Human immunodeficiency virus type 1 capsid formation in reticulocyte lysates.
The Gag polyprotein of human immunodeficiency virus (HIV) (Pr55Gag) contains sufficient information to direct particle assembly events when expressed within tissue culture cells. HIV Gag proteins normally form particles at a plasma membrane assembly site, in a manner analogous to that of the type C avian and mammalian leukemia/sarcoma viruses. It has not previously been demonstrated that immature HIV capsids can form without budding through an intact cellular membrane. In this study, a rabbit reticulocyte lysate translation reaction was used to recreate HIV capsid formation in vitro. Production of HIV-1 Pr55Gag and of a matrix-deleted Gag construct resulted in the formation of a subset of Gag protein structures with an equilibrium density of 1.15 g/ml. Gel filtration chromatography revealed these Gag protein structures to be larger than 2 x 10(6) Da, consistent with the formation of large multimers or capsids. These Gag protein structures were protease sensitive in the absence of detergent, indicating that they did not contain a complete lipid envelope. Spherical structures were detected by electron microscopy within the reticulocyte lysate reaction mixtures and appeared essentially identical to immature HIV capsids or retrovirus-like particles. These results demonstrate that the HIV Gag protein is capable of producing immature capsids in a cell-free reaction and that such capsids lack a complete lipid envelope.
Spearman
P
P
Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232-2581, USA.
Ratner
L
L
D000818:Animals; D002213:Capsid / Q000378:metabolism* / Q000648:ultrastructure; D002845:Chromatography; D003902:Detergents / Q000494:pharmacology; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D017830:Octoxynol / Q000494:pharmacology; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D014158:Transcription, Genetic; D014357:Trypsin / Q000378:metabolism; D019065:Virus Assembly
D003902:Detergents; D015683:Gene Products, gag; D011498:Protein Precursors; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D017830:Octoxynol; D014357:Trypsin