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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

Kinetics of p12 and CA loss from cells infected with Mo-MLV.Ten cells were randomly chosen (based solely on nuclear stain) and imaged using a spinning disk confocal microscope. The numbers of p12(myc) or CA puncta in each cell were determined. The mean number of puncta per cell was normalised to the mean number of CA or p12(myc) puncta present at time point zero for each infection. The amount of p12 (dashed lines) and CA (solid lines) puncta are displayed against time in the graphs for: (A) p12 mutant 5, (B) p12 mutant 6, (C) p12 mutant 7, (D) p12 mutant 8 and (E) p12 mutant 14. Wild type Mo-MLV data (p12, black dashed and CA, black solid lines) are also plotted in each graph as a reference. Each point indicates the mean with SEM error bars. (F) The normalised numbers of CA and p12(myc) puncta in cells two hours post-infection are displayed as a bar chart showing the mean with SEM error bars.
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ppat-1004474-g007: Kinetics of p12 and CA loss from cells infected with Mo-MLV.Ten cells were randomly chosen (based solely on nuclear stain) and imaged using a spinning disk confocal microscope. The numbers of p12(myc) or CA puncta in each cell were determined. The mean number of puncta per cell was normalised to the mean number of CA or p12(myc) puncta present at time point zero for each infection. The amount of p12 (dashed lines) and CA (solid lines) puncta are displayed against time in the graphs for: (A) p12 mutant 5, (B) p12 mutant 6, (C) p12 mutant 7, (D) p12 mutant 8 and (E) p12 mutant 14. Wild type Mo-MLV data (p12, black dashed and CA, black solid lines) are also plotted in each graph as a reference. Each point indicates the mean with SEM error bars. (F) The normalised numbers of CA and p12(myc) puncta in cells two hours post-infection are displayed as a bar chart showing the mean with SEM error bars.

Mentions: To quantify these observations, cells were chosen at random using the nuclear counterstain and the entire cell body was imaged using a spinning disk confocal microscope. Outlines of the cells were drawn and the number of p12(myc) and CA puncta within each cell determined. Table S1 shows the mean numbers of puncta measured at time zero and two hours post-infection. The mean number of p12(myc) and CA puncta at each time point for each infection was normalised to the mean number of puncta at the zero hour time point and plotted against time post-infection. Figure 7 shows the analysis from 10 cells containing high numbers of puncta (∼250–300 puncta per cell at the zero hour time point). Importantly, very similar results were obtained from analysis of 10–16 cells from separate infections containing lower numbers of puncta; 30–60 puncta per cell at the zero hour time point (unpublished data). These analyses clearly demonstrate that there was a rapid reduction of p12 puncta from all N-terminal p12 mutant infected cells (Fig. 7A–D, coloured dashed lines), with more than 75% of the p12 puncta lost by two hours post-infection (Fig. 7F). In contrast, less than 50% of the p12 puncta were lost in cells infected with either wild type Mo-MLV (Fig. 7A–E, black dashed line, 7F) or p12 mutant 14 (Fig. 7E, purple dashed line, 7F). Notably, the number of CA puncta was also reduced by 65–75% by two hours post-infection in cells infected with the N-terminal p12 mutants (Fig. 7F), while there was only a minor reduction of CA puncta for wild type and p12 mutant 14 infections (Fig. 7F). Statistical analysis (t-test) of the number of puncta in cells two hours after infection (Fig. 7F) showed highly significant differences for N-terminal p12 mutants compared to wild type infections. Specifically, comparing p12 puncta with wild type gave p values of 0.01, 0.002, 0.0038 and 0.0029 for p12 mutants 5, 6, 7 and 8 respectively, and comparing CA puncta with wild type gave p values of 0.0028, 0.0023, 0.0029 and 0.0036 for p12 mutants 5, 6, 7 and 8, respectively. Taken together, these results suggest that alteration to the N-terminus of p12 results in a rapid loss of both p12 and CA itself from incoming viral cores. This suggests that the N-terminal domain of p12 is required for the retention of p12 within the RTC, and for conservation of the MLV CA core in the target cell.


