The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity.
Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.
Kräusslich
H G
HG
Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Fäcke
M
M
Heuser
A M
AM
Konvalinka
J
J
Zentgraf
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism* / Q000472:pathogenicity; D006868:Hydrolysis; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014162:Transfection
D015683:Gene Products, gag; D010446:Peptide Fragments