation through 30% sucrose cushions, Pr55^gag was the major gag-related protein of VLP isolated from cells infected with PR-mutated vGAG/PR⁻ and vGAG/POL.PR⁻ (Fig. 2, lanes 9 and 11). p37^gag was not detected in VLP produced by the viruses with the D⁴³⁶V substitution. In contrast, p37^gag was the major VLP protein produced by vGAG/PR⁺ or vGAG/POL (Fig. 2, lanes 10 and 12). Smaller gag-related proteins were detected in cells infected with TED gag-containing viruses (Fig. 2, lanes 2 to 6). Because these proteins were not detected in VLP preparations (Fig. 2, lanes 8 to 12) and their abundance varied between experiments, it is likely that they represented Pr55^gag breakdown products.

Synthesis of in-frame TED gag-pol fusions.

Synthesis of the PR-containing Gag-Pol polyprotein Pr195^gag-pol requires a ribosomal frameshift in the −1 direction within the 43-bp overlap between TED gag and pol (Fig. 1A). The abundance of Gag proteins relative to Gag-Pol proteins produced by the PR-deficient baculovirus vectors (Fig. 2) suggested that the frameshift affects the stoichiometry of TED structural and enzymatic proteins, including PR. Thus, to investigate the potential regulatory role of the frameshift, we inserted 4 nucleotides immediately adjacent to the predicted site of ribosomal slippage (GGAUUUU), creating an in-frame gag-pol fusion (fs⁻) that eliminated the synthesis of Pr55^gag. Recombinant viruses vGAG/POL.fs⁻ and vGAG/POL.PR⁻fs⁻, with and without active PR, respectively, were generated (Fig. 1B). Consistent with an in-frame fusion, vGAG/POL.fs⁻ produced p37^gag but not the precursor, Pr55^gag, as indicated by analysis of the particulate fraction of infected cells (Fig. 3, lane 5). The level of p37^gag derived from vGAG/POL.fs⁻ was comparable to that of vGAG/POL (Fig. 3, lane 3), containing the natural −1 frameshift, and similar to that of vGAG³⁴⁹ (lane 2), containing a C-terminal truncation of Pr55^gag (see below). D⁴³⁶V inactivation of PR within the in-frame gag-pol fusion caused high levels of accumulation of Pr195^gag-pol (Fig. 3, lane 6) and yielded a smaller gag-related protein of unknown origin. The loss of p37^gag and the appearance of Pr195^gag-pol upon PR inactivation demonstrated that PR is active within the Gag-Pol polyprotein. Moreover, p37^gag can be derived by PR-mediated cleavage of Pr195^gag-pol.

FIG. 3.

Expression of TED gag-pol in-frame fusions. Triton X-100 extracts were prepared from SF21 cells 42 h after infection (MOI of 10) with recombinant vGAG, vGAG³⁴⁹, vGAG/POL, vGAG/POL.PR⁻, vGAG/POL.fs⁻, or vGAG/POL.PR⁻fs⁻. Particulate material was collected by centrifugation through a 30% (wt/vol) sucrose cushion, and samples (7.5 × 10⁴ cell equivalents) were subjected to immunoblot analysis by using α-Tg. Molecular mass markers (in kilodaltons) and TED proteins are indicated.

Kinetics of PR-mediated cleavages.

To validate the precursor-product relationships of TED gag proteins and confirm the requirement of TED PR in proteolytic processing, we conducted pulse-chase analyses in which immunoprecipitation with α-p55 antiserum monitored the fate of radiolabeled Gag proteins. Pr55^gag synthesized in cells infected with either vGAG (Fig. 4, lanes 1 to 3) or D⁴³⁶V-mutated vGAG/PR⁻ or vGAG/POL.PR⁻ (lanes 9 to 14) was relatively stable during the chase. Moreover, at no time was p37^gag detected. Polyproteins p80 and Pr195^gag-pol, synthesized by the PR-deficient viruses, were also detected (Fig. 4, lanes 9 to 14). When functional PR was synthesized by viruses vGAG/PR⁺ (Fig. 4, lanes 4 to 8) and vGAG/POL (lanes 15 to 19), Pr55^gag levels decreased whereas p37^gag levels increased. Although the level of immunoprecipitated Pr55^gag was reduced in PR-synthesizing cells, the rate of p37^gag appearance indicated that Pr55^gag had a half-life of ∼15 min, consistent with our previous findings (30). Collectively, these data demonstrated that p37^gag is derived from Pr55^gag. However, the smaller, sister fragment to p37^gag was not detected by immunoprecipitation with α-p55, despite the presence of Cys and Met residues for radiolabeling (see below).

