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Functional characterization of the putative hepatitis B virus core protein late domain using retrovirus chimeras.

Garcia ML, Reynolds TD, Mothes W, Robek MD - PLoS ONE (2013)

Bottom Line: The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP(138)) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells.We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production.These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA.

ABSTRACT
The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP(138)) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells. However, mutation of this region of Core typically disrupts nucleocapsid formation in the cytoplasm, making it difficult to ascertain if the Core PPAY motif constitutes a functional L-domain that mediates HBV release in the context of replicating virus. Since many viral L-domains are functionally interchangeable between different virus families, and such swapping experiments have been used as a tool to identify other viral sequences with L-domain activity, we generated chimeric constructs between murine leukemia virus (MLV) Gag and HBV Core to determine if the potential HBV L-domain motif is sufficient to stimulate virus release. We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production. Additionally, we found that overexpression of the cellular Nedd4 or WWP1 ubiquitin ligases stimulates release of a partially defective PPAY domain mutant, providing further evidence supporting a role for the Nedd4 ubiquitin ligase in promoting HBV release. These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells.

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Nedd4 and WWP1 overexpression rescues release of an MLV L-domain chimera mutant.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and GagPolPPAY/PNAP alone, GagPolPPAF/PNAP alone, or GagPolPPAF/PNAP with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding (A and B) Nedd4 or (C and D) WWP1. Released virus and HECT ligase protein levels were detected by western blot analysis with the indicated antibodies. (B and D) Protein levels for Pr65 Gag (immature; red bar) and p30 CA (mature; blue bar) were quantified as described above. The mean and standard error of two independent experiments are indicated. Infectious MLV virus produced from the harvested samples was measured and compared to the production of Gag protein products in the released virus samples. Percentages of infectious virus are relative to infectious virus produced without coexpression of the indicated HECT ligase.
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pone-0072845-g005: Nedd4 and WWP1 overexpression rescues release of an MLV L-domain chimera mutant.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and GagPolPPAY/PNAP alone, GagPolPPAF/PNAP alone, or GagPolPPAF/PNAP with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding (A and B) Nedd4 or (C and D) WWP1. Released virus and HECT ligase protein levels were detected by western blot analysis with the indicated antibodies. (B and D) Protein levels for Pr65 Gag (immature; red bar) and p30 CA (mature; blue bar) were quantified as described above. The mean and standard error of two independent experiments are indicated. Infectious MLV virus produced from the harvested samples was measured and compared to the production of Gag protein products in the released virus samples. Percentages of infectious virus are relative to infectious virus produced without coexpression of the indicated HECT ligase.

Mentions: The HECT ligases were next assayed for the ability to stimulate release of infectious virions and the mature p30 CA Gag product using the GagPolPPAF/PNAP partial release mutant. In the absence of HECT ligase overexpression, MLV GagPolPPAF/PNAP demonstrated a >80% reduction of released mature virions as indicated by p30 CA protein levels when compared to MLV GagPolPPAY/PNAP (Fig. 3). When Nedd4 (Fig. 5A and B) or WWP1 (Fig. 5C and D) were titrated in at increasing concentrations, the total level of Gag released by MLV GagPolPPAF/PNAP into the media maximally increased compared to MLV GagPolPPAF/PNAP in the absence of ligase (Fig. 5B and D). The total level of released Gag eventually decreased at higher concentrations of overexpressed HECT ligase most likely due to disruption of the endocytic trafficking pathways that are regulated by HECT ligase activity. Additionally, the level of released mature virions for MLV GagPolPPAF/PNAP increased to levels comparable to the MLV GagPolPPAY/PNAP chimera with co-expression of 500 ng of the indicated HECT ligase. The release of mature virions correlated with infectious MLV production, indicating that Nedd4 and WWP1 overexpression can functionally rescue the release defect in an MLV L-domain chimera mutant.


