Defects in cellular sorting and retroviral assembly induced by GGA overexpression.

Anjali Joshi,Kunio Nagashima,Eric O Freed

doi:10.1186/1471-2121-10-72

Abstract

BackgroundWe previously demonstrated that overexpression of Golgi-localized, γ-ear containing, Arf-binding (GGA) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs). GGA overexpression led to the formation of large, swollen vacuolar compartments, which in the case of GGA1 sequestered HIV-1 Gag.ResultsIn the current study, we extend our previous findings to characterize in depth the GGA-induced compartments and the determinants for retroviral Gag sequestration in these structures. We find that GGA-induced structures are derived from the Golgi and contain aggresome markers. GGA overexpression leads to defects in trafficking of transferrin receptor and recycling of cation-dependent mannose 6-phosphate receptor. Additionally, we find that compartments induced by GGA overexpression sequester Tsg101, poly-ubiquitin, and, in the case of GGA3, Hrs. Interestingly, brefeldin A treatment, which leads to the dissociation of endogenous GGAs from membranes, does not dissociate the GGA-induced compartments. GGA mutants that are defective in Arf binding and hence association with membranes also induce the formation of GGA-induced structures. Overexpression of ubiquitin reverses the formation of GGA-induced structures and partially rescues HIV-1 particle production. We found that in addition to HIV-1 Gag, equine infectious anemia virus Gag is also sequestered in GGA1-induced structures. The determinants in Gag responsible for sequestration map to the matrix domain, and recruitment to these structures is dependent on Gag membrane binding.ConclusionThese data provide insights into the composition of structures induced by GGA overexpression and their ability to disrupt endosomal sorting and retroviral particle production.

Highlights

We previously demonstrated that overexpression of Golgi-localized, J-ear containing, Arf-binding (GGA) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs)
The HIV-1 Gag precursor protein, Pr55Gag, contains in its p6 domain a Pro-Thr/Ser-Ala-Pro [P(T/S)adaptor proteins (AP)] motif that binds Tsg101, a component of the endosomal sorting complex required for transport-I (ESCRT-I) [7,8,9,10], and a Tyr-Pro-Xn-Leu (YPXnL, where X is any amino acid and n = 1-3 residues) motif that interacts with the ESCRT-associated factor ALG-2 interacting protein X (Alix) [11,12,13]
We demonstrated previously that overexpression of the Golgi-localized, J-ear containing, Arf-binding (GGA) proteins inhibits the production of HIV-1 and equine infectious anemia virus (EIAV) particles by impairing the association of Gag with membrane [37]

Summary

Introduction

We previously demonstrated that overexpression of Golgi-localized, J-ear containing, Arf-binding (GGA) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs). While the Gag precursor proteins are the sole viral determinants required for the production of immature viruslike particles (VLPs), a number of host factors have been implicated in various steps of the virus assembly and release pathway. Retroviral late domains are known to interact with components of the endosomal sorting machinery. The HIV-1 Gag precursor protein, Pr55Gag, contains in its p6 domain a Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif that binds Tsg101, a component of the endosomal sorting complex required for transport-I (ESCRT-I) [7,8,9,10], and a Tyr-Pro-Xn-Leu (YPXnL, where X is any amino acid and n = 1-3 residues) motif that interacts with the ESCRT-associated factor Alix [11,12,13]. Retroviral Gag proteins are ubiquitinated [21,22,23], and while ubiquitination of Gag does not appear to play an essential role in virus budding [24], in the absence of a functional late domain ubiquitin can serve to promote virus release [25]

Methods

Results

Discussion

Conclusion