Bashar Ghoujal1, Miroslav P Milev, Lara Ajamian, Karen Abel, Andrew J Mouland. 1. HIV-1 RNA Trafficking Laboratory, Lady Davis Institute at the Jewish General Hospital and the Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3T 1E2, Canada.
Abstract
BACKGROUND INFORMATION: Several host proteins play crucial roles in the HIV-1 replication cycle. The endosomal sorting complex required for transport (ESCRT) exemplifies a large, multi-component host machinery that is required by HIV-1 for viral budding. ESCRT promotes the inward budding of vesicles from the membranes of late endosomes to generate multi-vesicular bodies. However, HIV-1 co-opts the ESCRT to enable outwards budding of virus particles from the plasma membrane, a phenomenon that is topologically similar to multi-vesicular body biogenesis. A role for ESCRTII in mRNA trafficking has been established in Drosophila in which the ESCRT-II components, Vps22 and Vps36, promote the localisation of the bicoid mRNA in the fertilised egg. This is achieved via specific interactions with the Staufen protein. In this work, we investigated a possible implication of ESCRT-II in the HIV-1 replication cycle. RESULTS: Co-immunoprecipitation analyses and live cell tri-molecular fluorescence complementation assays revealed that interactions between EAP30 and Gag and another between EAP30 and Staufen1 occur in mammalian cells. We then depleted EAP30 (the orthologue for Vps22) by siRNA to target ESCRT-II in HIV-1 expressing cells. This treatment disrupted ESCRT-II function and leads to the degradation of the two other ESCRT-II complex proteins, EAP45 and EAP20, as well as the associated Rab7-interacting lysosomal protein. The depletion of EAP30 led to dramatically reduced viral structural protein Gag and virus production levels, without any effect on viral RNA levels. On the contrary, the overexpression of EAP30 led to a several-fold increase in virus production. Unexpec-tedly, siRNA-mediated depletion of EAP30 led to a block to HIV-1 genomic RNA trafficking and resulted in the accumulation of genomic RNA in the nucleus and juxtanuclear domains. CONCLUSIONS: Our data provide the first evidence that the Staufen1-ESCRT-II interaction is evolutionarily conserved from lower to higher eukaryotes and reveal a novel role for EAP30 in the control of HIV-1 RNA trafficking and gene expression.
BACKGROUND INFORMATION: Several host proteins play crucial roles in the HIV-1 replication cycle. The endosomal sorting complex required for transport (ESCRT) exemplifies a large, multi-component host machinery that is required by HIV-1 for viral budding. ESCRT promotes the inward budding of vesicles from the membranes of late endosomes to generate multi-vesicular bodies. However, HIV-1 co-opts the ESCRT to enable outwards budding of virus particles from the plasma membrane, a phenomenon that is topologically similar to multi-vesicular body biogenesis. A role for ESCRTII in mRNA trafficking has been established in Drosophila in which the ESCRT-II components, Vps22 and Vps36, promote the localisation of the bicoid mRNA in the fertilised egg. This is achieved via specific interactions with the Staufen protein. In this work, we investigated a possible implication of ESCRT-II in the HIV-1 replication cycle. RESULTS: Co-immunoprecipitation analyses and live cell tri-molecular fluorescence complementation assays revealed that interactions between EAP30 and Gag and another between EAP30 and Staufen1 occur in mammalian cells. We then depleted EAP30 (the orthologue for Vps22) by siRNA to target ESCRT-II in HIV-1 expressing cells. This treatment disrupted ESCRT-II function and leads to the degradation of the two other ESCRT-II complex proteins, EAP45 and EAP20, as well as the associated Rab7-interacting lysosomal protein. The depletion of EAP30 led to dramatically reduced viral structural protein Gag and virus production levels, without any effect on viral RNA levels. On the contrary, the overexpression of EAP30 led to a several-fold increase in virus production. Unexpec-tedly, siRNA-mediated depletion of EAP30 led to a block to HIV-1 genomic RNA trafficking and resulted in the accumulation of genomic RNA in the nucleus and juxtanuclear domains. CONCLUSIONS: Our data provide the first evidence that the Staufen1-ESCRT-II interaction is evolutionarily conserved from lower to higher eukaryotes and reveal a novel role for EAP30 in the control of HIV-1 RNA trafficking and gene expression.
Authors: Frances M Brodsky; R Thomas Sosa; Joel A Ybe; Theresa J O'Halloran Journal: Cold Spring Harb Perspect Biol Date: 2014-09-02 Impact factor: 10.005