Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA

Abstract

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

“…For NMR studies, the proteins were dialyzed against 50 mM sodium phosphate (pH 5.5), 100 mM NaCl, and 5 mM dithiothreitol (DTT) and then concentrated by Millipore ultrafiltration concentrators against the same buffer. Protein purities were evaluated after fractionation by SDS-PAGE (32,33,(57)(58)(59)(60)(61) by Coomassie Blue staining (32,(57)(58)(59). The HIV NC peptide was prepared commercially as reported previously (61).…”

Human Immunodeficiency Virus Type 1 Matrix Protein Assembles on Membranes as a Hexamer

“…Final liposome lipid concentrations were 0.4 mg/ml cholesterol and 1.6 mg/ml of either PC or PS, and liposomes were stored for up to 1 month under nitrogen at Ϫ20°C. Monolayer membrane binding assays were modified from the monolayer binding assays described by Zuber et al (63). One-hundred-microliter samples containing 200 to 300 nM protein in monolayer buffer (25 mM sodium phosphate [pH 7.8], 50 to 125 mM NaCl) were deposited into 15-mm-wide, 1.5-mm-deep glass depression (Boerner) slides.…”

“…While HIV-1 MA associates in solution and in 3D crystals as trimers, the capsid domains of retroviruses tend to organize into hexamer rings (3,4,6,7,25,27,30,31,32,34,37,39,40,63). These observations have prompted several researchers to incorporate MA trimers into PrGag hexamer models by postulating that trimers form nodes which interconnect hexamers and that two MA members from each of three trimer units position themselves in a p3 fashion over CA hexamers (15,48).…”

“…Moreover, in X-ray crystal structures, the exposed basic residues of MA trimers align in a way that could mediate binding to acidic phospholipids at the cytoplasmic faces of cellular membranes (23,48). Not surprisingly, a number of investigators have found that retroviral matrix domains preferentially bind to membranes containing the net negatively charged phospholipid phosphatidylserine (PS) versus the neutral, zwitterionic phospholipid phosphatidylcholine (PC) (11,14,53,57,60,61).While HIV-1 MA associates in solution and in 3D crystals as trimers, the capsid domains of retroviruses tend to organize into hexamer rings (3,4,6,7,25,27,30,31,32,34,37,39,40,63). These observations have prompted several researchers to incorporate MA trimers into PrGag hexamer models by postulating that trimers form nodes which interconnect hexamers and that two MA members from each of three trimer units position themselves in a p3 fashion over CA hexamers (15,48).…”

Cell biology of retroviral RNA packaging

“…It was shown that nucleic acid greatly increases the efficiency of particles assembly, that the size of these particles was dependent on the size of the RNA, and that treatment of particles with ribonuclease triggered disassembly. 81,82 Later studies performed on MLV and HIV-1 particles generated in mammalian cells have demonstrated that, in the absence of viral gRNA, VLPs specifically package cellular mRNAs randomly, as mRNA species detected in VLPs are in proportion to their cellular levels, 24,83,84 at the exception of some RNA species such as 7SL, and U6 RNAs, that are specifically enriched in wild-type HIV-1 and MLV particles and whose function remains unclear. 24,25,83,84 Interestingly, retroviral cores containing either viral or cellular RNAs were disassembled by RNase treatment, 83 confirming the importance of the RNA-Gag interaction in the formation and structure of retroviral particles.…”