The Noncanonical Gag Domains p8 and n Are Critical for Assembly and Release of Mouse Mammary Tumor Virus

Abstract

The mouse mammary tumor virus (MMTV) Gag contains the unique domains pp21, p3, p8, and n. We investigated the contribution of these domains to particle assembly and found that the region spanning the p8 and n domains is critical for shape determination and assembly. Deletion of pp21 and p3 reduced the number of released particles, but deletion of the n domain resulted in frequent formation of aberrant particles, while deletion of p8 severely impaired assembly. Further investigation of p8 revealed that both the basic and the proline-rich motifs within p8 contribute to MMTV assembly.

Mouse mammary tumor virus (MMTV), a member of the Betaretrovirus genus, exhibits B-type morphology and assembles immature particles within the cell cytoplasm prior to their transport to the plasma membrane. The MMTV Gag polyprotein contains the noncanonical domains pp21, p3, p8, and n of unknown function(s), which are located between the matrix (MA) and capsid (CA) proteins (7, 10). These domains have unique sequences with low similarity to other retroviral Gag proteins, including the betaretroviral Mason-Pfizer monkey virus (M-PMV) phosphoprotein pp24/16, which contains late (L) domain motifs (6, 20), and the p12 protein, which contains the internal scaffold domain (ISD) responsible for efficient Gag-Gag interactions (16, 18, 23). No previously described L domain motifs (3, 4, 11) are present within the noncanonical domains. Interestingly, the p8 domain consists of a highly basic N-terminal region and a C-terminal part that contains the proline-rich motif PPxxF. This motif is thought to function as an interaction sequence for proteins containing class II Homer/Vesl (EVH1) or wasp homology 1 (WH1) domains (1, 2) that mediate essential interactions with multiple cellular pathways (17) including regulation of actin dynamics (13).

To investigate the role of specific MMTV Gag domains in assembly and particle release, we prepared a series of Gag mutants containing deletions of individual domains (Δpp21, Δp3, Δp8, and Δn) or the p8 motifs (Δ1-p8, residues 1 to 17; Δ2-p8, residues 18 to 26) (Fig. 1 A). The Gag sequences were cloned into the proviral vectors pSMt-HYB and pSMt-HYB/D26A, which include a mutation of the protease active site (22). These chimeric constructs utilize the heterologous M-PMV 5′ long terminal repeat (LTR) sequence and produce assembly-competent but noninfectious MMTV. Transcription driven by heterologous LTR enabled measurable expression in transiently transfected human 293T cells, which have been shown to support the production of infectious MMTV particles (8, 14). The 293T cells were transfected using the FuGENE HD (Roche Molecular Biochemicals) reagent. Pulse-chase experiments using cells metabolically labeled with [³⁵S]methionine-[³⁵S]cysteine protein labeling mix and immunoprecipitation of viral proteins by rabbit polyclonal anti-MMTV CA antiserum were performed as described previously (22).

FIG. 1.

Synthesis, processing, and release of viral proteins from 293T cells expressing MMTV Gag deletion mutants. (A) Schematic representation of the MMTV Gag polyprotein and deletion mutants. The total number of amino acids (aa) in each polyprotein is specified. Gag domains are labeled as follows: MA, matrix protein; pp21, phosphoprotein; p3, protein p3; p8, protein p8; n, peptide n; CA, capsid protein; NC, nucleocapsid protein; Myr, myristic acid moiety. (B) Pulse-chase analysis of MMTV Gag deletion mutants expressed in 293T cells transiently transfected with pSMt-HYB (lanes 1 to 6)- and pSMt-HYB/D26A (lanes 7 to 15)-derived proviral constructs. Viral proteins were immunoprecipitated with polyclonal rabbit anti-CA serum, separated by SDS-12% PAGE, and subjected to phosphorimager analysis. The migration positions of prestained molecular size standards in kilodaltons are indicated on the left, the migration positions of the MMTV Gag and its individual deletion mutants are marked by asterisks, and the position of the mature CA is indicated on the right.

For each mutant the Gag, Gag-Pro, and Gag-Pro-Pol polyproteins were synthesized in the transfected cells (Fig. 1B). Following virus release, the Δpp21 and Δp3 Gag mutants were processed by protease efficiently and the levels of CA protein in released particles were comparable between these mutants and wild-type (wt) Gag (Fig. 1B, lanes 3 and 4). The Δn mutant produced particles at a significantly reduced but still detectable level (Fig. 1B, lane 6), while the deletion of the p8 domain resulted in failure for particle production in 293T cells.

Immature viral particles released from cells transfected with the pSMt-HYB/D26A-derived constructs were detected for wt and Δpp21, Δp3, and Δn mutants (Fig. 1B, lanes 8 to 10 and 12). The production of extracellular particles was reduced by approximately 50% for the Δpp21 and Δp3 mutants. Deletion of the n domain resulted in a significant decrease of particle release (by 80%), and no released particles were detected for the Δp8 mutant using autoradiography of labeled virus-associated proteins separated on SDS-polyacrylamide gels. Very low but detectable levels of released Gag were produced by Δ1-p8 (Fig. 1B, lane 14) and Δ2-p8 (Fig. 1B, line 15), suggesting that both parts of this Gag region contribute to the process of particle production.

