Ensamblaje in vitro de la cápsida del virus de la inmunodeficiencia humana y su inhibición por péptidos diseñados racionalmente

• Autores: Rebeca Bocanegra Rojo
• Directores de la Tesis: Mauricio García Mateu (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2011
• Idioma: español
• Tribunal Calificador de la Tesis: Luis Menéndez Arias (presid.), Iván Ventoso Bande (secret.), Jose Luis Neira Falairo (voc.), Miguel Ángel Martínez Sierra (voc.), C. López Galíndez (voc.), German Rivas Caballero (voc.), Juan Carlos Saíz Calahorra (voc.)
• Enlaces
  • Tesis en acceso abierto en: digitool-uam.greendata.es
• Resumen

  • Morphogenesis of the human immunodeficiency virus type 1 (HIV¿1) involves two stages. The first stage leads to the formation of an immature, non¿infectious viral particle that contains a spherical capsid made of multiple copies of the Gag polyprotein, which includes the capsid protein CA. The interfaces between Gag subunits are still not well defined. Recently, a mutated form of the C¿terminal domain (CTD) of CA has been shown to form in solution a domain¿swapped dimer. The domain¿swapped interface involves the highly conserved and functionally important MHR sequence, and has been proposed to have a role during immature capsid assembly. The second stage of HIV¿1 morphogenesis involves a dramatic structural rearrangement of the immature viral particle to yield a mature, infectious virion. During HIV¿1 maturation CA is released as an independent protein composed of two domains, the N¿terminal domain (NTD) and the CTD. In the maturing virion, CA self¿assembles to form a truncated cone¿shaped capsid.

    Assembly of the mature HIV¿1 capsid involves several well¿defined, discrete intersubunit interfaces in which one or both CA domains participate. NTD¿NTD and NTD¿CTD interfaces are involved in the formation of CA hexamers, and CTD¿CTD interfaces are involved in joining each hexamer to its neighbors through homodimerization. All of these interfaces are being structurally and functionally studied in detail and constitute attractive targets for the design of assembly inhibitors acting as new anti¿HIV¿1 agents. In the last few years, a few organic compounds and peptides identified by screening or combinatorial approaches have been shown to inhibit HIV¿1 capsid assembly; some of those compounds showed also antiviral activity in HIV¿1¿infected cells. The present work has focused on two related goals: first, to provide further insights into oligomerization interfaces involved in HIV¿1 capsid assembly; second, to develop a novel approach for inhibition of HIV¿1 capsid assembly based on the rational design of peptides aimed at mimicking different structural elements (helices) involved in distinct oligomerization interfaces in the mature HIV¿1 capsid; these peptides would act as interfacial inhibitors by binding the interfaces and sterically blocking the interactions between CA subunits. In the first part of this study we have explored the conditions needed for hexamerization of CA, focusing on the effect of macromolecular crowding on the oligomerization versus polymerization of this protein. Our results revealed that, contrary to polymerization of intact CA, hexamerization of CA with an inactivated CTD¿CTD interface cannot be promoted, even in a crowded medium. A conformational rearrangement of CTD that occurs only on its homodimerization through the CTD¿CTD interface is needed to generate in CTD the NTD¿binding epitope involved in the CTD¿NTD interfaces required, (together with the NTD¿NTD interfaces), for CA hexamerization and polymerization.

    In the second part of this study we have explored a prediction of macromolecular crowding theory that had not been experimentally tested before. Our results revealed that, as predicted by theory, the inhibitory activity of relatively small compounds on HIV¿ 1 capsid assembly is reduced in the presence of inert macromolecular crowding agents, that were used to mimic in vitro the crowded conditions found in physiological environm n 6 e ts, including cells and the HIV¿1 virion. In the third part of this study we have rationally designed and/or modified different interfacial peptides that represent helices 8 (peptide H8) or 9 (peptides CAC1 and its derivative CAC1M) located in the CTD domain, and respectively involved in the CTD¿NTD and CTD¿CTD oligomerization interfaces. These peptides were able to efficiently inhibit the in vitro assembly of the mature HIV¿1 capsid. In addition, cocktails of interfacial peptides including CAC1, CAC1M and/or H8 abolished mature capsid assembly using lower doses of each peptide, and showed a significant antiviral activity in HIV¿1¿infected cells. In the fourth part of this study, we have produced a library of combinatorial variants of the CTD domain, and showed that different structural solutions at the CTD dimeriza ce a tion interfa llow the preservation of the dimerization affinity. In the fifth and last part of this study, we have undertaken a biophysical and biochemical study of the domain¿swapped dimer of a variant form of CTD (CTD¿¿177).

    The results indicate that highly conserved residues that are part of the MHR are important for the conformational stability of the protein, but do not participate directly in domain¿swapped dimerization of the CTD.