Assembly of Human Immunodeficiency Virus Precursor Gag Proteins

Abstract

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 Å is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers. During human immunodeficiency virus 1 (HIV-1)1 assembly, the viral precursor Gag (PrGag) protein oligomerizes on cellular membranes and directs the budding of immature virus particles. Normally, during or after budding, PrGag proteins are processed into the mature Gag proteins: matrix (MA), capsid, nucleocapsid (NC), and p6 (1). PrGag processing is accompanied by a dramatic change in HIV-1 particle morphology. By conventional thin section electron microscopy, the electrondense protein shell of immature virions appears to reorganize into centrally located, conical, or cylindrical mature virus cores (1).Recently, we demonstrated that the major HIV-1 Gag protein capsid could assemble in vitro into two distinct arrangements (2). Both of these arrangements showed hexameric rings of capsid N-terminal domains (NTDs) linked via C-terminal domain (CTD) contacts but differed in that one showed tight packing of hexamers with adjacent NTD rings in apparent contact, whereas the other featured clearly separated NTD rings (2). Interestingly, the two in vitro arrangements appear to have in vivo counterparts. The tightly packed arrangement is similar to that observed in immature virions, where hexamer-to-hexamer spacing is in the range of 65 to 80 Å (2-5). In contrast, the loosely packed arrangement appears to correspond to the organization of capsid proteins in mature cores assembled in vitro and in vivo with a hexamer ring-to-ring spacing of 95-110 Å (6, 7). The similarity between our results and those observed in vivo prompted us to propose a model in which viral morphogenesis was accompanied by a shift from tightly packed hexamer rings to loosely packed rings, and we predicted that virions would have mo...

Domain-swapped dimerization of the HIV-1 capsid C-terminal domain

“…However, the sizes of the spheres vary significantly (from 50 to Ͼ300 nm), with the most common size being Ϸ200 nm. These spheres are not as uniform and are significantly larger than the particles reported for the wild-type p6-deleted Gag constructs (5,27,29). Intriguingly, the size distribution of the particles formed by the ⌬177 Gag in vitro is similar to the one observed in vivo for a Gag mutant with impaired CA-CTD dimerization (31).…”

“…Purified CA can assemble at high salt conditions into tubes and cones, mimicking the architecture of mature viral cores isolated from the live virus (22)(23)(24)(25)(26). On the other hand, longer Gag constructs, encompassing various portions of the matrix domain, CA, spacer peptide 1 (SP1), and nucleocapsid, were shown to assemble into spheres that are similar to the immature viral particles (5,(27)(28)(29). To probe the effects of the ⌬177 mutation on assembly, we introduced this deletion into the full-length CA protein (30) and the p6-deleted construct of Gag (5).…”

“…The C terminus of the CA-CTD construct was exactly at the C terminus of the mature CA (Leu-231). The CA and the p6-deleted construct of Gag (MACANCexact) were expressed and purified following published procedures (5,30). Sedimentation equilibrium experiments with the ⌬177 CA-CTD mutant were performed by using a Beckman XLA analytical centrifuge in the 50 mM sodium phosphate (pH 6.5)/50 mM NaCl/5 mM DTT buffer (Beckman Coulter, Fullerton, CA).…”

The structural biology of HIV assembly

“…Nevertheless, further studies will be required to confirm these models and provide molecular details of the intra-and interhexamer contacts in the immature HIV-1 Gag lattice. In this regard, considerable insight will likely be gleaned from higher resolution structural studies of immature HIV-1, MLV, and MPMV (Mazon-Pfizer monkey virus) Gag assemblies formed in vitro [62][63][64][65], and from deuterium-exchange protection experiments that map protein-protein interfaces in the immature lattice (P. Prevelige, pers. comm.…”

Une nouvelle vision de l’assemblage du VIH-1

“…Dans certaines cellules, cibles physiologiques de l'infection, les particules virales s'assembleraient et/ou s'accumuleraient dans des compartiments intracellulaires tels que les endosomes tardifs, pour être libérées dans le milieu extracellulaire, ou transmises directement à une cellule saine via une « synapse virale ». Une telle vision « à couvert » de l'assemblage du VIH- [5]. Dans Gag, l'hélice carboxy-terminale du CTD se prolonge dans un court peptide (SP1) libéré après maturation par la protéase et reliant CA à la nucléocapside (NC).…”