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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

Binding of purified p12 to N-MLV CA-coated lipid nanotubes.Multimeric arrays of wild type (WT) and P1G mutant N-MLV CA were generated by immobilising the His-tagged purified proteins on lipid nanotubes comprising the Ni2+-chelating lipid, DGS-NTA. Purified wild type p12 (lanes 2–4) or p12 Mutant 6 (lanes 6–8) were incubated with lipid nanotubes coated with WT CA (lanes 2 and 6), P1G CA (lanes 3 and 7) or no tubes (lanes 4 and 8) prior to centrifugation through a sucrose cushion. The pelleted material was resuspended in SDS-PAGE sample buffer and probed for CA and p12 by immunoblotting, using appropriate antibodies. Input, lanes 1 and 5, represents 1/40th dilution of the purified p12 proteins before incubation with CA-coated lipid nanotubes.
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ppat-1004474-g008: Binding of purified p12 to N-MLV CA-coated lipid nanotubes.Multimeric arrays of wild type (WT) and P1G mutant N-MLV CA were generated by immobilising the His-tagged purified proteins on lipid nanotubes comprising the Ni2+-chelating lipid, DGS-NTA. Purified wild type p12 (lanes 2–4) or p12 Mutant 6 (lanes 6–8) were incubated with lipid nanotubes coated with WT CA (lanes 2 and 6), P1G CA (lanes 3 and 7) or no tubes (lanes 4 and 8) prior to centrifugation through a sucrose cushion. The pelleted material was resuspended in SDS-PAGE sample buffer and probed for CA and p12 by immunoblotting, using appropriate antibodies. Input, lanes 1 and 5, represents 1/40th dilution of the purified p12 proteins before incubation with CA-coated lipid nanotubes.

Mentions: For p12 to be incorporated into the RTC, one would expect p12 to interact with core components. Indeed, a small amount of CA was previously immunoprecipitated from cells challenged with Mo-MLV using an antibody against myc-tagged p12, although this could not be recapitulated by immunoprecipitation of p12 from virions [12]. Given that p12 appears to influence the stability of the core, it is logical to predict that p12 binds directly to CA. However, a direct binding has never been shown, and most CA binding assays are hindered by the fact that the CA in the RTC is present in the form of a lattice, so monomeric CA may not recapitulate the binding surface present in an array. Fortunately, a protocol to form mature MLV CA lattice arrays on lipid nanotubes was previously established to study CA-Fv1 interactions [34]. We therefore used this approach to investigate whether p12 directly binds the CA lattice. Briefly, purified His-tagged N-MLV CA was immobilised on lipid nanotubes comprising the Ni2+-chelating lipid, DGS-NTA. These tubes were then incubated with purified p12 protein, and bound complexes were separated from unbound p12 by centrifugation through a sucrose cushion. The pelleted fraction was analysed for the presence of His-tagged CA or p12 proteins by immunoblotting with anti-His tag and anti-p12 polyclonal antibodies respectively. Fig. 8 shows representative immunoblots from 4 independent experiments that demonstrate detectable binding of wild type p12 protein to CA-coated lipid nanotubes (lane 2) but not p12 mutant 6 (lane 6). Importantly, we did not detect binding of either p12 protein to a version of CA that cannot form high density, regular arrays, CA-P1G [34] (lanes 3 and 7) showing there was little non-specific binding. Nor did either p12 protein pellet in the absence of CA-coated nanotubes under these conditions (lanes 4 and 8). In addition, cell lysates expressing either Fv1b or Fv1n were also incubated with the same CA-coated tubes as a positive and negative control for CA binding respectively [34]. Fig. S7 shows that we could detect binding of Fv1b to the nanotubes, but Fv1n had much reduced binding as expected (compare lanes 2 and 6), confirming that the CA was arranged in regular arrays that mimic true viral cores. Both Fv1 proteins showed weak binding to CA-P1G (lanes 3 and 7) indicating some non-specific binding. Together, this indicates that p12 does bind directly to the CA lattice and that the N-terminus of p12 is necessary for this interaction.


