Implications for Viral Capsid Assembly from Crystal Structures of HIV-1 Gag_1-278 and CA^N_133-278^,

Abstract

Gag, the major structural protein of retroviruses such as HIV-1, comprises a series of domains connected by flexible linkers. These domains drive viral assembly by mediating multiple interactions between adjacent Gag molecules and by binding to viral genomic RNA and host cell membranes. Upon viral budding, Gag is processed by the viral protease to liberate distinct domains as separate proteins. The first two regions of Gag are MA, a membrane-binding module, and CA, which is a two-domain protein that makes important Gag-Gag interactions, forms the cone-shaped outer shell of the core (the capsid) in the mature HIV-1 particle, and makes an important interaction with the cellular protein cyclophilin A (CypA). Here, we report crystal structures of the mature CA N-terminal domain (CA(N)(133-278)) and a MA-CA(N) fusion (Gag(1-278)) at resolutions/R(free) values of 1.9 A/25.7% and 2.2 A/25.8%, respectively. Consistent with earlier studies, a comparison of these structures indicates that processing at the MA-CA junction causes CA to adopt an N-terminal beta-hairpin conformation that seems to be required for capsid morphology and viral infectivity. In contrast with an NMR study (Tang, C., et al. (2002) Nat. Struct. Biol. 9, 537-543), structural overlap reveals only small relative displacements for helix 6, which is located between the beta-hairpin and the CypA-binding loop. These observations argue against the proposal that CypA binding is coupled with beta-hairpin formation and support an earlier surface plasmon resonance study (Yoo, S., et al. (1997) J. Mol. Biol. 269, 780-795), which concluded that beta-hairpin formation and CypA-binding are energetically independent events.

The structural biology of HIV assembly

“…High resolution structures show that the free MA and CA NTD domains are connected by a flexible linker that could easily span the distance between the MA and CA NTD layers of the immature virion [53,54]. The MA domain does not change structure when tethered to CA NTD , consistent with the idea that the membrane-binding "heads" of MA are connected via flexible linkers to the CA NTD hexamers below.…”

Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

“…Upon cleavage at the MAjCA junction, the first 13 N-terminal residues of CA (residues 133-145) fold into a β-hairpin (30), disruption of which leads to noninfectious virions with aberrant morphologies (31)(32)(33). The 1 D NH RDC values for these hairpin residues calculated from the X-ray coordinates of CA NTD (24), using the alignment tensor obtained from the SVD fits for the remaining CA NTD , however, do not match with their experimentally observed counterparts in ΔGag (Fig.…”

A Strongly Transdominant Mutation in the Human Immunodeficiency Virus Type 1 gag Gene Defines an Achilles Heel in the Virus Life Cycle

“…All orthoretroviruses encode a proline at the N terminus of CA (30). This proline plays a key structural role, as it anchors a salt bridge between its ring nitrogen and carboxylate group of a conserved aspartic acid (D51 in HIV-1 CA), creating a new loop with the N-terminal protein sequence that folds into a ␤-hairpin structure (21,28,38,42). Preventing cleavage at the MA/CA site would sequester the proline from the formation of this loop, inhibiting proper core assembly.…”

“…With the proteolysis of Gag, there is a dramatic structural rearrangement in which the CA proteins condense to form the cone-shaped capsid shell surrounding the NC/RNA nucleoprotein complex (43). During maturation, the released N terminus of the CA protein adopts a ␤-hairpin structure by forming a salt bridge between Pro1 and Asp51 of CA, which appears to be important for the assembly of conical capsid (21,28,38,42). Since proteolytic processing is essential for the formation of infectious virus, PR has been the target of a very successful group of inhibitors now in clinical use.…”