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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

The effect of p12 mutations on the biophysical properties of the Mo-MLV intra-virion CA core.(A) Purified wild type or p12 mutant Mo-MLV VLPs were spun through a layer of detergent into a 10–42% (w/w) linear sucrose gradient. Fractions were harvested and analysed by immunoblotting using an anti-CA antibody (note: fractions 1–3 are diluted by 1∶13 to reduce the immunoblot signal, *: this input was run on a separate gel from the gradient fractions). Representative immunoblots are shown for wild type and each of the p12 mutants. (B) For each experiment, the sucrose density of the fraction containing the peak CA signal was measured, and the change in density compared to peak CA fraction for wild type virions was calculated. The mean and range from two independent experiments are displayed in the histogram.
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ppat-1004474-g003: The effect of p12 mutations on the biophysical properties of the Mo-MLV intra-virion CA core.(A) Purified wild type or p12 mutant Mo-MLV VLPs were spun through a layer of detergent into a 10–42% (w/w) linear sucrose gradient. Fractions were harvested and analysed by immunoblotting using an anti-CA antibody (note: fractions 1–3 are diluted by 1∶13 to reduce the immunoblot signal, *: this input was run on a separate gel from the gradient fractions). Representative immunoblots are shown for wild type and each of the p12 mutants. (B) For each experiment, the sucrose density of the fraction containing the peak CA signal was measured, and the change in density compared to peak CA fraction for wild type virions was calculated. The mean and range from two independent experiments are displayed in the histogram.

Mentions: Both the abrogation and biophysical data presented thus far have highlighted that N-terminal p12 mutant RTCs are altered in infected cells. However, to assess whether virions themselves are intrinsically different, we attempted to isolate CA cores from whole VLPs. Concentrated wild type and p12 mutant Mo-MLV VLPs were spun through a layer of detergent into a 10–42% (w/w) equilibrium sucrose gradient. Fractions were collected and the presence of CA analysed by immunoblotting. Firstly, it should be noted that the majority of CA was found in the first three fractions at the top of the gradient (samples were diluted 1∶13 relative to the remaining fractions before gel electrophoresis). The high level of un-complexed CA suggests that the detergent extraction was detrimental to core integrity, as has been described previously [24], although it is likely that a proportion of the CA in particles never forms part of the core. As was seen for the RTCs from infected cells (Fig. 2C), the N-terminal p12 mutant CA assemblies had a different distribution in the gradient to those from wild type Mo-MLV (Fig. 3A). For all samples, there was a population of CA at the top of the gradient (fractions 1–3) and a second population of CA in the middle of the gradient. However, the peak of CA was detected in fractions 6 to 8 for wild type virions, but in fractions 4 to 6 for all N-terminal mutants (Fig. 3A), indicating a reduction in density for these mutants. Curiously, the C-terminal p12 mutant CA assemblies migrated to a higher sucrose density than wild type assemblies (Fig. 3A). The density of the fraction containing the peak CA signal was measured for each mutant and the change in peak density compared to wild type particles was calculated. The mean and range from multiple independent experiments are plotted in Fig. 3B.


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 intra-virion CA core.(A) Purified wild type or p12 mutant Mo-MLV VLPs were spun through a layer of detergent into a 10–42% (w/w) linear sucrose gradient. Fractions were harvested and analysed by immunoblotting using an anti-CA antibody (note: fractions 1–3 are diluted by 1∶13 to reduce the immunoblot signal, *: this input was run on a separate gel from the gradient fractions). Representative immunoblots are shown for wild type and each of the p12 mutants. (B) For each experiment, the sucrose density of the fraction containing the peak CA signal was measured, and the change in density compared to peak CA fraction for wild type virions was calculated. The mean and range from two independent experiments are displayed in the histogram.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004474-g003: The effect of p12 mutations on the biophysical properties of the Mo-MLV intra-virion CA core.(A) Purified wild type or p12 mutant Mo-MLV VLPs were spun through a layer of detergent into a 10–42% (w/w) linear sucrose gradient. Fractions were harvested and analysed by immunoblotting using an anti-CA antibody (note: fractions 1–3 are diluted by 1∶13 to reduce the immunoblot signal, *: this input was run on a separate gel from the gradient fractions). Representative immunoblots are shown for wild type and each of the p12 mutants. (B) For each experiment, the sucrose density of the fraction containing the peak CA signal was measured, and the change in density compared to peak CA fraction for wild type virions was calculated. The mean and range from two independent experiments are displayed in the histogram.
Mentions: Both the abrogation and biophysical data presented thus far have highlighted that N-terminal p12 mutant RTCs are altered in infected cells. However, to assess whether virions themselves are intrinsically different, we attempted to isolate CA cores from whole VLPs. Concentrated wild type and p12 mutant Mo-MLV VLPs were spun through a layer of detergent into a 10–42% (w/w) equilibrium sucrose gradient. Fractions were collected and the presence of CA analysed by immunoblotting. Firstly, it should be noted that the majority of CA was found in the first three fractions at the top of the gradient (samples were diluted 1∶13 relative to the remaining fractions before gel electrophoresis). The high level of un-complexed CA suggests that the detergent extraction was detrimental to core integrity, as has been described previously [24], although it is likely that a proportion of the CA in particles never forms part of the core. As was seen for the RTCs from infected cells (Fig. 2C), the N-terminal p12 mutant CA assemblies had a different distribution in the gradient to those from wild type Mo-MLV (Fig. 3A). For all samples, there was a population of CA at the top of the gradient (fractions 1–3) and a second population of CA in the middle of the gradient. However, the peak of CA was detected in fractions 6 to 8 for wild type virions, but in fractions 4 to 6 for all N-terminal mutants (Fig. 3A), indicating a reduction in density for these mutants. Curiously, the C-terminal p12 mutant CA assemblies migrated to a higher sucrose density than wild type assemblies (Fig. 3A). The density of the fraction containing the peak CA signal was measured for each mutant and the change in peak density compared to wild type particles was calculated. The mean and range from multiple independent experiments are plotted in Fig. 3B.

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