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

Long terminal repeat (LTR)-containing retrotransposons are a diverse group of elements found in a broad variety of eukaryotic hosts. Their structures and mechanisms of propagation are closely related to those of retroviruses. Both retroviruses and LTR-containing retrotransposons encode Gag, reverse transcriptase (RT), and integrase (IN) proteins. The Gag proteins assemble into the coats of virus and virus-like particles (VLPs), and the formation of these particles is required for reverse transcription. Once RT produces the full-length double-stranded cDNA, IN inserts the cDNA into the genome of a host cell. Most of what is known about the function of Gag proteins resulted from the study of retroviruses. With an estimated 1,500 Gag molecules per virion in the case of the Rous sarcoma virus (59), Gag constitutes the major component of viral particles. It is well established that Gag is able to form extracellular particles in the absence of the other viral proteins (10, 19, 23, 62). As a result of its role in particle formation, Gag is responsible for the size of virions, the process of budding, and the packaging of the other components of the virus, such as the genomic RNA, RT, IN, and envelope proteins (56). Once the particles have assembled, their maturation results from the processing of Gag by the viral protease (PR) into at least three major products, matrix (MA), capsid (CA), and nucleocapsid (NC) (33, 64). MA is derived from the amino terminus of Gag and directs the location of particle formation (for a review, see reference 58). CA, the largest cleavage product of Gag, forms the shell surrounding the ribonucleoprotein complex that contains the genomic RNA. CA has several separate functions that contribute to the assembly of particles, the release of particles from infected cells, and the reverse transcription of viral RNA. Numerous mutations in the CA domains of the murine leukemia virus and of human immunodeficiency virus type 1 have been found to block assembly and particle release (11, 24, 43, 67). Certain mutations in the CA sequences of several retroviruses have been found to destroy infectivity without affecting assembly or budding (27, 43, 55, 60). Strong evidence for the role of the CA in postentry replication comes from experiments with the murine leukemia virus that showed that specific mutations inhibit the synthesis of viral cDNA (3). Each of the functions of Gag is thought to require the major homology region (MHR), a motif of CA that is about 20 amino acids long and is highly conserved among Gag proteins (49a, 64). Mutations in the MHR of the Rous sarcoma virus can be deleterious to particle release or have an effect on cDNA synthesis (9, 14). Finally, the NC protein, located at the carboxy terminus of Gag, is tightly bound to the genomic RNA (18, 21). NC is basic and contains one or more zinc finger motifs involved in the packaging of the viral RNA (13). In biochemical experiments, NC appears to have chaperone activity resulting in the stimulation of template switching by RT (4, 17, 50, 65; for a review, see reference 51). The spumaretroviruses are replication-competent retroviruses that express a Gag protein that has no sequence similarity to the MA, CA, and NC of other retroviruses (for a review, see references 31 and 41). This dissimilarity includes the lack of an MHR motif in CA and of Zn fingers in NC (44, 52). In place of the Zn fingers, a glycine-arginine-rich motif near the carboxy terminus of the spumaretroviral Gag has been shown to have nucleic acid binding properties (66). Although less is known about the Gag proteins of LTR-containing retrotransposons, evidence from electron microscopy indicates that they form VLPs, as documented for Ty1 (22) and Ty3 (25) of Saccharomyces cerevisiae as well as for copia (46) of Drosophila melanogaster. Intracytoplasmic particles have also been detected for the endogenous retroviruses of Drosophila, gypsy (32) and ZAM (30), elements closely related to LTR-containing retrotransposons. The retrotransposon Ty3 encodes a CA protein that contains an MHR-like motif (48). However, no homologs of CA and no MHR motifs have been identified in elements such as Ty1 (12) or the Tf1 and Tf2 elements from Schizosaccharomyces pombe (37). Tf1 and Tf2 are two closely related LTR-containing retrotransposons (37), and their sequences encode single primary products of translation that have homologies to PR, RT, and IN (36, 37, 61). Although the amino acid sequences encoded by Tf1 and Tf2 are virtually identical for RT and IN, the sequences of Gag diverge. Tf1 and Tf2 have been studied as model retroviruses by using the techniques of yeast genetics. The expression of Tf1 RNA from an inducible promoter results in high levels of transposition (26, 34, 36). Mutations within the conserved residues of PR, RT, and IN greatly reduce the transposition of Tf1 and indicate that these enzymes are required for Tf1 activity (5, 6, 35). However, much less is known about whether a Gag-like protein is required for Tf1 transposition. A protein of 27 kDa, derived from the N terminus of the product of the open reading frame (ORF), cosediments in sucrose gradients with RT, IN, and cDNA of Tf1 (36). This protein also has a nuclear localization signal (NLS) that is required for nuclear localization and, as a result, for transposition activity (16). This evidence is consistent with the possibility that the 27-kDa protein may function as the Gag of Tf1. Nevertheless, its sequence has no similarity to that of any known Gag. It lacks the MHR motif, and the 27-kDa protein is not processed into smaller species, such as MA, CA, and NC. In addition, it is not known whether this putative Gag protein assembles to form the shell of a particle or even if it is required for transposition. We tested the Gag of Tf1 for functions associated with Gag proteins. 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 characterization of these mutant transposons revealed that Gag expression was required for packaging of RNA, particle formation, and reverse transcription. These observations were possible because we report here for the first time that Tf1 is able to assemble into intracytoplasmic particles easily visible by electron microscopy. In fact, the expression of Gag alone was sufficient for the formation of VLPs. Taken together, these results indicate that the Gag of Tf1 functions similarly to the Gag proteins of retroviruses.