HIV Gag-Leucine Zipper Chimeras Form ABCE1-Containing Intermediates and RNase-Resistant Immature Capsids Similar to Those Formed by Wild-Type HIV-1 Gag

Abstract

During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly. NC is also critical for Gag multimerization and RNA binding. Previous studies of GagZip chimeric proteins in which NC was replaced with a heterologous leucine zipper that promotes protein dimerization but not RNA binding established that the RNA binding properties of NC are dispensable for capsid formation per se . Here we utilized GagZip proteins to address the question of whether the RNA binding properties of NC are required for ABCE1 binding and for the formation of ABCE1-containing capsid assembly intermediates. We found that assembly-competent HIV-1 GagZip proteins formed ABCE1-containing intermediates, while assembly-incompetent HIV-1 GagZip proteins harboring mutations in residues critical for leucine zipper dimerization did not. Thus, these data suggest that ABCE1 does not bind to NC directly or through an RNA bridge, and they support a model in which dimerization of Gag, mediated by NC or a zipper, results in exposure of an ABCE1-binding domain located elsewhere in Gag, outside NC. Additionally, we demonstrated that immature capsids formed by GagZip proteins are insensitive to RNase A, as expected. However, unexpectedly, immature HIV-1 capsids were almost as insensitive to RNase A as GagZip capsids, suggesting that RNA is not a structural element holding together immature wild-type HIV-1 capsids.

The biological regulation of ABCE1

“…NC does not bind to ABCE1 protein directly. Instead, NC mediates dimerization of Gag, resulting in exposure of an ABCE1 protein binding domain located elsewhere in Gag, outside NC, recruiting ABCE1 protein (27). It was just proved precisely what a study demonstrated: RLI (ABCE1) is induced during HIV-1 infection and downregulates the 2-5A/RNase L pathway in human T cells (28).…”

Rustless translation

“…ABCE1 is found in assembly intermediates at the plasma membrane but not in mature capsids (Zimmerman et al , 2002 ;Lingappa et al , 2006 ). Hereby, the nucleocapsid (NC) domain of Gag binds viral genomic RNA and is also required for interaction with ABCE1, albeit indirectly (Klein et al , 2011 ).…”

“…Hereby, functions of ABCE1 in both termination and splitting, but also its interactions with translation factors, could be modulated or exploited by the virus. Because the interaction of Gag with ABCE1 requires Gag dimerization (Klein et al , 2011 ), formation of large assembly intermediates could specifically sequester ABCE1 to downregulate initiation in cis , but other models are also possible. Therefore, it will be interesting to investigate whether, for example, Gag and ABCE1 interact at ribosomes, at ribosomal subunits or alternatively whether Gag prevents binding of ABCE1 to ribosomes or its interaction with translation factors.…”

“…Here, ABCE1 interacts with the Gag polypeptide, which is the only viral protein required to assemble and release virus like particles (Zimmerman et al , 2002 ;Dooher and Lingappa , 2004 ;Lingappa et al , 2006 ;Klein et al , 2011 ). After its synthesis from translation of full length viral genomic RNA, 3000 -5000 Gag polypeptides assemble in a stepwise process at the plasma membrane, forming buds that produce immature capsids (Briggs and Krausslich , 2011 ).…”

Targeting viral capsid assembly

“…the captured rabies assembly intermediates bind a biotinylated secondary antibody (II[c(1)]), which is used to produce a fluorescent readout that indicates capsid assembly (II[d (1) the viral capsid has recently been shown by Jaisri Lingappa and others to form via a pathway catalyzed by a host assembly complex. [2][3][4] Thus, directly targeting the proteins comprising the host assembly complex could be a way to block formation of the viral capsid and prevent viral infection.…”