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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

Immunofluorescence of p12 and CA in cells infected with Mo-MLV p12 mutants.U/R cells were challenged with ecotropic wild type or p12 mutant Mo-MLV VLPs, containing a myc-tag in p12, by cold spinoculation (MOI 3). Cells were fixed at various times post-infection and stained with either an (A) anti-myc or (B) anti-CA antibody followed by a Cy3 (A) or FITC (B) -conjugated secondary antibody. The nuclear DNA was counterstained using DAPI (blue). Images from the time course were captured using a spinning disk confocal microscope and representative images of cells from each time point are shown. All images are three dimensional acquisitions projected on a two dimensional plane. Images were processed using SlideBook. Scale bars are 10 µm.
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ppat-1004474-g006: Immunofluorescence of p12 and CA in cells infected with Mo-MLV p12 mutants.U/R cells were challenged with ecotropic wild type or p12 mutant Mo-MLV VLPs, containing a myc-tag in p12, by cold spinoculation (MOI 3). Cells were fixed at various times post-infection and stained with either an (A) anti-myc or (B) anti-CA antibody followed by a Cy3 (A) or FITC (B) -conjugated secondary antibody. The nuclear DNA was counterstained using DAPI (blue). Images from the time course were captured using a spinning disk confocal microscope and representative images of cells from each time point are shown. All images are three dimensional acquisitions projected on a two dimensional plane. Images were processed using SlideBook. Scale bars are 10 µm.

Mentions: For wild type Mo-MLV, an apparent decrease in the number of p12 puncta could be seen with time (Fig. 6A, top row). Traditionally, it has been thought that the rate of CA uncoating for MLV is slower than HIV-1, with significant amounts of CA remaining with the MLV PIC during transit to the nucleus [24], [33]. In line with these observations, we could see that cells challenged with wild type Mo-MLV contained large numbers of CA puncta, which remained for the length of the time course (Fig. 6B, top row). Thus, during infection with wild type Mo-MLV, some proportion of the viral p12 is shed from the PIC faster than CA, mirroring our earlier immunoblot observations (Fig. 2B). Interestingly, all of the N-terminal p12 mutants exhibited a differing phenotype to wild type Mo-MLV: A significant reduction in the number of p12 puncta could be observed very early, 0.5–1 hour post-infection (Fig. 6A and S6A). Furthermore, the number of CA puncta in N-terminal p12 mutant challenged cells also decreased with time (Fig. 6B and S6B). Consistent with our biophysical data, cells infected with the C-terminal p12 mutant 14 showed a similar pattern of puncta to wild type infections (Fig. S6A and B, bottom row).


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)

Immunofluorescence of p12 and CA in cells infected with Mo-MLV p12 mutants.U/R cells were challenged with ecotropic wild type or p12 mutant Mo-MLV VLPs, containing a myc-tag in p12, by cold spinoculation (MOI 3). Cells were fixed at various times post-infection and stained with either an (A) anti-myc or (B) anti-CA antibody followed by a Cy3 (A) or FITC (B) -conjugated secondary antibody. The nuclear DNA was counterstained using DAPI (blue). Images from the time course were captured using a spinning disk confocal microscope and representative images of cells from each time point are shown. All images are three dimensional acquisitions projected on a two dimensional plane. Images were processed using SlideBook. Scale bars are 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004474-g006: Immunofluorescence of p12 and CA in cells infected with Mo-MLV p12 mutants.U/R cells were challenged with ecotropic wild type or p12 mutant Mo-MLV VLPs, containing a myc-tag in p12, by cold spinoculation (MOI 3). Cells were fixed at various times post-infection and stained with either an (A) anti-myc or (B) anti-CA antibody followed by a Cy3 (A) or FITC (B) -conjugated secondary antibody. The nuclear DNA was counterstained using DAPI (blue). Images from the time course were captured using a spinning disk confocal microscope and representative images of cells from each time point are shown. All images are three dimensional acquisitions projected on a two dimensional plane. Images were processed using SlideBook. Scale bars are 10 µm.
Mentions: For wild type Mo-MLV, an apparent decrease in the number of p12 puncta could be seen with time (Fig. 6A, top row). Traditionally, it has been thought that the rate of CA uncoating for MLV is slower than HIV-1, with significant amounts of CA remaining with the MLV PIC during transit to the nucleus [24], [33]. In line with these observations, we could see that cells challenged with wild type Mo-MLV contained large numbers of CA puncta, which remained for the length of the time course (Fig. 6B, top row). Thus, during infection with wild type Mo-MLV, some proportion of the viral p12 is shed from the PIC faster than CA, mirroring our earlier immunoblot observations (Fig. 2B). Interestingly, all of the N-terminal p12 mutants exhibited a differing phenotype to wild type Mo-MLV: A significant reduction in the number of p12 puncta could be observed very early, 0.5–1 hour post-infection (Fig. 6A and S6A). Furthermore, the number of CA puncta in N-terminal p12 mutant challenged cells also decreased with time (Fig. 6B and S6B). Consistent with our biophysical data, cells infected with the C-terminal p12 mutant 14 showed a similar pattern of puncta to wild type infections (Fig. S6A and B, bottom row).

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