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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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Substitution of HIV-1 matrix with other membrane targeting domains rescued PRE-dependent HIV-1 Gag budding in human cells.(A) Schematic diagram of plasmids expressing HA tagged PRE-dependent HIV-1 Gag mutants (upper panel) and EIAV Gag mutants (lower panel). (B) Budding of PRE-dependent HIV-1 and EIAV Gag mutants. 293T cells were transfected with the indicated PRE-dependent HIV-1 and EIAV Gag mutants described in (A). At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using HA antibody. Data are representative of three independent experiments.
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pone-0006551-g005: Substitution of HIV-1 matrix with other membrane targeting domains rescued PRE-dependent HIV-1 Gag budding in human cells.(A) Schematic diagram of plasmids expressing HA tagged PRE-dependent HIV-1 Gag mutants (upper panel) and EIAV Gag mutants (lower panel). (B) Budding of PRE-dependent HIV-1 and EIAV Gag mutants. 293T cells were transfected with the indicated PRE-dependent HIV-1 and EIAV Gag mutants described in (A). At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using HA antibody. Data are representative of three independent experiments.

Mentions: We first constructed a panel of PRE-dependent HIV-1 Gag and EIAV Gag MA mutants (Fig. 5A). 293T cells were transfected with this panel of HA tagged PRE-dependent HIV-1 Gag and EIAV Gag constructs, followed by Western blotting of cell lysates and pelleted VLPs at 24 h post transfection. Replacing the HIV MA with the v-Src myristoylation signal rescued PRE-dependent HIV-1 Gag budding (Fig. 5B, compare lane 1 and lane 2). Consistent with our previous observations [14], and in contrast to PRE-dependent HIV-1 Gag, PRE-dependent EIAV Gag could bud efficiently from human cells (Fig. 5B, compare lane 1 and lane 4). Interestingly, MA swapping reversed this phenotype. PRE-dependent chimeric HIV-1 Gag containing EIAV MA budded as efficiently as PRE-dependent EIAV Gag (Fig. 5B, compare lane 3 and lane 5), whereas PRE-dependent chimeric EIAV Gag containing HIV MA failed to bud efficiently (Fig. 5B, lane 6). The PRE-dependent chimeric EIAV Gag budding deficiency seemed to be specific for the HIV MA, because replacing EIAV MA with the v-Src myristoylation signal did not interfere with chimeric EIAV Gag budding (Fig. 5B, lane 5).


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)

Substitution of HIV-1 matrix with other membrane targeting domains rescued PRE-dependent HIV-1 Gag budding in human cells.(A) Schematic diagram of plasmids expressing HA tagged PRE-dependent HIV-1 Gag mutants (upper panel) and EIAV Gag mutants (lower panel). (B) Budding of PRE-dependent HIV-1 and EIAV Gag mutants. 293T cells were transfected with the indicated PRE-dependent HIV-1 and EIAV Gag mutants described in (A). At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using HA antibody. Data are representative of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2717210&req=5

pone-0006551-g005: Substitution of HIV-1 matrix with other membrane targeting domains rescued PRE-dependent HIV-1 Gag budding in human cells.(A) Schematic diagram of plasmids expressing HA tagged PRE-dependent HIV-1 Gag mutants (upper panel) and EIAV Gag mutants (lower panel). (B) Budding of PRE-dependent HIV-1 and EIAV Gag mutants. 293T cells were transfected with the indicated PRE-dependent HIV-1 and EIAV Gag mutants described in (A). At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using HA antibody. Data are representative of three independent experiments.
Mentions: We first constructed a panel of PRE-dependent HIV-1 Gag and EIAV Gag MA mutants (Fig. 5A). 293T cells were transfected with this panel of HA tagged PRE-dependent HIV-1 Gag and EIAV Gag constructs, followed by Western blotting of cell lysates and pelleted VLPs at 24 h post transfection. Replacing the HIV MA with the v-Src myristoylation signal rescued PRE-dependent HIV-1 Gag budding (Fig. 5B, compare lane 1 and lane 2). Consistent with our previous observations [14], and in contrast to PRE-dependent HIV-1 Gag, PRE-dependent EIAV Gag could bud efficiently from human cells (Fig. 5B, compare lane 1 and lane 4). Interestingly, MA swapping reversed this phenotype. PRE-dependent chimeric HIV-1 Gag containing EIAV MA budded as efficiently as PRE-dependent EIAV Gag (Fig. 5B, compare lane 3 and lane 5), whereas PRE-dependent chimeric EIAV Gag containing HIV MA failed to bud efficiently (Fig. 5B, lane 6). The PRE-dependent chimeric EIAV Gag budding deficiency seemed to be specific for the HIV MA, because replacing EIAV MA with the v-Src myristoylation signal did not interfere with chimeric EIAV Gag budding (Fig. 5B, lane 5).

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