FIG. 4.

Pulse-chase analysis of TED Gag processing. Cells infected 42 h previously with the indicated AcMNPV recombinants (MOI of 10) were radiolabeled for 20 min with [³⁵S]methionine-cysteine. The radiolabel was replaced with medium containing excess unlabeled methionine-cysteine, and cell lysates were prepared 0, 0.25, 0.5, 1, and 3 h thereafter. TED gag-related proteins were immunoprecipitated by using α-p55 and were subjected to polyacrylamide gel electrophoresis and fluorography. Molecular mass standards (in kilodaltons) and TED-specific proteins are indicated.

Pr55^gag→p37^gag cleavage site.

Inspection of the predicted amino acid sequence of Pr55^gag revealed a C-terminal region (residues 332 to 386) that is highly acidic and devoid of basic residues (Fig. 5A). This unusual domain is conserved among the gypsy retroelements tom and 17.6. The acidic domain of TED is joined to an NC-like region (residues 199 to 314) that is basic and proline rich and that lacks acidic residues. Since proteolytic cleavage between the NC-like region and the acidic domain would yield a protein similar in size to p37^gag, we predicted that processing occurs at or near the predicted junction of these dissimilar segments. To test this possibility, we first inserted the 9-amino-acid HA epitope from influenza virus HA at residue 355 within the acidic domain of Pr55^gag (Fig. 5A). The presence of the HA tag in Pr55^gag was verified by immunoblot analysis (with HA-specific serum) of cells infected with the resulting AcMNPV recombinant vGAG^HA/POL (data not shown). However, p37^gag was not detected by the HA-specific serum after cleavage, nor were smaller HA-containing cleavage products detected. These findings suggested that the acidic domain was proteolytically removed from the C terminus of Pr55^gag and that the sister cleavage fragment was unstable.

FIG. 5.

PR-mediated cleavage of C-terminal TED Gag truncations. (A) Comparison of gypsy element Gag precursors. The predicted N-terminal CA-like (CA), basic NC-like (NC) (shaded), and C-terminal acidic (striped) domains of TED (11) and analogous domains of the predicted Gag proteins of Drosophila gypsy elements tom and 17.6 (37, 46) are shown. Tick marks indicate basic or acidic amino acid residues. The HA epitope (▾) was inserted at residue 355 within the acidic domain of TED Pr55^gag. Predicted PR cleavage sites are indicated (vertical arrows). (B) TED gag sequences. AcMNPV vectors contain the full-length gag open reading frame (vGAG) or 3′ truncations that are designated by the number of codons extending from the initiation codon to the introduced stop codon (•). Synthesized Gag proteins are indicated on the right. Restriction site abbreviations are listed in the legend to Fig. 1. (C) Immunoblot analysis. Cells were infected with wild-type (WT) AcMNPV (lane 1), vGAG²²⁹ (lane 2), vGAG³³⁸ (lane 3), vGAG³⁴⁹ (lane 4), vGAG/POL (lane 5), or vGAG/POL.PR⁻ (lane 9) alone. Cells infected with vGAG²²⁹, vGAG³³⁸, and vGAG³⁴⁹ were also coinfected with vGAG/POL (lanes 6 to 8) or vGAG/POL.PR⁻ (lanes 10 to 12). SDS lysates were prepared 42 h after infection and subjected to immunoblot analysis (3 × 10⁴ cell equivalents) by using α-Tg. Molecular mass standards (in kilodaltons), TED-specific proteins, and gag-related degradation proteins (★) are indicated.

