SEQUENTIAL STEPS IN HUMAN-IMMUNODEFICIENCY-VIRUS PARTICLE MATURATION REVEALED BY ALTERATIONS OF INDIVIDUAL GAG POLYPROTEIN CLEAVAGE SITES

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase ( PR), Extracellular maturation leads to condensation of the spherical c ore to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins, CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on th e human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and rele ased by sequential PR-mediated cleavages. To assess the role of indivi dual cleavages in maturation, we constructed point mutations abolishin g cleavage at these sites. either alone or in combination, When all th ree sites between CA and NC were mutated, immature particles containin g stable CA NC were observed, with no apparent effect on other cleavag es, Delayed maturation with irregular morphology of the ribonucleoprot ein core was observed when cleavage of SP1 from NC was prevented. Bloc king the release of SP1 from CA, on the other hand, yielded normal con densation of the ribonucleoprotein core but prevented capsid condensat ion. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable again st detergent treatment than wild-type HIV-1. We suggest that HIV matur ation is a sequential profess controlled by the rate of cleavage at in dividual sites, Initial rapid cleavage at the C terminus of SP1 releas es the RNA binding NC protein and leads to condensation of the ribonuc leoprotein core, Subsequently, CA is separated from the membrane by cl eavage between the matrix protein and CA and late release of SP1 from CA is required for capsid condensation.