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HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

Ko LJ, Yu FH, Huang KJ, Wang CT - FEBS Open Bio (2015)

Bottom Line: Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly.To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues.Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan ; Institute of Public Health, National Yang-Ming University School of Medicine, Taipei, Taiwan.

ABSTRACT
Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

No MeSH data available.


Related in: MedlinePlus

VLP release and processing kinetics 293T cells grown in 10 cm culture dishes in triplicate were transfected with the designated plasmid, pooled, and divided equally onto four dish plates. At 16 h post-transfection, supernatants were collected and fed medium containing 30 μg/ml of cycloheximide. Cells and supernatants were collected at 0, 4, 8 and 12 h following cycloheximide treatment. Supernatants were pelleted through 20% sucrose cushions and subjected to Western immunoblotting (panel A). Gag proteins were quantified by scanning p24gag-associated band densities from immunoblots. Densitometric units representing total Gag proteins in medium and the ratio of p24gag to Pr55gag band densities were plotted against time (panels B and C, respectively).
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f0020: VLP release and processing kinetics 293T cells grown in 10 cm culture dishes in triplicate were transfected with the designated plasmid, pooled, and divided equally onto four dish plates. At 16 h post-transfection, supernatants were collected and fed medium containing 30 μg/ml of cycloheximide. Cells and supernatants were collected at 0, 4, 8 and 12 h following cycloheximide treatment. Supernatants were pelleted through 20% sucrose cushions and subjected to Western immunoblotting (panel A). Gag proteins were quantified by scanning p24gag-associated band densities from immunoblots. Densitometric units representing total Gag proteins in medium and the ratio of p24gag to Pr55gag band densities were plotted against time (panels B and C, respectively).

Mentions: Although NC15A and ΔMA/NC15A both exhibited similar levels of unprocessed Gag precursor at 48 h post-transfection (Fig. 2A, lanes 4 and 5), results from a time-course analysis of Gag processing suggest that removal of the MA globular domain significantly enhanced NC15A Gag processing efficiency (Fig. 3). It is likely that the Gag mutation that disrupts VLP assembly also affects Gag–Pol molecular interaction, which in turn impairs PR activation. To further determine if MA removal impacts VLP release and maturation, accumulated VLPs in supernatants were collected at different time intervals following treatment with cycloheximide. Our results indicate significant increases in wt virus-associated Gag accumulation at 4 and 8 h host-treatment, but not at 12 h, suggesting that most VLP assembly and release in the Gag molecules was completed by 8 h post-treatment (Fig. 4). Likewise, we did not observe significant changes in virus-associated Gag cleavage profiles, suggesting that PR quickly mediates virus maturation following virus budding. Mutations at HIV-1 NC basic residues can lead to PR-dependent virion instability, with virions tending to fall apart with little recovery [28,35]. This may partly explain why ΔMA/NC15A exhibited slight accumulation decreases rather than increases at 8 and 12 h. Some of mature wt VLPs might also be unstable after release, which leads to a decrease accumulated VLPs at 12 h. A faster release rate in ΔMA compared to the wt was not observed in repeat independent experiments. However, we consistently observed that ΔMA/NC15A exhibited faster virus release and processing rates compared to NC15A.


HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

Ko LJ, Yu FH, Huang KJ, Wang CT - FEBS Open Bio (2015)

VLP release and processing kinetics 293T cells grown in 10 cm culture dishes in triplicate were transfected with the designated plasmid, pooled, and divided equally onto four dish plates. At 16 h post-transfection, supernatants were collected and fed medium containing 30 μg/ml of cycloheximide. Cells and supernatants were collected at 0, 4, 8 and 12 h following cycloheximide treatment. Supernatants were pelleted through 20% sucrose cushions and subjected to Western immunoblotting (panel A). Gag proteins were quantified by scanning p24gag-associated band densities from immunoblots. Densitometric units representing total Gag proteins in medium and the ratio of p24gag to Pr55gag band densities were plotted against time (panels B and C, respectively).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4402288&req=5

f0020: VLP release and processing kinetics 293T cells grown in 10 cm culture dishes in triplicate were transfected with the designated plasmid, pooled, and divided equally onto four dish plates. At 16 h post-transfection, supernatants were collected and fed medium containing 30 μg/ml of cycloheximide. Cells and supernatants were collected at 0, 4, 8 and 12 h following cycloheximide treatment. Supernatants were pelleted through 20% sucrose cushions and subjected to Western immunoblotting (panel A). Gag proteins were quantified by scanning p24gag-associated band densities from immunoblots. Densitometric units representing total Gag proteins in medium and the ratio of p24gag to Pr55gag band densities were plotted against time (panels B and C, respectively).
Mentions: Although NC15A and ΔMA/NC15A both exhibited similar levels of unprocessed Gag precursor at 48 h post-transfection (Fig. 2A, lanes 4 and 5), results from a time-course analysis of Gag processing suggest that removal of the MA globular domain significantly enhanced NC15A Gag processing efficiency (Fig. 3). It is likely that the Gag mutation that disrupts VLP assembly also affects Gag–Pol molecular interaction, which in turn impairs PR activation. To further determine if MA removal impacts VLP release and maturation, accumulated VLPs in supernatants were collected at different time intervals following treatment with cycloheximide. Our results indicate significant increases in wt virus-associated Gag accumulation at 4 and 8 h host-treatment, but not at 12 h, suggesting that most VLP assembly and release in the Gag molecules was completed by 8 h post-treatment (Fig. 4). Likewise, we did not observe significant changes in virus-associated Gag cleavage profiles, suggesting that PR quickly mediates virus maturation following virus budding. Mutations at HIV-1 NC basic residues can lead to PR-dependent virion instability, with virions tending to fall apart with little recovery [28,35]. This may partly explain why ΔMA/NC15A exhibited slight accumulation decreases rather than increases at 8 and 12 h. Some of mature wt VLPs might also be unstable after release, which leads to a decrease accumulated VLPs at 12 h. A faster release rate in ΔMA compared to the wt was not observed in repeat independent experiments. However, we consistently observed that ΔMA/NC15A exhibited faster virus release and processing rates compared to NC15A.

Bottom Line: Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly.To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues.Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan ; Institute of Public Health, National Yang-Ming University School of Medicine, Taipei, Taiwan.

ABSTRACT
Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

No MeSH data available.


Related in: MedlinePlus