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)

Binding of purified p12 to N-MLV CA-coated lipid nanotubes.Multimeric arrays of wild type (WT) and P1G mutant N-MLV CA were generated by immobilising the His-tagged purified proteins on lipid nanotubes comprising the Ni2+-chelating lipid, DGS-NTA. Purified wild type p12 (lanes 2–4) or p12 Mutant 6 (lanes 6–8) were incubated with lipid nanotubes coated with WT CA (lanes 2 and 6), P1G CA (lanes 3 and 7) or no tubes (lanes 4 and 8) prior to centrifugation through a sucrose cushion. The pelleted material was resuspended in SDS-PAGE sample buffer and probed for CA and p12 by immunoblotting, using appropriate antibodies. Input, lanes 1 and 5, represents 1/40th dilution of the purified p12 proteins before incubation with CA-coated lipid nanotubes.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004474-g008: Binding of purified p12 to N-MLV CA-coated lipid nanotubes.Multimeric arrays of wild type (WT) and P1G mutant N-MLV CA were generated by immobilising the His-tagged purified proteins on lipid nanotubes comprising the Ni2+-chelating lipid, DGS-NTA. Purified wild type p12 (lanes 2–4) or p12 Mutant 6 (lanes 6–8) were incubated with lipid nanotubes coated with WT CA (lanes 2 and 6), P1G CA (lanes 3 and 7) or no tubes (lanes 4 and 8) prior to centrifugation through a sucrose cushion. The pelleted material was resuspended in SDS-PAGE sample buffer and probed for CA and p12 by immunoblotting, using appropriate antibodies. Input, lanes 1 and 5, represents 1/40th dilution of the purified p12 proteins before incubation with CA-coated lipid nanotubes.
Mentions: For p12 to be incorporated into the RTC, one would expect p12 to interact with core components. Indeed, a small amount of CA was previously immunoprecipitated from cells challenged with Mo-MLV using an antibody against myc-tagged p12, although this could not be recapitulated by immunoprecipitation of p12 from virions [12]. Given that p12 appears to influence the stability of the core, it is logical to predict that p12 binds directly to CA. However, a direct binding has never been shown, and most CA binding assays are hindered by the fact that the CA in the RTC is present in the form of a lattice, so monomeric CA may not recapitulate the binding surface present in an array. Fortunately, a protocol to form mature MLV CA lattice arrays on lipid nanotubes was previously established to study CA-Fv1 interactions [34]. We therefore used this approach to investigate whether p12 directly binds the CA lattice. Briefly, purified His-tagged N-MLV CA was immobilised on lipid nanotubes comprising the Ni2+-chelating lipid, DGS-NTA. These tubes were then incubated with purified p12 protein, and bound complexes were separated from unbound p12 by centrifugation through a sucrose cushion. The pelleted fraction was analysed for the presence of His-tagged CA or p12 proteins by immunoblotting with anti-His tag and anti-p12 polyclonal antibodies respectively. Fig. 8 shows representative immunoblots from 4 independent experiments that demonstrate detectable binding of wild type p12 protein to CA-coated lipid nanotubes (lane 2) but not p12 mutant 6 (lane 6). Importantly, we did not detect binding of either p12 protein to a version of CA that cannot form high density, regular arrays, CA-P1G [34] (lanes 3 and 7) showing there was little non-specific binding. Nor did either p12 protein pellet in the absence of CA-coated nanotubes under these conditions (lanes 4 and 8). In addition, cell lysates expressing either Fv1b or Fv1n were also incubated with the same CA-coated tubes as a positive and negative control for CA binding respectively [34]. Fig. S7 shows that we could detect binding of Fv1b to the nanotubes, but Fv1n had much reduced binding as expected (compare lanes 2 and 6), confirming that the CA was arranged in regular arrays that mimic true viral cores. Both Fv1 proteins showed weak binding to CA-P1G (lanes 3 and 7) indicating some non-specific binding. Together, this indicates that p12 does bind directly to the CA lattice and that the N-terminus of p12 is necessary for this interaction.

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