Defects in cellular sorting and retroviral assembly induced by GGA overexpression.
BACKGROUND
We previously demonstrated that overexpression of Golgi-localized, gamma-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.


RESULTS
In 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.


CONCLUSIONS
These data provide insights into the composition of structures induced by GGA overexpression and their ability to disrupt endosomal sorting and retroviral particle production.
Virus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Maryland, USA.
Anjali
A
Joshi
Kunio
K
Nagashima
Eric O
EO
Freed
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D012190:Retroviridae / Q000502:physiology*; D054875:Ubiquitination; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000502:physiology
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D017527:Receptor, IGF Type 2; D011990:Receptors, Transferrin; D054301:gag Gene Products, Human Immunodeficiency Virus; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors