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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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Generation of MLV GagCore L-domain chimeras.(A) HBV Core encodes a putative L-domain motif suggesting a potential docking site for HECT Ub ligases that direct HBV release. (B) L-domain chimeric proteins between MLV Gag and Core were generated by replacement of the endogenous MLV PPPY motif with the Core PPAY/PNAP motif and flanking residues. The location of the MLV Gag PPPY motif in the p12 region is italicized. The Gag4A mutant contains four alanine amino acid substitutions (AAAA) that abolish Gag L-domain activity. The inserted Core sequence is underlined in the MLV L-domain chimeras. The L-domain motif and L-domain motif mutations are italicized. Chimeric Gag protein was subcloned into the MLV GagPol expression plasmid to generate GagPol, GagPol4A, GagPolPPAY/PNAP, GagPolPPAY, GagPolPNAP, GagPolLPAY/PNAP, GagPolPPAF/PNAP, and GagPolPPAA/PNAP expression constructs. The MLV Pr65 Gag protein contains matrix (MA), capsids (CA), and nucleocapsid (NC) domains. Asterisks indicate PR cleavage sites.
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pone-0072845-g001: Generation of MLV GagCore L-domain chimeras.(A) HBV Core encodes a putative L-domain motif suggesting a potential docking site for HECT Ub ligases that direct HBV release. (B) L-domain chimeric proteins between MLV Gag and Core were generated by replacement of the endogenous MLV PPPY motif with the Core PPAY/PNAP motif and flanking residues. The location of the MLV Gag PPPY motif in the p12 region is italicized. The Gag4A mutant contains four alanine amino acid substitutions (AAAA) that abolish Gag L-domain activity. The inserted Core sequence is underlined in the MLV L-domain chimeras. The L-domain motif and L-domain motif mutations are italicized. Chimeric Gag protein was subcloned into the MLV GagPol expression plasmid to generate GagPol, GagPol4A, GagPolPPAY/PNAP, GagPolPPAY, GagPolPNAP, GagPolLPAY/PNAP, GagPolPPAF/PNAP, and GagPolPPAA/PNAP expression constructs. The MLV Pr65 Gag protein contains matrix (MA), capsids (CA), and nucleocapsid (NC) domains. Asterisks indicate PR cleavage sites.

Mentions: To determine if the Core 129PPAYRPPNAP138 sequence constitutes a genuine L-domain motif that directs release of a heterologous virus, expression constructs were generated to encode an MLV GagPolCore chimera protein where the endogenous PPPY L-domain of Gag was replaced with the putative Core L-domain motif (Fig. 1A and B). An MLV GagPolPPAY/PNAP chimera was synthesized by insertion of a Core 18 amino acid sequence fragment containing both the PPAY and PNAP motifs, as well as two MLV GagPol constructs that contained a 10 amino acid sequence with the PPAY or the PNAP sequence motif, GagPolPPAY and GagPolPNAP, respectively. To produce quantifiable infectious MLV particles containing chimeric protein, 293T cells were transfected with plasmids encoding the Friend MLV envelope protein, the LTR-LacZ reporter construct and the GagPol plasmids encoding either wild-type Gag, a Gag4A mutant that lacks L-domain activity, GagPPAY/PNAP, GagPPAY, or GagPNAP. Media and cellular lysates were harvested from virus-replicating cells 1 day post-transfection and resolved by SDS-PAGE to detect virion-associated Gag or cell-associated Gag protein, respectively, by western blot (Fig. 2A). Additionally, DFJ8 target cells that stably express MCAT-1, a surface receptor that is recognized by the Friend MLV envelope protein, were incubated with serial dilutions of the collected media for 24 h and subsequently stained for LacZ activity to quantify the amount of infectious MLV particles produced by the virus-replicating cells (Fig. 2B). As previously reported [39], [42], the AAAA (4A) mutation in Gag strongly inhibited infectious MLV release to 3% of wild-type MLV (Fig. 2A and B). Interestingly, the 18 amino acid sequence of Core that contains the PPAY and PNAP motif restored release of infectious MLV to 75% of wild-type MLV. Functional replacement of the endogenous Gag PPPY motif by the Core sequence can be attributed to the PPAY motif since that sequence alone can induce MLV release at levels comparable to wild-type Gag while the PNAP sequence alone lowers MLV release to levels similar to that observed for the 4A mutation (Fig. 2A and B).


