Ty3 Nucleocapsid Controls Localization of Particle Assembly

Ty3 is a long-terminal-repeat (LTR) retrotransposon in Saccharomyces cerevisiae that encodes a Gag3 protein with domains similar to those of retrovirus capsid (CA) and nucleocapsid (NC) (51, 59). Our previous studies showed that Ty3 protein, RNA, and viruslike particles (VLPs) accumulate in association with RNA processing bodies (P bodies), and these are proposed to be the sites of assembly (4). The present study was undertaken to determine the role of the Ty3 NC domain in the association of Ty3 protein and RNA with P bodies and in the assembly of VLPs. The similarity of retrovirus and Ty3 CA and NC domains suggests that they perform similar functions in their respective assembly contexts. The major structural proteins of retroviruses include matrix (MA), CA, and NC domains, as well as other less-conserved domains (12). The MA domain concentrates precursor polyproteins at the membrane (reviewed in references 1, 19, 46, 54, and 60) or the microtubule organizing center (65, 76) assembly sites. The Gag NC zinc-binding residues are directly implicated in the recognition of genomic RNA (17). In addition, the NC domain mediates less-specific interactions of Gag with RNA via patches of basic amino acids. Binding to RNA in turn facilitates Gag-Gag interactions within the CA domain that are required for assembly. Hence, these basic NC residues together with the spacer (SP) domain found between CA and NC domains of some retrovirus Gag proteins are designated the interaction or I domain (5, 6, 11, 13, 37, 42). Genetic studies with murine leukemia virus (MLV) (49) and Rous sarcoma virus (RSV) (37) show that mutations in NC are associated with a late phenotype, but the molecular basis of that phenotype is not known. The NC domain also mediates interactions with several host factors, some of which are related to intracellular localization and assembly. Interactions have been documented with the structural protein actin (41, 47, 55, 73), elongation factor 1 alpha (10), ABCE1 (40), APOBEC3G (61), and nucleolin (2). Despite the central role played by NC in RNA packaging and assembly, it is not completely clear when and how the first critical contacts occur between Gag and genomic RNA (8). There is evidence that Gag interactions with genomic RNA occur prior to arrival at the final assembly site. However, retroviruses differ in the mode of nuclear export of genomic RNA, the cis or trans nature of Gag association with template and/or genomic RNA, and localization of the assembly site. Human immunodeficiency virus type 1 (HIV-1) RNA and Gag concentrate early during expression at a pericentriolar site (38, 53). Delivery of RNA to the pericentriolar site is promoted by association with the A2 mRNA nuclear export protein (38). Interestingly, packaging efficiency is also partially dependent upon the HIV-1-encoded genomic RNA nuclear export factor, Rev (7, 68). These observations are consistent with the lack of bias for HIV-1 genomic RNA packaging by the proteins from which it is translated (50). Recent studies showed that mutations in the zinc-binding domain of HIV-1 NC disrupted trafficking of both Gag and genomic RNA to the plasma membrane assembly site (25). Association of Gag and genomic RNA prior to assembly occurs for other retroviruses as well. The MLV genomic RNA which originates from a nontranslated RNA pool (39) also associates intracellularly with Gag and Gag-Pol for trafficking to the plasma membrane assembly site (3). In cells infected by simian foamy virus (64) and RSV (9) Gag proteins traffic to the nucleus, and it has been proposed that they could capture genomic RNA for delivery to the assembly site. However, early nuclear localization of simian foamy virus Gag is not absolutely required for replication of virus in tissue culture cells. In the case of RSV, host factors have been identified as affecting nuclear export and packaging of genomic RNA (34). Thus, in both of these cases, the extent to which Gag or its NC domain is required for targeting the majority of genomic RNA to the assembly site remains unclear. Despite the prevalence of LTR retrotransposons throughout eukaryotes, intracellular assembly of endogenous retroviruses and retrotransposons is relatively unexplored. Clusters of VLPs are observed by electron microscopy (EM) in cells expressing endogenous retroviruses, including murine MusD (56) and ovine enJSRV (48). Retrotransposons in Drosophila, such as copia (45), in S. cerevisiae, such as Ty1 (21) and Ty3 (26), and in Schizosaccharomyces pombe, such as Tf1 (70) are also observed in clusters. However, the localization of assembly and identity of determinants that target both RNA and protein to those assembly sites are relatively unexplored. Ty3 is 5.4 kb in length and encodes Gag3 and Gag3-Pol3 precursor polyproteins (reviewed in reference 59) (Fig. ​(Fig.1A)1A) which form VLPs of 40 to 50 nm in diameter. Gag3, the major structural protein, is processed by Ty3 protease (PR) into 24-kDa CA, p27, and 57-amino-acid (aa) NC. A 26-aa SP3 between CA and NC domains is inferred from positions of processing sites but has not been observed (31, 32). Processing of Gag3-Pol3, in addition to CA and SP3, produces a 76-aa NC, which spans the frameshift of Gag3-Pol3, protease (PR), 10-kDa junction fragment, reverse transcriptase (RT), and integrase (IN). Expression of Ty3 under a synthetic GAL1-10 promoter or the native pheromone-inducible promoter results in the formation of VLP clusters (4, 26). VLPs are associated with Ty3 RNA and, after longer times of induction, with cDNA. Ty3 protein, RNA, and VLPs are concentrated in association with P bodies, the proposed site of assembly (4). Two lines of evidence support a role for NC in the formation and function of VLPs. First, mutations in the conserved zinc-binding motif of the Gag3 NC domain were previously shown to cause decreased production and processing of VLPs (51). Second, in vitro data support a role for Ty3 NC in annealing of the primer tRNAMet to Ty3 at 5′ and 3′ sites (20) and in priming Ty3 RT (14). FIG. 1. Ty3 Gag3. (A) Ty3 genome and polyprotein organization. Protein domains are as marked; SP3, a predicted junction fragment of about 10 kDa between PR and RT, and the differentially processed ends of IN are indicated in that order as empty boxes. (B) Mutagenesis ... The present study was undertaken to determine the role of Ty3 NC in the localization of Ty3 polyproteins and RNA with P-body components. The Ty3 NC domain was shown to be important for RNA cluster formation and for association of Ty3 proteins with P-body components. A subset of mutations disrupted particle formation and resulted in Gag3 nuclear localization. Our data support the model that NC mediates Ty3 RNA and protein association with P-body components and that this association occurs during assembly.