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)

Kinetics of p12 and CA loss from cells infected with Mo-MLV.Ten cells were randomly chosen (based solely on nuclear stain) and imaged using a spinning disk confocal microscope. The numbers of p12(myc) or CA puncta in each cell were determined. The mean number of puncta per cell was normalised to the mean number of CA or p12(myc) puncta present at time point zero for each infection. The amount of p12 (dashed lines) and CA (solid lines) puncta are displayed against time in the graphs for: (A) p12 mutant 5, (B) p12 mutant 6, (C) p12 mutant 7, (D) p12 mutant 8 and (E) p12 mutant 14. Wild type Mo-MLV data (p12, black dashed and CA, black solid lines) are also plotted in each graph as a reference. Each point indicates the mean with SEM error bars. (F) The normalised numbers of CA and p12(myc) puncta in cells two hours post-infection are displayed as a bar chart showing the mean with SEM error bars.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004474-g007: Kinetics of p12 and CA loss from cells infected with Mo-MLV.Ten cells were randomly chosen (based solely on nuclear stain) and imaged using a spinning disk confocal microscope. The numbers of p12(myc) or CA puncta in each cell were determined. The mean number of puncta per cell was normalised to the mean number of CA or p12(myc) puncta present at time point zero for each infection. The amount of p12 (dashed lines) and CA (solid lines) puncta are displayed against time in the graphs for: (A) p12 mutant 5, (B) p12 mutant 6, (C) p12 mutant 7, (D) p12 mutant 8 and (E) p12 mutant 14. Wild type Mo-MLV data (p12, black dashed and CA, black solid lines) are also plotted in each graph as a reference. Each point indicates the mean with SEM error bars. (F) The normalised numbers of CA and p12(myc) puncta in cells two hours post-infection are displayed as a bar chart showing the mean with SEM error bars.
Mentions: To quantify these observations, cells were chosen at random using the nuclear counterstain and the entire cell body was imaged using a spinning disk confocal microscope. Outlines of the cells were drawn and the number of p12(myc) and CA puncta within each cell determined. Table S1 shows the mean numbers of puncta measured at time zero and two hours post-infection. The mean number of p12(myc) and CA puncta at each time point for each infection was normalised to the mean number of puncta at the zero hour time point and plotted against time post-infection. Figure 7 shows the analysis from 10 cells containing high numbers of puncta (∼250–300 puncta per cell at the zero hour time point). Importantly, very similar results were obtained from analysis of 10–16 cells from separate infections containing lower numbers of puncta; 30–60 puncta per cell at the zero hour time point (unpublished data). These analyses clearly demonstrate that there was a rapid reduction of p12 puncta from all N-terminal p12 mutant infected cells (Fig. 7A–D, coloured dashed lines), with more than 75% of the p12 puncta lost by two hours post-infection (Fig. 7F). In contrast, less than 50% of the p12 puncta were lost in cells infected with either wild type Mo-MLV (Fig. 7A–E, black dashed line, 7F) or p12 mutant 14 (Fig. 7E, purple dashed line, 7F). Notably, the number of CA puncta was also reduced by 65–75% by two hours post-infection in cells infected with the N-terminal p12 mutants (Fig. 7F), while there was only a minor reduction of CA puncta for wild type and p12 mutant 14 infections (Fig. 7F). Statistical analysis (t-test) of the number of puncta in cells two hours after infection (Fig. 7F) showed highly significant differences for N-terminal p12 mutants compared to wild type infections. Specifically, comparing p12 puncta with wild type gave p values of 0.01, 0.002, 0.0038 and 0.0029 for p12 mutants 5, 6, 7 and 8 respectively, and comparing CA puncta with wild type gave p values of 0.0028, 0.0023, 0.0029 and 0.0036 for p12 mutants 5, 6, 7 and 8, respectively. Taken together, these results suggest that alteration to the N-terminus of p12 results in a rapid loss of both p12 and CA itself from incoming viral cores. This suggests that the N-terminal domain of p12 is required for the retention of p12 within the RTC, and for conservation of the MLV CA core in the target cell.

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