Quenching protein dynamics interferes with HIV capsid maturation

Abstract

Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA–SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA–SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix–coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions.

Identification of a Structural Element in HIV-1 Gag Required for Virus Particle Assembly and Maturation

“…This result implies that changes in the PPIP(122-125) motif do not prevent the formation of the six-helix bundle at the CA-SP1 junction and thus do not abrogate hexamer assembly. Interestingly, there does appear to be some cross talk between the PPIP(122-125) motif and SP1, as a mutation at residue 8 of SP1 (SP1-T8I [44,45]) results in conformational changes in the PPIP(122-125) loop (46).…”

All-atom virus simulations

“…During maturation, proteolytic processing of Gag yields cleavage products that reassemble to produce mature virions [123]. All-atom simulations proved invaluable in determining the structures of the immature HTLV-1 [124] and RSV [125] Gag lattices, as well as the HIV-1 capsid protein-SP1 peptide maturation intermediate (CA-SP1) [126]. Notably, simulations revealed that the CA-SP1 six-helix bundle exists in a dynamic helix-coil equilibrium, and that maturation-inhibiting drugs and mutations act by stabilizing a helical form of the bundle [126].…”

“…All-atom simulations proved invaluable in determining the structures of the immature HTLV-1 [124] and RSV [125] Gag lattices, as well as the HIV-1 capsid protein-SP1 peptide maturation intermediate (CA-SP1) [126]. Notably, simulations revealed that the CA-SP1 six-helix bundle exists in a dynamic helix-coil equilibrium, and that maturation-inhibiting drugs and mutations act by stabilizing a helical form of the bundle [126]. MD studies of CA-SP1 bound to inositol phosphates showed that these small molecules facilitate the formation of the six-helix bundle and, thus, promote assembly of the Gag lattice [127].…”

Strukturelle Untersuchung subviraler Partikel durch die Kombination von zellfreier Proteinherstellung mit 110 kHz MAS‐NMR‐Spektroskopie

“…Damit reduziert sich die erforderliche Probenmenge um fast zwei Größenordnungen und es eröffnet sich die Möglichkeit, die Proteine in den Mengen, wie sie typischerweise in zellfreier Herstellung gewonnen werden, zu analysieren. Während >100 kHz MAS‐NMR‐Spektroskopie, zellfreie Proteinherstellung, NMR‐Spektroskopie an großen Komplexen und von Membranproteinen schon in der Literatur beschrieben wurden, zeigen wir hier, dass trotz der bereits hohen Komplexität jedes einzelnen dieser Elemente steht, deren Verbindung möglich ist und uns erlaubt hat, NMR‐Daten der DHBV‐SVPs mit guter Auflösung und Empfindlichkeit zu erhalten. Dies sollte die Untersuchungen dieser und ähnlich großer Membranproteinkomplexe, deren Strukturen bisher nicht aufgeklärt werden konnten, ermöglichen.…”