Mutations in SP and adjoining sequences in Rous sarcoma virus Gag lead to tubular budding

Dept. of Molecular Biology and Genetics, Biotechnology Bldg, Cornell University, Ithaca, NY 14853; Dept of Molecular Microbiology and Immunology, Life Science Center, University of Missouri Medical School, Columbia, MO 65211

^* To whom correspondence should be addressed. Email: vmv1{at}cornell.edu.

	Abstract

All ortho-retroviruses 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 immediately upstream residues are known to be critical for proper assembly. Using mutagenesis and electron microscopy analysis of insect cells or chicken cells over-expressing RSV Gag, we defined the SP assembly domain to include the last eight residues of CA, all twelve residues of SP, and the first four 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.