To map the location of the Pr55^gag cleavage site, recombinant viruses with 3′ deletions of TED gag were constructed (Fig. 5B). Since cleavage occurred near the Pr55^gag C terminus, we predicted that removal of these residues would eliminate processing by PR and thereby provide the location of the cleavage site. Immunoblot analysis of infected cells demonstrated that vGAG²²⁹, vGAG³³⁸, and vGAG³⁴⁹ produced gag truncation proteins p25, p38, and p40, respectively (Fig. 5C, lanes 2 to 4). When provided in trans, functional PR had no effect on the size or accumulation of p25, as shown by coinfection of vGAG²²⁹ with PR-containing vGAG/POL or PR-deficient vGAG/POL.PR⁻ (Fig. 5C, lanes 6 and 10). Thus, cleavage occurs on the C-terminal side of residue 229. In contrast, coinfection of vGAG³⁴⁹ with vGAG/POL reduced the intracellular level of truncation protein p40 and yielded p37^gag (Fig. 5C; compare lanes 4 and 8). Since p40 cleavage was not detected upon coinfection with vGAG/POL.PR⁻ (Fig. 5C, lane 12), PR was responsible for the cleavage of p40. Little, if any, change in the size or level of truncation protein p38 was detected upon coinfection with either vGAG/POL or vGAG/POL.PR⁻ (Fig. 5C, lanes 7 and 11). Taken together, these findings indicate that Pr55^gag cleavage occurred on the N-terminal side of residue 349 at or near residue 338.

Dispensability of the acidic domain for Pr55^gag cleavage.

To determine whether the C-terminal acidic domain was required for Gag cleavage, we constructed an AcMNPV vector, vGAG-P35, in which a portion of TED gag was fused in frame with the heterologous gene p35 and placed under the control of the polh promoter. Expression of the gag-p35 chimera produced Gag-P35 (Fig. 6A), which contained the first 338 residues of Pr55^gag (p38) fused to P35, a 35-kDa cytosolic protein encoded by AcMNPV (16). Infection with vGAG-P35 yielded the full-length Gag-P35 fusion, as detected by using Gag-specific α-Tg and P35-specific α-P35NF antisera (Fig. 6A, lanes 3 and 8). Upon infection of vGAG-P35 with PR-expressing vGAG/POL, Gag-P35 was cleaved to produce P35′, a protein detected exclusively by α-P35NF (Fig. 6A, lane 9). Due to the presence of Gag-derived residues, P35′ was larger than wild-type P35 detected in all recombinant AcMNPV-infected cells (Fig. 6A, lanes 6 to 10). The absence of P35′ in cells coinfected with vGAG-P35 and PR-deficient vGAG/POL.PR⁻ (Fig. 6A, lane 10) verified that P35′ was generated by PR-mediated cleavage of Gag-P35. However, the abundance of the Gag-P35 fusion protein in PR-synthesizing cells (Fig. 6A, lanes 4 and 9) suggested that cleavage of Gag-P35 was less efficient than that of Pr55^gag. These data demonstrated that PR can cleave a heterologous Gag fusion protein lacking the acidic domain and indicated that cleavage is N terminal to Gag residue 338.

FIG. 6.

PR-mediated cleavage of TED Gag-P35. (A) Immunoblot analysis of cell lysates. Cells were infected with vGAG/POL (POL) (lanes 1 and 6), vGAG/POL.PR⁻ (PR⁻) (lanes 2 and 7), or vGAG-P35 (−) (lanes 3 and 8) alone. In addition, cells were coinfected with vGAG-P35 and vGAG/POL (lanes 4 and 9) or with vGAG-P35 and vGAG/POL.PR⁻ (lanes 5 and 10). SDS lysates (3 × 10⁴ cell equivalents) were subjected to immunoblot analysis by using α-Tg or α-P35NF. (B) VLP analysis. The VLP from cells 42 h after infection with vGAG-P35 were collected by centrifugation through a 30% sucrose cushion. Triton X-100 (TX-100) extract from 2 × 10⁴ cells (lane 1) and VLP from 6 × 10⁴ cells (lane 2) were subjected to immunoblot analysis by using α-P35NF.

Dispensability of the acidic domain for VLP assembly.

