Structural and Dynamics Studies of the D54A Mutant of Human T Cell Leukemia Virus-1 Capsid Protein

Fadila Bouamr,Claudia C Cornilescu,Stephen P Goff,Nico Tjandra,Carol A Carter

doi:10.1074/jbc.m408119200

Abstract

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.

Highlights

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal ␤-hairpin
Since studies with other retroviruses suggest that the ␤-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus ␤-hairpin by disrupting the salt bridge between Pro1 and Asp54 that stabilizes the ␤-hairpin
The mature virion is conical for human immunodeficiency virus type 1 (HIV-1) and other lentiviruses and spherical or irregularly polyhedral for human T cell leukemia virus type 1 (HTLV-1) and other members of the retrovirus group, including Rous sarcoma virus and Moloney murine leukemia virus

Summary

Introduction

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal ␤-hairpin. A substitution in helix 3 (Asp54-Leu to Arg-Ser) produced a 10-fold greater defect than substitutions elsewhere in the NTD and blocked particle assembly almost completely [15] These observations suggest that the NTD and C-terminal domain in the HIV-1 and HTLV-1 CA proteins do not function in the same manner. An insertion next to the conserved Asp51–Leu residues in the NTD of the HIV-1 CA protein had no detectable effect on viral particle formation [13] This difference suggests that structurally equivalent regions can form functionally distinct assembly interfaces during capsid assembly, despite conservation of the secondary and tertiary structures. The fact that much of the Gag precursor incorporated into the viral particles was processed aberrantly to truncated CA protein suggests that the mutation exposed the N terminus to inappropriate proteolysis and possibly accounts for the reduced assembly efficiency

Methods

Results

Conclusion