Mutations in the Spacer Peptide and Adjoining Sequences in Rous Sarcoma Virus Gag Lead to Tubular Budding

Abstract

All orthoretroviruses encode a single structural protein, Gag, which is necessary and sufficient for the assembly and budding of enveloped virus-like particles from the cell. The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) contain a short spacer peptide (SP or SP1, respectively) separating the capsid (CA) and nucleocapsid (NC) domains. SP or SP1 and the residues immediately upstream are known to be critical for proper assembly. Using mutagenesis and electron microscopy analysis of insect cells or chicken cells overexpressing RSV Gag, we defined the SP assembly domain to include the last 8 residues of CA, all 12 residues of SP, and the first 4 residues of NC. Five-or two-amino acid glycine-rich insertions or substitutions in this critical region uniformly resulted in the budding of abnormal, long tubular particles. The equivalent SP1-containing HIV-1 Gag sequence was unable to functionally replace the RSV sequence in supporting normal RSV spherical assembly. According to secondary structure predictions, RSV and HIV-1 SP/SP1 and adjoining residues may form an alpha helix, and what is likely the functionally equivalent sequence in murine leukemia virus Gag has been inferred by mutational analysis to form an amphipathic alpha helix. However, our alanine insertion mutagenesis did not provide evidence for an amphipathic helix in RSV Gag. Taken together, these results define a short assembly domain between the folded portions of CA and NC, which is essential for formation of the immature Gag shell.

In Vitro Assembly of Virus-Like Particles of a Gammaretrovirus, the Murine Leukemia Virus XMRV

“…Structural and biochemical data suggest that the ordered arrays of CA are regulated by the presence (␤ ϩ ) or absence (␤ Ϫ ) of the ␤-hairpin at the N terminus and the presence (SP ϩ ) or absence (SP Ϫ ) of the SP sequence at the CA C terminus, thus determining the particle morphology (23,28). By this convention, within the immature virus, CA is in the form ␤ Ϫ /SP ϩ , and within the mature virus, it is in the form ␤ ϩ /SP Ϫ .…”

“…In vitro and in vivo assembly studies on HIV CANC and RSV CANC show that the presence of both N-and C-terminal deter- minants (␤ ϩ /SP ϩ ) of CA results in mature-like tubular (and, in the case of HIV-1, also conical) particle assembly (8,18,22). The HIV-1 ⌬MACA⌬SP1NC and RSV ⌬PRGag proteins, which do not form the ␤-hairpin and in which SP is mutated or deleted (␤ Ϫ /SP Ϫ ), assemble into tubular (and, in the case of HIV-1, also conical) particles (23,28). The mature CA proteins (␤ ϩ /SP Ϫ ) of HIV and RSV assemble in vitro into tubular structures; however, the conditions under which these structures are formed (ϳ1 M NaCl) suggest different types of CA-CA interactions (14,22,30).…”

“…Both HIV-1 and RSV SP sequences were also suggested to function as molecular switches, because the deletion of SP1 of HIV-1 Gag led to the formation of tubular instead of spherical particles (23). Similarly, mutations within the SP sequence of RSV Gag change spherical-particle formation to tubular-particle formation (28).…”

“…In the alpharetroviruses (e.g., Rous sarcoma virus [RSV]) and lentiviruses (e.g., HIV-1), a short spacer peptide (SP) (12 or 14 amino acids, respectively) separates CA from NC. This SP domain plays a critical role in assembly, as mutations or deletions in the SP influence immature-sphericalparticle assembly (1,28,33,37). Both HIV-1 and RSV SP sequences were also suggested to function as molecular switches, because the deletion of SP1 of HIV-1 Gag led to the formation of tubular instead of spherical particles (23).…”

Functional Virus-Like Particles Production Using Silkworm and Their Application in Life Science

“…This property makes them an ideal candidate for presenting antigenic proteins in a form that closely resembles the native state and thus provides a method to produce vaccines [26]. Gag proteins usually form VLPs of fixed size but this shape and size can be manipulated by addition of spacer peptides or deletion of specific regions [27]. Almost all the proteins have to interact with cell membranes and many are associated with membranes.…”