In vitro identification and characterization of an early complex linking HIV-1 genomic RNA recognition and Pr55Gag multimerization.
The minimal protein requirements that drive virus-like particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mGag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mGag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th alpha-helix of CA and the proposed alpha-helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mGag and a model RNA spanning the region from +223 to +506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mGag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study.
Roldan
Ariel
A
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada.
Russell
Rodney S
RS
Marchand
Bruno
B
Götte
Matthias
M
Liang
Chen
C
Wainberg
Mark A
MA
D019281:Dimerization; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D066298:In Vitro Techniques; D019590:Nucleocapsid Proteins / Q000235:genetics; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism*
D015683:Gene Products, gag; D019590:Nucleocapsid Proteins; D011498:Protein Precursors; D012367:RNA, Viral; C059767:p55 gag precursor protein, Human immunodeficiency virus 1