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)

Generation of MLV GagCore L-domain chimeras.(A) HBV Core encodes a putative L-domain motif suggesting a potential docking site for HECT Ub ligases that direct HBV release. (B) L-domain chimeric proteins between MLV Gag and Core were generated by replacement of the endogenous MLV PPPY motif with the Core PPAY/PNAP motif and flanking residues. The location of the MLV Gag PPPY motif in the p12 region is italicized. The Gag4A mutant contains four alanine amino acid substitutions (AAAA) that abolish Gag L-domain activity. The inserted Core sequence is underlined in the MLV L-domain chimeras. The L-domain motif and L-domain motif mutations are italicized. Chimeric Gag protein was subcloned into the MLV GagPol expression plasmid to generate GagPol, GagPol4A, GagPolPPAY/PNAP, GagPolPPAY, GagPolPNAP, GagPolLPAY/PNAP, GagPolPPAF/PNAP, and GagPolPPAA/PNAP expression constructs. The MLV Pr65 Gag protein contains matrix (MA), capsids (CA), and nucleocapsid (NC) domains. Asterisks indicate PR cleavage sites.
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pone-0072845-g001: Generation of MLV GagCore L-domain chimeras.(A) HBV Core encodes a putative L-domain motif suggesting a potential docking site for HECT Ub ligases that direct HBV release. (B) L-domain chimeric proteins between MLV Gag and Core were generated by replacement of the endogenous MLV PPPY motif with the Core PPAY/PNAP motif and flanking residues. The location of the MLV Gag PPPY motif in the p12 region is italicized. The Gag4A mutant contains four alanine amino acid substitutions (AAAA) that abolish Gag L-domain activity. The inserted Core sequence is underlined in the MLV L-domain chimeras. The L-domain motif and L-domain motif mutations are italicized. Chimeric Gag protein was subcloned into the MLV GagPol expression plasmid to generate GagPol, GagPol4A, GagPolPPAY/PNAP, GagPolPPAY, GagPolPNAP, GagPolLPAY/PNAP, GagPolPPAF/PNAP, and GagPolPPAA/PNAP expression constructs. The MLV Pr65 Gag protein contains matrix (MA), capsids (CA), and nucleocapsid (NC) domains. Asterisks indicate PR cleavage sites.
Mentions: To determine if the Core 129PPAYRPPNAP138 sequence constitutes a genuine L-domain motif that directs release of a heterologous virus, expression constructs were generated to encode an MLV GagPolCore chimera protein where the endogenous PPPY L-domain of Gag was replaced with the putative Core L-domain motif (Fig. 1A and B). An MLV GagPolPPAY/PNAP chimera was synthesized by insertion of a Core 18 amino acid sequence fragment containing both the PPAY and PNAP motifs, as well as two MLV GagPol constructs that contained a 10 amino acid sequence with the PPAY or the PNAP sequence motif, GagPolPPAY and GagPolPNAP, respectively. To produce quantifiable infectious MLV particles containing chimeric protein, 293T cells were transfected with plasmids encoding the Friend MLV envelope protein, the LTR-LacZ reporter construct and the GagPol plasmids encoding either wild-type Gag, a Gag4A mutant that lacks L-domain activity, GagPPAY/PNAP, GagPPAY, or GagPNAP. Media and cellular lysates were harvested from virus-replicating cells 1 day post-transfection and resolved by SDS-PAGE to detect virion-associated Gag or cell-associated Gag protein, respectively, by western blot (Fig. 2A). Additionally, DFJ8 target cells that stably express MCAT-1, a surface receptor that is recognized by the Friend MLV envelope protein, were incubated with serial dilutions of the collected media for 24 h and subsequently stained for LacZ activity to quantify the amount of infectious MLV particles produced by the virus-replicating cells (Fig. 2B). As previously reported [39], [42], the AAAA (4A) mutation in Gag strongly inhibited infectious MLV release to 3% of wild-type MLV (Fig. 2A and B). Interestingly, the 18 amino acid sequence of Core that contains the PPAY and PNAP motif restored release of infectious MLV to 75% of wild-type MLV. Functional replacement of the endogenous Gag PPPY motif by the Core sequence can be attributed to the PPAY motif since that sequence alone can induce MLV release at levels comparable to wild-type Gag while the PNAP sequence alone lowers MLV release to levels similar to that observed for the 4A mutation (Fig. 2A and B).

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