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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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Effect of HECT ligase overexpression on infectious MLV production.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and (A) GagPol, (B) GagPol4A, (C) GagPolPPAY/PNAP alone or with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding Nedd4, Nedd4L, WWP1, WWP2, Smurf1, Smurf2 or Itch. Percentages of infectious virus are relative to infectious virus produced when not co-expressed with the indicated HECT ligase.
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pone-0072845-g004: Effect of HECT ligase overexpression on infectious MLV production.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and (A) GagPol, (B) GagPol4A, (C) GagPolPPAY/PNAP alone or with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding Nedd4, Nedd4L, WWP1, WWP2, Smurf1, Smurf2 or Itch. Percentages of infectious virus are relative to infectious virus produced when not co-expressed with the indicated HECT ligase.

Mentions: Analysis of the effect of HECT ligase overexpression on infectious MLV release demonstrated that the seven HECT ligases tested did not enhance, and at high concentrations at which cytotoxic effects are expected, reduced infectious MLV production in wild-type MLV GagPol expressing cells (Fig. 4A). This result is to be expected as wild-type MLV GagPol contains a functional L-domain and is efficiently released at normal cellular levels of HECT ligase expression. In GagPol4A expressing cells, both Nedd4L and Itch overexpression enhanced the production of infectious virus indicating that they are functioning in an L-domain independent manner (Fig. 4B), similar to previous reports [43]. In the context of cells expressing the GagPolPPAY/PNAP L-domain chimera, six of the seven HECT ligases screened had no effect on the production of infectious MLV (Fig. 4C), similar to MLV GagPol. The exception was Nedd4L, which similar to the effect observed in cells expressing GagPol4A enhanced infectious MLV production, presumably in an L-domain independent manner.


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)

Effect of HECT ligase overexpression on infectious MLV production.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and (A) GagPol, (B) GagPol4A, (C) GagPolPPAY/PNAP alone or with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding Nedd4, Nedd4L, WWP1, WWP2, Smurf1, Smurf2 or Itch. Percentages of infectious virus are relative to infectious virus produced when not co-expressed with the indicated HECT ligase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756966&req=5

pone-0072845-g004: Effect of HECT ligase overexpression on infectious MLV production.Virion production by 293T cells co-transfected with plasmids Friend MLV Env, MLV LTR-LacZ, and (A) GagPol, (B) GagPol4A, (C) GagPolPPAY/PNAP alone or with increasing concentrations (0.125 µg to 2 µg) of a YFP fusion expression construct encoding Nedd4, Nedd4L, WWP1, WWP2, Smurf1, Smurf2 or Itch. Percentages of infectious virus are relative to infectious virus produced when not co-expressed with the indicated HECT ligase.
Mentions: Analysis of the effect of HECT ligase overexpression on infectious MLV release demonstrated that the seven HECT ligases tested did not enhance, and at high concentrations at which cytotoxic effects are expected, reduced infectious MLV production in wild-type MLV GagPol expressing cells (Fig. 4A). This result is to be expected as wild-type MLV GagPol contains a functional L-domain and is efficiently released at normal cellular levels of HECT ligase expression. In GagPol4A expressing cells, both Nedd4L and Itch overexpression enhanced the production of infectious virus indicating that they are functioning in an L-domain independent manner (Fig. 4B), similar to previous reports [43]. In the context of cells expressing the GagPolPPAY/PNAP L-domain chimera, six of the seven HECT ligases screened had no effect on the production of infectious MLV (Fig. 4C), similar to MLV GagPol. The exception was Nedd4L, which similar to the effect observed in cells expressing GagPol4A enhanced infectious MLV production, presumably in an L-domain independent manner.

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