In Vitro Assembly Properties of Purified Bacterially Expressed Capsid Proteins of Human Immunodeficiency Virus

Abstract

The Gag polyprotein of retroviruses is sufficient for embly antl budding of virus-like particles from the host cell. In the case of human immunodeficiency virus (HTV), Gag contains the domains matrix, capsid (CA), nucleocapsid (NC) and p6 which are separated by the viral proteinase inside the nascent virion, leading to morphological maturation to yield an infectious virus. In the mature virus, CA forms a capsid shell surrounding the ribonucleoprotein core consisting of NC and the genomic RNA. To define requirements for particle assembly and functional contributions of individual domains, we expressed domains of HIV Gag in Escherichiu coli and purified the products to near homogeneity. In vitro assembly of CA, with or without the C-terminally adjacent spacer peptide, yielded tubular structures with a diameter of approximately 55 nm and heterogeneous length. Efficient particle formation required high protein Concentration, high salt and neutral to alkaline pH. In contrast, in iitro assembly of CA-NC occurred at a 20-fold lower protein concentration and in low salt, but required addition of RNA. These results suggest that hydrophobic interactions of capsid proteins are sufficient for particle formation while the RNAbinding iiucleocapsid domain may concentrate and align structural protcins on the viral genome.

Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication

“…Within the constraints of an HIV-1 particle, the concentration of CA is quite high, favoring CA multimerization. Results from our study suggest relatively weak CA-CA interactions, as seen for CA assemblies in vitro (16,17,23,37), since detergent treatment of virions induced the solubilization of the majority of the virion-associated CA. Similarly, fusion of the virus with a target cell would release the volume constraints on the particle.…”

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly

“…Similar HIV-1 Gag-derived proteins assemble narrow tubular arrays (Fig. 1A) (19,20,24), which have a mature-like arrangement of CA. Prior studies had indicated that certain HIV-1 Gag-derived proteins can assemble tubular or spherical particles depending on the conditions (22) and that certain mutations in the CA-SP1 region converted the assembly from spherical to tubular or aberrant sheet-like structures (12,25).…”

“…The helical symmetry of the M-PMV ΔProCANC tubes allowed application of a hybrid cryo-electron tomography (ET) and cryo-EM image processing approach for structure determination (11). HIV-1 Gag derivatives that assemble tubular arrays adopt a mature-like arrangement of CA (19,20), and have indeed been important tools in determining the structure of the mature CA core (9,21). In contrast, HIV-1 Gag derivatives known to assemble into immature-like arrays form approximately spherical structures (22,23) that are unsuitable for application of high-resolution helical reconstruction methods.…”

The structural biology of HIV assembly

“…Pure recombinant HIV-1 CA proteins and CA-SP1-NC/RNA complexes can form cylindrical and conical shells in vitro, implying that all of the information necessary to form a conical capsid resides within the CA polypeptide [89,[99][100][101]. Mature HIV-1 and RSV CA proteins form hexagonal arrays of hexameric CA NTD rings, with each ring connected to its six nearest neighbors via CA CTD dimer interactions [90,91,97].…”