Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly.
Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the > 1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.
Morikawa
Y
Y
The Kitasato Institute, Minato-ku, Tokyo 108-8642, Japan. ymorikawa@kitasato.or.jp
Hockley
D J
DJ
Nermut
M V
MV
Jones
I M
IM
D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D017931:DNA Primers; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D008855:Microscopy, Electron, Scanning; D019814:Myristic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D018411:Spodoptera / Q000648:ultrastructure
D001704:Biopolymers; D017931:DNA Primers; D015683:Gene Products, gag; D019814:Myristic Acid