Basic Residues in the Nucleocapsid Domain of Gag Are Required for Interaction of HIV-1 Gag with ABCE1 (HP68), a Cellular Protein Important for HIV-1 Capsid Assembly

Jaisri R Lingappa,Julia E Dooher,Michael A Newman,Patti K Kiser,Kevin C Klein

doi:10.1074/jbc.m507255200

Jaisri R Lingappa, Julia E Dooher + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m507255200

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Abstract

During human immunodeficiency virus, type 1 (HIV-1) assembly, Gag polypeptides multimerize into immature HIV-1 capsids. The cellular ATP-binding protein ABCE1 (also called HP68 or RNase L inhibitor) appears to be critical for proper assembly of the HIV-1 capsid. In primate cells, ABCE1 associates with Gag polypeptides present in immature capsid assembly intermediates. Here we demonstrate that the NC domain of Gag is critical for interaction with endogenous primate ABCE1, whereas other domains in Gag can be deleted without eliminating the association of Gag with ABCE1. NC contains two Cys-His boxes that form zinc finger motifs and are responsible for encapsidation of HIV-1 genomic RNA. In addition, NC contains basic residues known to play a critical role in nonspecific RNA binding, Gag-Gag interactions, and particle formation. We demonstrate that basic residues in NC are needed for the Gag-ABCE1 interaction, whereas the cysteine and histidine residues in the zinc fingers are dispensable. Constructs that fail to interact with primate ABCE1 or interact poorly also fail to form capsids and are arrested at an early point in the immature capsid assembly pathway. Whereas others have shown that basic residues in NC bind nonspecifically to RNA, which in turn scaffolds or nucleates assembly, our data demonstrate that the same basic residues in NC act either directly or indirectly to recruit a cellular protein that also promotes capsid formation. Thus, in cells, basic residues in NC appear to act by two mechanisms, recruiting both RNA and a cellular ATPase in order to facilitate efficient assembly of HIV-1 capsids.

Highlights

Priate name for this protein, which is the sole member of the ATPbinding cassette protein family E and is present in many species that do not encode RNase L [16]
Co-immunoprecipitation with antibody to endogenous ABCE1 demonstrates that ABCE1 is associated with Gag polypeptides present in assembly intermediates isolated from primate cells expressing HIV-1 or other primate lentiviruses [1, 4]
Progressive Truncation in NC Leads to a Corresponding Reduction in the Gag-ABCE1 Interaction—Previously, we demonstrated that an antiserum directed against endogenous primate ABCE1 (␣-ABCE1) co-immunoprecipitates wild-type HIV-1 Gag from primate cells infected with HIV-1 or transfected with an HIV-1 proviral construct

Summary

Introduction

Priate name for this protein, which is the sole member of the ATPbinding cassette protein family E and is present in many species that do not encode RNase L [16]. Our findings reveal that the basic residues in NC that bind nonspecifically to RNA are important for the interaction of Gag with the cellular protein ABCE1, which facilitates HIV-1 capsid formation.

Results

Conclusion