Functional characterization of the putative hepatitis B virus core protein late domain using retrovirus chimeras.

Garcia ML, Reynolds TD, Mothes W, Robek MD - PLoS ONE (2013)

Nedd4 and WWP1 overexpression rescues release of an MLV L-domain chimera mutant.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and GagPolPPAY/PNAP alone, GagPolPPAF/PNAP alone, or GagPolPPAF/PNAP with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding (A and B) Nedd4 or (C and D) WWP1. Released virus and HECT ligase protein levels were detected by western blot analysis with the indicated antibodies. (B and D) Protein levels for Pr65 Gag (immature; red bar) and p30 CA (mature; blue bar) were quantified as described above. The mean and standard error of two independent experiments are indicated. Infectious MLV virus produced from the harvested samples was measured and compared to the production of Gag protein products in the released virus samples. Percentages of infectious virus are relative to infectious virus produced without coexpression of the indicated HECT ligase.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756966&req=5

pone-0072845-g005: Nedd4 and WWP1 overexpression rescues release of an MLV L-domain chimera mutant.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and GagPolPPAY/PNAP alone, GagPolPPAF/PNAP alone, or GagPolPPAF/PNAP with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding (A and B) Nedd4 or (C and D) WWP1. Released virus and HECT ligase protein levels were detected by western blot analysis with the indicated antibodies. (B and D) Protein levels for Pr65 Gag (immature; red bar) and p30 CA (mature; blue bar) were quantified as described above. The mean and standard error of two independent experiments are indicated. Infectious MLV virus produced from the harvested samples was measured and compared to the production of Gag protein products in the released virus samples. Percentages of infectious virus are relative to infectious virus produced without coexpression of the indicated HECT ligase.
Mentions: The HECT ligases were next assayed for the ability to stimulate release of infectious virions and the mature p30 CA Gag product using the GagPolPPAF/PNAP partial release mutant. In the absence of HECT ligase overexpression, MLV GagPolPPAF/PNAP demonstrated a >80% reduction of released mature virions as indicated by p30 CA protein levels when compared to MLV GagPolPPAY/PNAP (Fig. 3). When Nedd4 (Fig. 5A and B) or WWP1 (Fig. 5C and D) were titrated in at increasing concentrations, the total level of Gag released by MLV GagPolPPAF/PNAP into the media maximally increased compared to MLV GagPolPPAF/PNAP in the absence of ligase (Fig. 5B and D). The total level of released Gag eventually decreased at higher concentrations of overexpressed HECT ligase most likely due to disruption of the endocytic trafficking pathways that are regulated by HECT ligase activity. Additionally, the level of released mature virions for MLV GagPolPPAF/PNAP increased to levels comparable to the MLV GagPolPPAY/PNAP chimera with co-expression of 500 ng of the indicated HECT ligase. The release of mature virions correlated with infectious MLV production, indicating that Nedd4 and WWP1 overexpression can functionally rescue the release defect in an MLV L-domain chimera mutant.

Bottom Line: The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP(138)) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells.We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production.These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA.

ABSTRACT
The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP(138)) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells. However, mutation of this region of Core typically disrupts nucleocapsid formation in the cytoplasm, making it difficult to ascertain if the Core PPAY motif constitutes a functional L-domain that mediates HBV release in the context of replicating virus. Since many viral L-domains are functionally interchangeable between different virus families, and such swapping experiments have been used as a tool to identify other viral sequences with L-domain activity, we generated chimeric constructs between murine leukemia virus (MLV) Gag and HBV Core to determine if the potential HBV L-domain motif is sufficient to stimulate virus release. We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production. Additionally, we found that overexpression of the cellular Nedd4 or WWP1 ubiquitin ligases stimulates release of a partially defective PPAY domain mutant, providing further evidence supporting a role for the Nedd4 ubiquitin ligase in promoting HBV release. These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells.

Show MeSH
Related in: MedlinePlus