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

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 (TEDFP-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. ​(Fig.1A),1A), TEDFP-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 ... 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 Pr55gag (30). Consistent with Gag function, Pr55gag assembles to produce 60-nm-diameter VLP. Expression of TED gag-pol, which requires a −1 ribosomal frameshift (Fig. ​(Fig.1A),1A), yields VLP composed of the major CA protein p37gag, traces of Pr55gag, and RT activity. The appearance of p37gag correlated with expression of the 5′ portion of TED pol with sequence similarity to retroviral proteases (PR). Thus, it was proposed that p37gag is derived by PR-mediated processing of Pr55gag (30). Moreover, PR cleavage of the TED Gag-Pol polyprotein Pr195gag-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 Pr55gag is cleaved to produce the major VLP protein p37gag. Moreover, we report here that cleavage is mediated by the TED pol-encoded PR, which is generated from the frameshifted polyprotein Pr195gag-pol. The Gag cleavage site was mapped to the junction of a central NC-like domain and a C-terminal acidic domain within Pr55gag. 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 p37gag 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.