Limits...
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

The effect of p12 mutations on the biophysical properties of the Mo-MLV RTC.(A) D17 cells were infected for four hours with wild type Mo-MLV, and cell lysates were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient. Fractions were analysed by qPCR with primers targeted to strong stop viral cDNA and proteins were precipitated and analysed by immunoblotting using anti-CA (top blot) and anti-p12 monoclonal (bottom blot) antibodies (Fraction 1 is the top of gradient). The graph displays the copies of viral DNA per microliter (black line) and the percentage of total CA in each fraction (grey line). (B) Wild type Mo-MLV was used to challenge D17 cells and after four hours the cells were lysed (cell lysate). An aliquot of the Mo-MLV VLPs used for the infection was also lysed (virus) and both samples were analysed by immunoblotting with anti-CA (top panel) and anti-p12 (bottom panel) monoclonal antibodies. Two repeats of this experiment are shown in the immunoblot. (C) Infected cell lysates from D17 cells challenged with wild type or p12 mutant Mo-MLV VLPs were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient and analysed by immunoblotting for CA. (D) For each experiment, the intensity of the CA signal on the immunoblot was determined and the relative shift in the peak CA position compared to wild type RTCs was calculated. The mean and range of at least three independent experiments are displayed.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214797&req=5

ppat-1004474-g002: The effect of p12 mutations on the biophysical properties of the Mo-MLV RTC.(A) D17 cells were infected for four hours with wild type Mo-MLV, and cell lysates were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient. Fractions were analysed by qPCR with primers targeted to strong stop viral cDNA and proteins were precipitated and analysed by immunoblotting using anti-CA (top blot) and anti-p12 monoclonal (bottom blot) antibodies (Fraction 1 is the top of gradient). The graph displays the copies of viral DNA per microliter (black line) and the percentage of total CA in each fraction (grey line). (B) Wild type Mo-MLV was used to challenge D17 cells and after four hours the cells were lysed (cell lysate). An aliquot of the Mo-MLV VLPs used for the infection was also lysed (virus) and both samples were analysed by immunoblotting with anti-CA (top panel) and anti-p12 (bottom panel) monoclonal antibodies. Two repeats of this experiment are shown in the immunoblot. (C) Infected cell lysates from D17 cells challenged with wild type or p12 mutant Mo-MLV VLPs were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient and analysed by immunoblotting for CA. (D) For each experiment, the intensity of the CA signal on the immunoblot was determined and the relative shift in the peak CA position compared to wild type RTCs was calculated. The mean and range of at least three independent experiments are displayed.

Mentions: TRIM5alpha targets the CA shell of N-MLV. The failure of N-terminal p12 mutants to abrogate restriction factors therefore suggests an intriguing possibility that the CA shell is perturbed in these mutants. Thus, we investigated what effect N-terminal p12 mutations had on CA. We previously showed that CA expressed from gag containing an upstream mutation in p12 could be incorporated into the CA core and be recognised by restriction factors, indicating that mutations in p12 do not affect the function of CA molecules per se[10]. Therefore, we studied the effect of p12 mutations on CA complexes using a modified fate-of-CA assay. D17 cells were challenged with wild type or p12 mutant Mo-MLV VLPs for 4 hours and cell lysates were separated on 10–42% (w/w) linear sucrose gradients by velocity sedimentation. The presence of CA in each gradient fraction was assessed by immunoblotting (Fig. 2).


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)

The effect of p12 mutations on the biophysical properties of the Mo-MLV RTC.(A) D17 cells were infected for four hours with wild type Mo-MLV, and cell lysates were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient. Fractions were analysed by qPCR with primers targeted to strong stop viral cDNA and proteins were precipitated and analysed by immunoblotting using anti-CA (top blot) and anti-p12 monoclonal (bottom blot) antibodies (Fraction 1 is the top of gradient). The graph displays the copies of viral DNA per microliter (black line) and the percentage of total CA in each fraction (grey line). (B) Wild type Mo-MLV was used to challenge D17 cells and after four hours the cells were lysed (cell lysate). An aliquot of the Mo-MLV VLPs used for the infection was also lysed (virus) and both samples were analysed by immunoblotting with anti-CA (top panel) and anti-p12 (bottom panel) monoclonal antibodies. Two repeats of this experiment are shown in the immunoblot. (C) Infected cell lysates from D17 cells challenged with wild type or p12 mutant Mo-MLV VLPs were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient and analysed by immunoblotting for CA. (D) For each experiment, the intensity of the CA signal on the immunoblot was determined and the relative shift in the peak CA position compared to wild type RTCs was calculated. The mean and range of at least three independent experiments are displayed.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004474-g002: The effect of p12 mutations on the biophysical properties of the Mo-MLV RTC.(A) D17 cells were infected for four hours with wild type Mo-MLV, and cell lysates were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient. Fractions were analysed by qPCR with primers targeted to strong stop viral cDNA and proteins were precipitated and analysed by immunoblotting using anti-CA (top blot) and anti-p12 monoclonal (bottom blot) antibodies (Fraction 1 is the top of gradient). The graph displays the copies of viral DNA per microliter (black line) and the percentage of total CA in each fraction (grey line). (B) Wild type Mo-MLV was used to challenge D17 cells and after four hours the cells were lysed (cell lysate). An aliquot of the Mo-MLV VLPs used for the infection was also lysed (virus) and both samples were analysed by immunoblotting with anti-CA (top panel) and anti-p12 (bottom panel) monoclonal antibodies. Two repeats of this experiment are shown in the immunoblot. (C) Infected cell lysates from D17 cells challenged with wild type or p12 mutant Mo-MLV VLPs were separated using velocity sedimentation through a 10–42% (w/w) sucrose gradient and analysed by immunoblotting for CA. (D) For each experiment, the intensity of the CA signal on the immunoblot was determined and the relative shift in the peak CA position compared to wild type RTCs was calculated. The mean and range of at least three independent experiments are displayed.
Mentions: TRIM5alpha targets the CA shell of N-MLV. The failure of N-terminal p12 mutants to abrogate restriction factors therefore suggests an intriguing possibility that the CA shell is perturbed in these mutants. Thus, we investigated what effect N-terminal p12 mutations had on CA. We previously showed that CA expressed from gag containing an upstream mutation in p12 could be incorporated into the CA core and be recognised by restriction factors, indicating that mutations in p12 do not affect the function of CA molecules per se[10]. Therefore, we studied the effect of p12 mutations on CA complexes using a modified fate-of-CA assay. D17 cells were challenged with wild type or p12 mutant Mo-MLV VLPs for 4 hours and cell lysates were separated on 10–42% (w/w) linear sucrose gradients by velocity sedimentation. The presence of CA in each gradient fraction was assessed by immunoblotting (Fig. 2).

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