monolayer cell line  (ATCC)

Journal: Journal of Virology

Article Title: Positive correlation between structural disorder of the HIV-1 Gag N-terminal segment and progeny virus particle formation

doi: 10.1128/jvi.00887-25

Figure Lengend Snippet: Effects of Gag mutations on virion production and viral infectivity and relation between the disordered state and viral phenotype. ( A ) HEK293T cells were transfected with the indicated full-length proviral clones. The culture supernatants were collected at 24 h post-transfection to measure virion production by reverse transcriptase (RT) assays. RT activity is shown relative to that of NL4-3. Equal amounts of viruses, as determined by RT assays, were inoculated into TZM-bl cells. Cell lysates were prepared on day 2 post-infection for luciferase assays. Infectivity is presented as luciferase activity relative to that of NL4-3. Mean values ± SE from at least four independent experiments are shown. Significance relative to NL4-3 was determined by the Welch’s t -test (** P < 0.01; * P < 0.05). Gag-MA-G2A and Gag-CA-WMAA mutant clones were used as controls. ( B ) The relation between the mean disorder scores of the MA-NTD segment and the values of relative viral particle production. The mean disorder scores of the first nine amino acid residues of the Gag MA N-terminus were obtained for individual MA-9 mutants using the data from (AIUPred ; left panel) and (PONDR VL-XT [ ]; right panel). The values were used to assess the relation between the disorder level and viral production in (orange bars). R : Pearson’s correlation coefficient. ( C ) The relation between the mean disorder scores of the MA-NTD segment and relative viral infectivity values. The mean disorder scores were used to assess the relation between the level of disorder and viral infectivity values in (gray bars).

Article Snippet: Monolayer cell lines HEK293T (ATCC CRL-1573), HeLa (ATCC CCL-2), and HeLa-derived reporter TZM-bl ( ) were cultured and maintained in Eagle’s minimal essential medium containing 10% heat-inactivated fetal bovine serum.

Techniques: Infection, Transfection, Clone Assay, Reverse Transcription, Activity Assay, Luciferase, Mutagenesis

Journal: Journal of Virology

Article Title: Positive correlation between structural disorder of the HIV-1 Gag N-terminal segment and progeny virus particle formation

doi: 10.1128/jvi.00887-25

Figure Lengend Snippet: Effect of Gag-MA-S9 mutations on Env incorporation into virions. ( A ) The supernatants from HEK293T cells transfected with the indicated proviral clones were ultracentrifuged through a sucrose cushion, and the pellet samples were harvested for monitoring Env and Gag-p24 in virions. Equal amounts of samples (10 ng p24 for Env and 0.5 ng p24 for Gag) were analyzed by the Western blotting method. ( B ) Representative immunoblotting data from three independent experiments (performed twice for both S9R and S9E) are presented. Env incorporation into virions is presented as the normalized signal intensity (Env/p24) of each sample relative to that of NL4-3 (mean ± SE). The statistical analysis was conducted by Welch’s t -test. ** P < 0.01; * P < 0.05. ( C ) The correlation between the relative Env incorporation and relative infectivity is indicated by the R -score. ( D ) Three-dimensional locations of the MA-S9 amino acid residue in the immature HIV-1 MA model. The immature HIV-1 MA trimer structure is derived from PDB code 7OVQ . ( E ) Molecular interactions between two MA trimers. The magenta-colored bars indicate hydrogen bonds.

Article Snippet: Monolayer cell lines HEK293T (ATCC CRL-1573), HeLa (ATCC CCL-2), and HeLa-derived reporter TZM-bl ( ) were cultured and maintained in Eagle’s minimal essential medium containing 10% heat-inactivated fetal bovine serum.

Techniques: Transfection, Clone Assay, Western Blot, Infection, Residue, Derivative Assay

Journal: Journal of Virology

Article Title: Positive correlation between structural disorder of the HIV-1 Gag N-terminal segment and progeny virus particle formation

doi: 10.1128/jvi.00887-25

Figure Lengend Snippet: Effect of Gag-MA mutations on N-myristoylation of Gag proteins. ( A ) HEK293T cells were transfected with the indicated Gag-MA mutants derived from pNL4-3ΔPro/ΔEnv. At 24 h post-transfection, biotinylation reactions to detect any myristoylated proteins in cells were performed by using the Click-IT-based method (for details, see Materials and Methods). After the reactions, free biotin was removed from the samples, and equal amounts of samples (3.0 µg of total protein) were used for Western blotting analysis with horseradish peroxidase-conjugate streptavidin. Representative immunoblotting data from three independent experiments are shown. ( B ) Relative Myr-Gag/Gag is presented as described in Materials and Methods (mean ± SE). Significance relative to control NL4-3 was determined by Welch’s t -test. ** P < 0.01; * P < 0.05. Myr-Gag, Myristoylated Gag; NL, NL4-3 WT; pUC, negative control.

Article Snippet: Monolayer cell lines HEK293T (ATCC CRL-1573), HeLa (ATCC CCL-2), and HeLa-derived reporter TZM-bl ( ) were cultured and maintained in Eagle’s minimal essential medium containing 10% heat-inactivated fetal bovine serum.

Techniques: Transfection, Derivative Assay, Western Blot, Control, Negative Control