A Temporospatial Map That Defines Specific Steps at Which Critical Surfaces in the Gag MA and CA Domains Act during Immature HIV-1 Capsid Assembly in Cells

Abstract

During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCEWhile much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used biochemical and ultrastructural analyses to generate a temporospatial map showing the precise order in which four critical surfaces in Gag act during immature capsid formation in provirus-expressing cells. Because three of these surfaces make important contacts in the hexameric lattices that are found in the completed immature capsid, these data allow us to propose a model for the sequence of events leading to formation of the hexameric lattices. By providing a dynamic view of when and where critical Gag-Gag contacts form during the assembly process and how those contacts function in the nascent capsid, our study provides novel insights into how an immature capsid is built in infected cells.

How HIV-1 Gag assembles in cells: Putting together pieces of the puzzle

“…Shortly after translation, Gag co-opts a cytoplasmic host complex that contains RNA granule proteins (also called processing body or P body proteins), (Reed et al, 2012). Two of the proteins in this complex are host enzymes that facilitate assembly - ABCE1 and DDX6 (Reed et al, 2012; Robinson et al, 2014; Zimmerman et al, 2002). A third co-opted host protein, Staufen, is an RNA binding protein that facilitates Gag multimerization and RNA packaging [reviewed in (Cochrane et al, 2006)].…”

“…These studies have revealed that Gag progresses through a sequential pathway of assembly intermediates, which are described by their sedimentation values (∼10S, ∼80S, ∼150S, and ∼500S). The high-molecular-mass assembly intermediates (the ∼80S, ∼150S, and ∼500S complexes) contain gRNA and other viral proteins, as well as cellular proteins, such as ABCE1 and DDX6, that dissociate when assembly is completed (Dooher et al, 2007; Reed et al, 2012; Robinson et al, 2014; Zimmerman et al, 2002). Consistent with this model, assembly-defective Gag mutants are arrested at specific steps in this assembly pathway, resulting in accumulation of the assembly intermediates that precede the point of blockade (Fig.…”

“…Such interfaces are present in the fully assembled immature capsid and likely form when Gag multimerizes. Notably, many of the residues that form CA-CA interfaces in the immature capsid are also required for efficient immature capsid assembly (Bharat et al, 2014; Bharat et al, 2012; Robinson et al, 2014). Recent biochemical studies in cells have defined when these and other critical residues act during assembly, leading to a model for the temporal sequence in when each of these critical CA-CA interfaces are formed (Robinson et al, 2014).…”

Movements of HIV-1 genomic RNA-APOBEC3F complexes and PKR reveal cytoplasmic and nuclear PKR defenses and HIV-1 evasion strategies

“…In addition, DDX6 binds extensively onto mRNAs and has been reported to chaperone gRNA-Gag associations (Ernoult-Lange et al, 2012; Reed et al, 2012; Robinson et al, 2014; Yu et al, 2011). Moreover, EDC4 functions as a binding platform that recruits important PB proteins including the decapping enzymes Dcp1 and 2, thereby enhancing their activities (Chang et al, 2014).…”

Deficiency of Functional Iron-Sulfur Domains in ABCE1 Inhibits the Proliferation and Migration of Lung Adenocarcinomas By Regulating the Biogenesis of Beta-Actin In Vitro

“…ABCE1 codes for ribonuclease L inhibitor, which regulates various biological functions, such as ribosome biosynthesis and recycling, as well as HIV capsid assembly [23, 24]. …”