Abstract
The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.
Highlights
The assembly of the HIV particle is orchestrated by the HIV-1 Gag precursor protein (Pr55Gag)
HIV use the oligomeric states of HIV Pr55Gag proteins and the RNA sequences as means to regulate the viral assembly process
Using the entire form of recombinant Pr55Gag in isothermal titration calorimetry (ITC) studies, we present the first thermodynamic analysis of how HIV Pr55Gag and RNA regulate assembly
Summary
The assembly of the HIV particle is orchestrated by the HIV-1 Gag precursor protein (Pr55Gag). The nucleocapsid (NC) domain is responsible for genomic RNA packaging and the capsid (CA) domain mediates the key interactions to promote Pr55Gag-Pr55Gag oligomerization (for review see [1,2,3]) While both Pr55Gag-nucleic acid and Pr55Gag-Pr55Gag interactions have been identified as major contributors to the assembly process [4], the mechanistic details of how they regulate HIV assembly are not well defined, in the context of full length Pr55Gag. It is generally accepted that nucleic acid binds to Pr55Gag and acts as a scaffold for Pr55Gag oligomerization [5,6,7], short oligonucleotides (10 nucleotides or less) are not sufficient to bridge multiple Pr55Gag molecules to support assembly [8, 9]. The energy requirements and thermodynamic properties that drive these two basic components to facilitate the formation of the viral particle are currently not known In this regard, a thermodynamic analysis of the assembly process will provide critical information (such as, the affinity and the stoichiometry between ligands and substrates, plus the energetics involved in these reactions) to define the mechanisms of the process. Enthalpic and entropic components of the binding reaction derived from the analysis will enable us to gain insight into the mechanisms of the interactions that take place
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