Examination of the particulate fraction derived from vGAG-P35-infected cells (Fig. 6B) indicated that the Gag-P35 fusion protein retained the capacity to assemble VLP. This finding suggested that the acidic domain is not required for particle assembly. To define Pr55^gag residues involved in VLP assembly and to further investigate TED gag functions, we tested the effects of the 3′ gag truncations (Fig. 5B) on VLP yields produced by AcMNPV vectors. Deletion of 61 (vGAG³⁴⁹) or 72 (vGAG³³⁸) residues at the Pr55^gag C terminus had no effect on particle assembly, since the yield of p40-containing VLP (Fig. 7, lane 12) or p38-containing VLP (lane 11) was comparable to that of vGAG/POL-infected cells (lane 9). VLP composed of either p38 (vGAG³³⁸) or p40 (vGAG³⁴⁹) were stable to sucrose gradient purification (Fig. 7, lanes 15 and 16) and exhibited a density (1.19 g/ml) comparable to that of VLP composed of full-length Pr55^gag. Electron microscopic examination also indicated that negatively stained VLP from vGAG³⁴⁹ resembled negatively stained Pr55^gag-containing VLP (data not shown). In contrast, removal of 181 residues (vGAG²²⁹) that included residues within the basic, NC-like domain eliminated recovery of VLP (Fig. 7, lane 10) without altering protein (p25) stability (lane 4). Thus, Pr55^gag residues from 229 to 338 contribute to VLP assembly or stability, or both.

FIG. 7.

Effect of TED gag truncations on VLP assembly. SF21 cells were harvested 42 h after infection (MOI of 10) with wild-type (WT) AcMNPV, vGAG, vGAG/POL (POL), vGAG²²⁹ (229), vGAG³³⁸ (338), or vGAG³⁴⁹ (349). Triton X-100 (T X-100) extract (2.5 × 10⁴ cell equivalents) (lanes 1 to 6) and the VLP-containing (particulate) fraction (7.5 × 10⁴ cell equivalents) collected from extracts by centrifugation through a 30% sucrose cushion (lanes 7 to 12) were subjected to immunoblot analysis by using α-Tg. Threefold less sample was used from vGAG-infected cells (lanes 2 and 8). VLP from equivalent numbers of vGAG/POL-, vGAG²²⁹-, vGAG³³⁸-, and vGAG³⁴⁹-infected cells were sedimented on 20 to 70% (wt/vol) sucrose density gradients and analyzed similarly (lanes 13 to 16).

DISCUSSION

Model for TED VLP assembly and maturation.

Our data indicate that the assembly and maturation of TED Gag and Gag-Pol proteins resemble those of the simple vertebrate retroviruses. The primary translation product of TED gag, Pr55^gag, first assembles with the less abundant Gag-Pol polyprotein Pr195^gag-pol to yield immature particles. PR-mediated processing of both precursors then generates mature VLP containing the major CA protein p37^gag, the pol-encoded enzymes RT and IN, and genomic RNA. By analogy to other retroelements (20, 23, 32, 36, 44), it is expected that the maturation process activates RT and IN for reverse transcription and subsequent integration of TED sequences into the host T. ni genome. Thus, proper proteolytic processing of VLP is an essential step in transposition. The intracellular site for assembly and processing of TED VLP and whether VLP bud from the plasma membrane remain to be determined.

PR processing of Pr55^gag and Pr195^gag-pol.

By using baculovirus vectors and TED gag-specific antisera, we have demonstrated that the major VLP structural protein p37^gag of TED is derived from precursor Pr55^gag, confirming earlier studies (30). As expected, p37^gag is also generated from Pr195^gag-pol (Fig. 3). On the basis of the fact that replacement of the critical aspartate (D⁴³⁶V) within the consensus active site of PR abolished processing (Fig. 2), TED PR is responsible for these cleavages. Although the D⁴³⁶V loss-of-function mutation did not affect VLP formation (Fig. 2), it is likely that loss of PR disrupts TED transposition, since PR is required for retrotransposition and retrovirus replication (20, 23, 43, 51). Interestingly, coexpression of TED gag-pol and PR-deficient gag-pol inhibited Pr55^gag processing by the wild-type PR (12). This dominant inhibition of PR by PR-deficient Pr195^gag-pol is consistent with the requirement of Gag-Pol polyprotein dimerization for PR activity in a manner analogous to that of the vertebrate retroviruses (reviewed in references 5 and 49).

CA and NC functions of TED p37^gag.

