Late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis.
Retroviral Gag polyproteins contain regions that promote the separation of virus particles from the plasma membrane and from each other. These Gag regions are often referred to as late assembly (L) domains. The L domain of human immunodeficiency virus type 1 (HIV-1) is in the C-terminal p6(gag) domain and harbors an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses are in the N-terminal half of the Gag precursor and have a PPXY core motif. We recently observed that L domains induce the ubiquitination of a minimal HIV-1 Gag construct and that point mutations which abolish L domain activity prevent Gag ubiquitination. In that study, a peptide from the Ebola virus L domain with overlapping P(T/S)APP and PPXY motifs showed exceptional activity in promoting Gag ubiquitination and the release of virus-like particles. We now show that a substitution which disrupts the PPXY motif but leaves the P(T/S)APP motif intact abolishes L domain activity in the minimal Gag context, but not in the context of a near full-length HIV-1 Gag precursor. Our results reveal that the P(T/S)APP motif does not function autonomously and indicate that the HIV-1 nucleocapsid-p1 region, which is proximal to p6(gag), can cooperate with the conserved L domain core motif. We have also examined the effects of ubiquitin mutants on virus-like particle production, and the results indicate that residues required for the endocytosis function of ubiquitin are also involved in virus budding.
Strack
Bettina
B
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Calistri
Arianna
A
Göttlinger
Heinrich G
HG
D020816:Amino Acid Motifs; D004705:Endocytosis / Q000502:physiology*; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D014771:Virion; D019065:Virus Assembly / Q000502:physiology*; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D054301:gag Gene Products, Human Immunodeficiency Virus; C083868:gag protein p1, Human immunodeficiency virus; C083002:p6 gag protein, Human immunodeficiency virus 1