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Changes in cellular proteins associated with the expression of human immunodeficiency virus type 1 trans-activator
P N Ranganathan1, S Ranganathan, A Srinivasan
1Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA 19107.
Abstract:
Earlier studies have revealed a distinct class of regulatory proteins known as trans-activator proteins in diverse biological systems. These proteins have been shown to act on both homologous and heterologous promoter targets. Activation of heterologous targets is speculated to be an integral part of virus-induced pathogenesis. To verify this hypothesis, stable Tat-producing human rhabdomyosarcoma (RD) cell lines were generated. These cell lines produced significant levels of functional Tat, as measured by transfection with the reporter plasmid pLTR-CAT. Tat-producing cells, although morphologically similar to the control, exhibited a slower growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cellular proteins from control (tat-) and tat+ cells revealed increased quantities of 34- and 40-kD proteins along with the appearance of a new 74-kD protein in tat+ cells. Subsequent two-dimensional gel analysis revealed several additional differences. Tat+ cell lines produced two proteins of M(r) 19.5 and 44 kD anew, while proteins with M(r) 14.5, 42, and 52.5 kD were in greater abundance. Interestingly, a 26-kD protein that was originally present in the G418+/tat- (control) sample disappeared in the presence of Tat. These data support a possible modulator role for Tat in cellular gene expression.
Insights
Trans-activator protein (Tat) can alter cellular gene expression. Tat-producing cells showed altered protein profiles and slower growth, suggesting Tat modulates cellular processes.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Trans-activator proteins (Tat) regulate gene expression in various biological systems.
- Tat proteins can target both homologous and heterologous promoters.
- Heterologous target activation by Tat is implicated in virus-induced pathogenesis.
Purpose of the Study:
- To investigate the role of Tat in cellular gene expression and pathogenesis.
- To generate and characterize stable Tat-producing human rhabdomyosarcoma (RD) cell lines.
- To verify the hypothesis that Tat activation of heterologous targets is integral to virus-induced pathogenesis.
Main Methods:
- Generation of stable Tat-producing RD cell lines (tat+).
- Transfection with reporter plasmid pLTR-CAT to measure Tat functionality.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis to analyze cellular protein profiles.
Main Results:
- Tat-producing cells exhibited a slower growth rate compared to control cells.
- SDS-PAGE revealed altered protein quantities (increased 34, 40 kD; new 74 kD) in tat+ cells.
- Two-dimensional gel analysis showed novel proteins (19.5, 44 kD) and differential abundance of others (14.5, 42, 52.5 kD) in tat+ cells, with one protein (26 kD) disappearing.
Conclusions:
- The observed changes in protein expression support a role for Tat in modulating cellular gene expression.
- Tat may influence cellular processes beyond viral gene activation.
- Further research is warranted to elucidate the precise mechanisms of Tat-mediated cellular modulation.