We further examined the phenotype of MMTV Gag mutants observed in 293T cells in the RBA rat mammary cell line. It has been reported elsewhere that rat cells are susceptible to MMTV infection (5, 15) and transfection by some MMTV hybrid constructs (19). Unfortunately, in contrast to 293T cells, the mammary gland cells do not support episomal replication of simian virus 40 (SV40) origin-containing plasmids like the pSMt-HYB-derived constructs. The MMTV expression in RBA cells was barely detectable using metabolic labeling, and therefore, we analyzed the synthesis of cell-associated viral and virus-associated proteins by Western blotting and ultrasensitive chemiluminescent detection (Fig. 2 A). RBA cells (ATCC) grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) on 100-mm plates were transfected with the pSMt-HYB/D26A-derived constructs using Lipofectamine LTX reagent (Invitrogen). The medium was replaced 6 h after transfection with fresh DMEM and incubated for 36 h to allow virus production. The culture medium was then filtered through a 0.45-μm filter, and particles were collected by centrifugation through a 20% sucrose cushion. The proteins were separated by SDS-10% PAGE and detected by a rabbit anti-MMTV CA polyclonal antibody. Under these conditions we were able to detect virus-associated protein production for the deletion mutants as well as wt (Fig. 2). We detected a very low level of released particles for Δp8, which we have not found in the human cells. Therefore, we retested the Δp8 mutant in 293T cells under the conditions used for this mutant in the rat cells and we also detected a low level of released Δp8 Gag by Western blot analysis (Fig. 2B, lane 11), indicating that the cell type does not influence the phenotype of this Gag mutant.

FIG. 2.

Western blot analysis of MMTV Gag mutants produced by rat mammary cells (RBA) (lanes 1 to 8) (A) and 293T cells (lanes 9 to 11) (B) transiently transfected with pSMt-HYB/D26A-derived constructs. At 36 h posttransfection, cell-associated viral proteins and virion-associated proteins were separated by SDS-10% PAGE and detected by Western blot assay using polyclonal anti-MMTV CA antiserum. The migration positions of molecular size standards in kilodaltons are indicated on the left, and the migration positions of the MMTV Gag and its individual deletion mutants are marked by asterisks.

To determine the effect of the various Gag deletions on particle morphology, 293T cells transfected with pSMt-HYB/D26A-based constructs were analyzed by transmission electron microscopy (Fig. 3). For the full-length Gag construct, we observed assembled particles within the cytoplasm and few particles assembling at the plasma membrane with C-type-like morphology (Fig. 3, WT). The mean diameter of released immature MMTV particles was approximately 110 ± 10 nm (n = 26). A lower number of similar particles was found also for the Δpp21 and Δp3 mutants. In cells transfected with the Δp8 mutant, we did not observe any preassembled structures in the cytoplasm and no particles were found at the cell membrane in the process of budding and release. However, we did find a very low level of budding particles for both the Δ1-p8 and the Δ2-p8 mutants. Deletion of the p8 basic motif (Δ1-p8) yielded structures assembling with C-type-like morphology at the plasma membrane. The shape of the few released particles found was similar to that of wt MMTV. In contrast, the Δ2-p8 mutant produced a limited number of elongated aberrant particles seen both in the process of budding and as apparently released particles. The effects of the Δp8 and Δ2-p8 mutants and the loss of the PPxxF EVH1 motif (WASP) within these mutants imply that EVH1-regulated actin dynamics may be involved in the intracellular trafficking of retroviral Gag polyproteins. However, further experiments are necessary to confirm the role of actin in MMTV Gag transport. Removal of the very short n domain adjacent to the CA region in MMTV Gag also resulted in formation of aberrant particle structures (Fig. 3). We observed preassembled as well as released particles with both tubular and round shapes with a diameter of about 80 ± 5 nm (n = 20), indicating the important role of the n domain in proper particle shape determination. A similar tubular phenotype was observed when 25 residues in the Rous sarcoma virus p10 domain were deleted. This specific sequence was shown to be critical for Gag-Gag interactions and for the correct shape of immature particles (9, 12). Filamentous structures were also observed for a mutant of Moloney murine leukemia virus, in which the positionally analogous p12 domain was deleted (21).

FIG. 3.

Thin-section electron microscopy analysis of immature MMTV particles produced by Gag deletion mutants in transiently transfected 293T cells. Scale bars, 200 nm.

Taken together, our results define an MMTV Gag-specific assembly region, comprising domains p8 and n, located immediately upstream of the CA domain. The distinct motifs within the p8 domain and the n domain appear to work in an additive and synergistic manner, since all three are required for a wt level of assembly and particle release. However, they each may possess distinct functions either potentially in transport or for the correct shape of immature particles. Although the molecular and cellular mechanisms by which MMTV achieves virion production remain to be more fully characterized, we provide here an initial assessment of the noncanonical Gag domains in these processes. Despite similarities in its morphogenetic pathway with M-PMV, MMTV appears to employ different mechanisms for intracytoplasmic assembly, budding, and release. Consistent with previously published data (23), the present results support the idea that the potential to support particle assembly is spread over individual noncanonical MMTV domains, which contribute to this process in a cumulative manner.