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HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

Jin J, Sturgeon T, Weisz OA, Mothes W, Montelaro RC - PLoS ONE (2009)

Bottom Line: PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles.Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains.Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC) assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

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Coexpression of membrane targeting and assembly competent Rev-dependent HIV-1 Gag rescues PRE-dependent HIV-1 Gag budding.(A) Schematic diagram of plasmids expressing HA tagged Rev-dependent HIV-1 Gag mutants. (B) Budding of PRE-dependent HIV-1 Gag-GFP upon co-expression with PRE-dependent or Rev-dependent HIV-1 Gag-HA. PRE-dependent HIV-1 Gag-GFP was co-transfected at an equal molar ratio into 293T cells with empty vector, PRE-dependent HIV-1 Gag-HA, or the indicated Rev-dependent HIV-1 Gag-HA constructs described in A. At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using GFP and HA antibody. Data are representative of three independent experiments.
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pone-0006551-g004: Coexpression of membrane targeting and assembly competent Rev-dependent HIV-1 Gag rescues PRE-dependent HIV-1 Gag budding.(A) Schematic diagram of plasmids expressing HA tagged Rev-dependent HIV-1 Gag mutants. (B) Budding of PRE-dependent HIV-1 Gag-GFP upon co-expression with PRE-dependent or Rev-dependent HIV-1 Gag-HA. PRE-dependent HIV-1 Gag-GFP was co-transfected at an equal molar ratio into 293T cells with empty vector, PRE-dependent HIV-1 Gag-HA, or the indicated Rev-dependent HIV-1 Gag-HA constructs described in A. At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using GFP and HA antibody. Data are representative of three independent experiments.

Mentions: Since Rev-dependent HIV-1 Gag could efficiently co-assemble with PRE-dependent Gag at the plasma membrane, we next tested whether this co-assembly can rescue PRE-dependent HIV-1 Gag budding in human cells (Fig. 4B). PRE-dependent HIV-1 Gag-GFP was co-transfected with empty vector, HA-tagged PRE-dependent, or Rev-dependent HIV-1 Gag in 293T cells. At 24 h post transfection, cell lysates and supernatant pellets (VLPs) were subjected to SDS-PAGE and Western Blotting to determine budding efficiencies. Co-expression with Rev-dependent HIV-1 Gag-HA enhanced PRE-dependent HIV-1 Gag-GFP budding by about 30 fold (Fig. 4B, compare lanes 1 and 3). In contrast, this enhancement was not observed upon co-expression of PRE-dependent HIV-1 Gag-HA (Fig. 4B, compare lanes 1 and 2).


HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

Jin J, Sturgeon T, Weisz OA, Mothes W, Montelaro RC - PLoS ONE (2009)

Coexpression of membrane targeting and assembly competent Rev-dependent HIV-1 Gag rescues PRE-dependent HIV-1 Gag budding.(A) Schematic diagram of plasmids expressing HA tagged Rev-dependent HIV-1 Gag mutants. (B) Budding of PRE-dependent HIV-1 Gag-GFP upon co-expression with PRE-dependent or Rev-dependent HIV-1 Gag-HA. PRE-dependent HIV-1 Gag-GFP was co-transfected at an equal molar ratio into 293T cells with empty vector, PRE-dependent HIV-1 Gag-HA, or the indicated Rev-dependent HIV-1 Gag-HA constructs described in A. At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using GFP and HA antibody. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2717210&req=5

pone-0006551-g004: Coexpression of membrane targeting and assembly competent Rev-dependent HIV-1 Gag rescues PRE-dependent HIV-1 Gag budding.(A) Schematic diagram of plasmids expressing HA tagged Rev-dependent HIV-1 Gag mutants. (B) Budding of PRE-dependent HIV-1 Gag-GFP upon co-expression with PRE-dependent or Rev-dependent HIV-1 Gag-HA. PRE-dependent HIV-1 Gag-GFP was co-transfected at an equal molar ratio into 293T cells with empty vector, PRE-dependent HIV-1 Gag-HA, or the indicated Rev-dependent HIV-1 Gag-HA constructs described in A. At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using GFP and HA antibody. Data are representative of three independent experiments.
Mentions: Since Rev-dependent HIV-1 Gag could efficiently co-assemble with PRE-dependent Gag at the plasma membrane, we next tested whether this co-assembly can rescue PRE-dependent HIV-1 Gag budding in human cells (Fig. 4B). PRE-dependent HIV-1 Gag-GFP was co-transfected with empty vector, HA-tagged PRE-dependent, or Rev-dependent HIV-1 Gag in 293T cells. At 24 h post transfection, cell lysates and supernatant pellets (VLPs) were subjected to SDS-PAGE and Western Blotting to determine budding efficiencies. Co-expression with Rev-dependent HIV-1 Gag-HA enhanced PRE-dependent HIV-1 Gag-GFP budding by about 30 fold (Fig. 4B, compare lanes 1 and 3). In contrast, this enhancement was not observed upon co-expression of PRE-dependent HIV-1 Gag-HA (Fig. 4B, compare lanes 1 and 2).

Bottom Line: PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles.Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains.Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC) assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

Show MeSH
Related in: MedlinePlus