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The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

Wight DJ, Boucherit VC, Wanaguru M, Elis E, Hirst EM, Li W, Ehrlich M, Bacharach E, Bishop KN - PLoS Pathog. (2014)

Bottom Line: Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores.We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions.These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.

ABSTRACT
The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

No MeSH data available.


Related in: MedlinePlus

Abrogation of TRIM5alpha restriction by mixed p12 mutant particles.(A) Schematic representation of the Gag-Pol expression plasmid and p12 substitution mutants used in this study showing the amino acid sequence of Mo-MLV and N-MLV p12. (B) LacZ-encoding N-MLV VLPs containing either wild type p12, p12 mutant 6 or p12 mutant 14 (triangles), or a mixture of both mutants (circles) were synthesised. The percentage of p12 mutant 14 Gag-Pol expression plasmid in the transfection mix is indicated in brackets for the mixed mutants. Serial dilutions of these VLPs were used to challenge TE671 cells. Four hours later, cells were challenged with a fixed dose of GFP-encoding N-MLV VLPs, and after a further three days, the number of GFP positive cells was measured by flow cytometry. The percentage of GFP positive cells is plotted against LacZ-virus dilution. (C) D17 cells were challenged with equivalent RT-units of the VLPs used in (B). Infectivity was measured by detection of beta-galactosidase activity in a chemiluminescent reporter assay and plotted as a percentage of wild type N-MLV infectivity. These data are representative of multiple independent experiments.
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ppat-1004474-g001: Abrogation of TRIM5alpha restriction by mixed p12 mutant particles.(A) Schematic representation of the Gag-Pol expression plasmid and p12 substitution mutants used in this study showing the amino acid sequence of Mo-MLV and N-MLV p12. (B) LacZ-encoding N-MLV VLPs containing either wild type p12, p12 mutant 6 or p12 mutant 14 (triangles), or a mixture of both mutants (circles) were synthesised. The percentage of p12 mutant 14 Gag-Pol expression plasmid in the transfection mix is indicated in brackets for the mixed mutants. Serial dilutions of these VLPs were used to challenge TE671 cells. Four hours later, cells were challenged with a fixed dose of GFP-encoding N-MLV VLPs, and after a further three days, the number of GFP positive cells was measured by flow cytometry. The percentage of GFP positive cells is plotted against LacZ-virus dilution. (C) D17 cells were challenged with equivalent RT-units of the VLPs used in (B). Infectivity was measured by detection of beta-galactosidase activity in a chemiluminescent reporter assay and plotted as a percentage of wild type N-MLV infectivity. These data are representative of multiple independent experiments.

Mentions: The Gag protein p12 has important roles during both the early and late stages of murine leukaemia virus (MLV) infection [8]. It harbours the PPPY late-domain (L-domain), essential for recruiting HECT ubiquitin ligases to manipulate the ESCRT pathway for efficient budding [8], [9]. Additionally, seven mutants have been defined in Mo-MLV p12, four in the N-terminus and three in the C-terminus, which have a potent block during the early stages of infection (Fig. 1A) [8], [10]. The replication defects of these mutants fall into three groups: (i) mutants defective in reverse transcription in vivo (mutant 6), (ii) mutants defective in reverse transcription in certain cell lines (mutant 8) and (iii) mutants competent for reverse transcription but failing to integrate their viral DNA (mutant 5, 7, 13, 14 and 15) [8], [10]. We have recently shown that the N- and C- terminal regions of p12, mapped out by these mutants, are actually two sequentially acting domains, both of which are required to be active on the same p12 molecule for the transduction of target cells [10].


The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

Wight DJ, Boucherit VC, Wanaguru M, Elis E, Hirst EM, Li W, Ehrlich M, Bacharach E, Bishop KN - PLoS Pathog. (2014)

Abrogation of TRIM5alpha restriction by mixed p12 mutant particles.(A) Schematic representation of the Gag-Pol expression plasmid and p12 substitution mutants used in this study showing the amino acid sequence of Mo-MLV and N-MLV p12. (B) LacZ-encoding N-MLV VLPs containing either wild type p12, p12 mutant 6 or p12 mutant 14 (triangles), or a mixture of both mutants (circles) were synthesised. The percentage of p12 mutant 14 Gag-Pol expression plasmid in the transfection mix is indicated in brackets for the mixed mutants. Serial dilutions of these VLPs were used to challenge TE671 cells. Four hours later, cells were challenged with a fixed dose of GFP-encoding N-MLV VLPs, and after a further three days, the number of GFP positive cells was measured by flow cytometry. The percentage of GFP positive cells is plotted against LacZ-virus dilution. (C) D17 cells were challenged with equivalent RT-units of the VLPs used in (B). Infectivity was measured by detection of beta-galactosidase activity in a chemiluminescent reporter assay and plotted as a percentage of wild type N-MLV infectivity. These data are representative of multiple independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214797&req=5

ppat-1004474-g001: Abrogation of TRIM5alpha restriction by mixed p12 mutant particles.(A) Schematic representation of the Gag-Pol expression plasmid and p12 substitution mutants used in this study showing the amino acid sequence of Mo-MLV and N-MLV p12. (B) LacZ-encoding N-MLV VLPs containing either wild type p12, p12 mutant 6 or p12 mutant 14 (triangles), or a mixture of both mutants (circles) were synthesised. The percentage of p12 mutant 14 Gag-Pol expression plasmid in the transfection mix is indicated in brackets for the mixed mutants. Serial dilutions of these VLPs were used to challenge TE671 cells. Four hours later, cells were challenged with a fixed dose of GFP-encoding N-MLV VLPs, and after a further three days, the number of GFP positive cells was measured by flow cytometry. The percentage of GFP positive cells is plotted against LacZ-virus dilution. (C) D17 cells were challenged with equivalent RT-units of the VLPs used in (B). Infectivity was measured by detection of beta-galactosidase activity in a chemiluminescent reporter assay and plotted as a percentage of wild type N-MLV infectivity. These data are representative of multiple independent experiments.
Mentions: The Gag protein p12 has important roles during both the early and late stages of murine leukaemia virus (MLV) infection [8]. It harbours the PPPY late-domain (L-domain), essential for recruiting HECT ubiquitin ligases to manipulate the ESCRT pathway for efficient budding [8], [9]. Additionally, seven mutants have been defined in Mo-MLV p12, four in the N-terminus and three in the C-terminus, which have a potent block during the early stages of infection (Fig. 1A) [8], [10]. The replication defects of these mutants fall into three groups: (i) mutants defective in reverse transcription in vivo (mutant 6), (ii) mutants defective in reverse transcription in certain cell lines (mutant 8) and (iii) mutants competent for reverse transcription but failing to integrate their viral DNA (mutant 5, 7, 13, 14 and 15) [8], [10]. We have recently shown that the N- and C- terminal regions of p12, mapped out by these mutants, are actually two sequentially acting domains, both of which are required to be active on the same p12 molecule for the transduction of target cells [10].

Bottom Line: Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores.We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions.These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.

ABSTRACT
The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

No MeSH data available.


Related in: MedlinePlus