IN-VITRO ASSEMBLY PROPERTIES OF PURIFIED BACTERIALLY EXPRESSED CAPSIDPROTEINS OF HUMAN-IMMUNODEFICIENCY-VIRUS

The Gag polyprotein of retroviruses is sufficient nt for assembly and budding of virus-like particles fi-om the host cell, In the case of hu man immunodeficiency virus (HIV), Gag contains the domains matrix, cap sid (CA), nucleocapsid (NC) and p6 which are separated by the viral pr oteinase inside the nascent virion, leading to morphological maturatio n to yield an infectious virus. In the mature virus, CA forms a capsid shell surrounding the ribonucleoprotein core consisting of NC and the genomic RNA, To define requirements for particle assembly and functio nal contributions of individual domains, we expressed domains of HIV G ag in Escherichia coli and purified the products to near homogeneity. ill vitro assembly of CA. with or without the C-terminally adjacent sp acer peptide, yielded tubular structures with a diameter of approximat ely 55 nm and heterogeneous length. Efficient particle formation requi red high protein concentration, high salt and neutral to alkaline pH. In contrast, in vitro assembly of CA-NC occurred at a 20-fold lower pr otein concentration and in low sail, but required addition of RNA. The se results suggest that hydrophobic interactions of capsid proteins ar e sufficient for particle formation while the RNA-binding nucleocapsid domain may concentrate and align structural proteins on the viral gen ome.