For the retroviruses, proteolytic cleavage releases the MA, CA, and NC domains from the Gag precursor, thereby irreversibly preparing the virus particle for reverse transcription and subsequent uncoating (reviewed in references 7, 49 and 50). In the case of TED, our studies indicated that Gag precursor Pr55^gag is cleaved to yield the single structural protein p37^gag, which may provide both CA and NC functions. PR-mediated cleavage occurred between Gag residues 305 and 325, as shown by the use of C-terminal Gag truncations (Fig. 5) and PR cleavage of the fusion protein Gag-P35 (Fig. 6). Cleavage removed the 9-kDa acidic domain from the C terminus of Pr55^gag and placed the basic, proline-rich NC domain at the C terminus of p37^gag (Fig. 5A). Thus, the position of the predicted NC (residues 199 to 314) of TED is analogous to that of the NC of other retroelements (reviewed in references 5, 7 and 38).

Unlike many retroelements, including the gypsy-related retrotransposon Ty3, TED Gag lacks retroviruslike major homology region and zinc finger (C-X₂-C-X₄-H-X₄-C) motifs, located within CA and NC domains, respectively. In this respect, TED Gag resembles the Gag protein of the retroviral foamy viruses (spumavirus genus), which also undergoes limited proteolysis at the C terminus only (reviewed in references 24 and 52). The predicted NC of TED is distinguished by its unusually high proline content (23%), lack of acidic residues, and abundance of basic residues (pI, 12.7). We have noted a striking similarity between the NC of TED and the corresponding region within gag of Drosophila elements tom and 17.6 (Fig. 5A). The abundance of proline (22 and 14% in tom and 17.6, respectively) and basic residues (pI values, 11.4 and 11.6, respectively), plus the absence of a zinc finger, suggests that NC function is conserved among these insect gypsy elements. The potential role of the basic residues in the NC for RNA packaging by TED remains to be determined. Nonetheless, the proline-rich NC also contributes to VLP assembly or stability. Stable VLP were not recovered when a C-terminal deletion removed a large portion of the TED NC-producing truncation protein p25 (Fig. 7). In contrast, selective deletion of the C-terminal acidic domain, which produced a p37^gag-like protein, had no effect on VLP yields. Collectively, these findings suggest that TED p37^gag has both NC and CA properties.

Function of the TED acidic domain.

Gypsy retroelements TED, tom, and 17.6 each contain acidic domains (Fig. 5A) analogous in size and charge (pI values, 3.4, 3.3, and 3.3, respectively). This striking similarity also suggests that the function of the acidic domain is conserved. Due to its acidic nature, it is unlikely that this domain participates directly in RNA binding. Although this domain is not required for TED VLP assembly or PR-mediated proteolysis, it may regulate these events. Alternatively, since the acidic domain is adjacent to PR within Pr195^gag-pol, it may contribute to PR function from within the Gag-Pol precursor. Interestingly, by using immunoprecipitations with α-p55 serum (Fig. 4) or epitope tagging (data not shown), we have failed to detect a separate acidic-domain-containing fragment. Thus, upon release by cleavage, the domain may have a short half-life or may no longer be recognized by these sera.

MA function for TED Gag?

As a potential retrovirus, TED gag is expected to encode an MA domain that promotes interaction between Gag proteins and the host cell membrane for virus budding (reviewed in reference 5). Indeed, TED env encodes a 75-kDa glycoprotein (gp75^env) that localizes to membrane fractions of env-expressing cells (35). In T. ni cells, resident copies of TED actively produce spliced, polyadenylated RNAs that encode gp75^env (12). Thus, TED VLP could generate enveloped, gp75^env-containing particles upon budding. However, due to low levels of TED expression in T. ni, we have not yet detected budded VLP, with or without gp75^env. Thus, the role of TED Gag proteins in promoting membrane association remains to be determined.

Production of TED Gag fusion proteins in insect cells.

When fused to the heterologous protein P35, TED Gag retained the capacity to form VLP (Fig. 6). Moreover, baculovirus-directed expression produced high yields of hybrid VLP that were readily purified by single-step centrifugation. In yeast, the retrotransposon Ty1 has been used to express Gag fusion proteins that self-assemble, facilitating the purification of recombinant proteins (1, 3). The efficiency of VLP assembly of TED Gag fusion proteins in insect culture, combined with the convenience and high level of expression afforded by the baculovirus vector system (34), provides another advantageous strategy for production of biologically important proteins in eukaryotes.