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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

Velocity sedimentation analysis of cytoplasmic Gag precursor complexes. 293T cells were transfected with 20 μg of the PR-inactivated versions of the indicated plasmids. Two days post-transfection, cells were homogenized and their extracted cytoplasmic lysates centrifuged through 25%, 35% and 45% sucrose step gradients at 130,000g for 1 h. Fractions were collected from gradient tops, and fraction aliquots were subjected to 10% SDS–PAGE and probed with a monoclonal antibody directed at p24CA. Total Gag proteins were quantified by scanning the immunoblot band densities of fractions 1–5. Multimerized Gag protein percentages were determined by dividing multimerized Gag protein density units (fractions 3–5) by total Gag density units (fractions 1–5) and normalizing the results to that of the wt.
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f0025: Velocity sedimentation analysis of cytoplasmic Gag precursor complexes. 293T cells were transfected with 20 μg of the PR-inactivated versions of the indicated plasmids. Two days post-transfection, cells were homogenized and their extracted cytoplasmic lysates centrifuged through 25%, 35% and 45% sucrose step gradients at 130,000g for 1 h. Fractions were collected from gradient tops, and fraction aliquots were subjected to 10% SDS–PAGE and probed with a monoclonal antibody directed at p24CA. Total Gag proteins were quantified by scanning the immunoblot band densities of fractions 1–5. Multimerized Gag protein percentages were determined by dividing multimerized Gag protein density units (fractions 3–5) by total Gag density units (fractions 1–5) and normalizing the results to that of the wt.

Mentions: We performed velocity sedimentation centrifugation analyses of cellular Gag molecules, using a myristylation-deficient (myr-) Gag mutant (defective in membrane binding and incapable of multimerizing into high-molecular weight complexes) as a control [34]. Results indicate that wt Gag and the assembly-competent mutant DMA tended to be found in high-sucrose density fractions, while most myr- Gag was recovered in fractions 1 and 2 (Fig. 5); this is in agreement with previously reported results [41,42]. We consistently observed substantial shifts of NC15A to higher sucrose density fractions following MA deletion (Fig. 5, panel 3 versus 4 from top), suggesting that MA removal mitigates the NC15A multimerization defect, which is compatible with improved VLP yields (Fig. 2). It should be noted that some slightly overexposed blots were purposely presented to make it easier for readers to see the detected signals. Not all of the Western blots shown in the figures were used for quantification.


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)

Velocity sedimentation analysis of cytoplasmic Gag precursor complexes. 293T cells were transfected with 20 μg of the PR-inactivated versions of the indicated plasmids. Two days post-transfection, cells were homogenized and their extracted cytoplasmic lysates centrifuged through 25%, 35% and 45% sucrose step gradients at 130,000g for 1 h. Fractions were collected from gradient tops, and fraction aliquots were subjected to 10% SDS–PAGE and probed with a monoclonal antibody directed at p24CA. Total Gag proteins were quantified by scanning the immunoblot band densities of fractions 1–5. Multimerized Gag protein percentages were determined by dividing multimerized Gag protein density units (fractions 3–5) by total Gag density units (fractions 1–5) and normalizing the results to that of the wt.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0025: Velocity sedimentation analysis of cytoplasmic Gag precursor complexes. 293T cells were transfected with 20 μg of the PR-inactivated versions of the indicated plasmids. Two days post-transfection, cells were homogenized and their extracted cytoplasmic lysates centrifuged through 25%, 35% and 45% sucrose step gradients at 130,000g for 1 h. Fractions were collected from gradient tops, and fraction aliquots were subjected to 10% SDS–PAGE and probed with a monoclonal antibody directed at p24CA. Total Gag proteins were quantified by scanning the immunoblot band densities of fractions 1–5. Multimerized Gag protein percentages were determined by dividing multimerized Gag protein density units (fractions 3–5) by total Gag density units (fractions 1–5) and normalizing the results to that of the wt.
Mentions: We performed velocity sedimentation centrifugation analyses of cellular Gag molecules, using a myristylation-deficient (myr-) Gag mutant (defective in membrane binding and incapable of multimerizing into high-molecular weight complexes) as a control [34]. Results indicate that wt Gag and the assembly-competent mutant DMA tended to be found in high-sucrose density fractions, while most myr- Gag was recovered in fractions 1 and 2 (Fig. 5); this is in agreement with previously reported results [41,42]. We consistently observed substantial shifts of NC15A to higher sucrose density fractions following MA deletion (Fig. 5, panel 3 versus 4 from top), suggesting that MA removal mitigates the NC15A multimerization defect, which is compatible with improved VLP yields (Fig. 2). It should be noted that some slightly overexposed blots were purposely presented to make it easier for readers to see the detected signals. Not all of the Western blots shown in the figures were used for quantification.

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