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Dramatic increase in poly(A) synthesis after infection of Molt-3 cells with HIV
H C Schröder1, M Rottmann, R Wenger
1Institut für Physiologische Chemie, Abteilung, Angewandte Molekularbiologie, Universität Mainz, F.R.G.
Abstract:
Infection of Molt-3 cells with human immunodeficiency virus-1 (HIV-1) was found to cause a rapid increase in extractable poly(A) polymerase activity, while the activity of poly(A) degrading endoribonuclease IV strongly decreased at the same time. The increase in poly(A) polymerase activity seems not to be due to a change in the actual number of enzyme molecules, but rather to posttranslational enzyme modification, most likely caused by phosphorylation by nuclear protein kinase NI or protein kinase C. Both kinases were found to be able to phosphorylate poly(A) polymerase in vitro [homogeneous enzyme as well as poly(A) polymerase in intact nuclei]. Phosphoamino acid analysis revealed an incorporation of phosphate into serine and, to a lower extent, into threonine residues of the enzyme protein; no phosphotyrosine could be detected. In the nucleus, the poly(A) polymerase and the endoribonuclease IV are bound to the nuclear matrix. The phosphorylation related enhancement of nuclear poly(A) polymerase activity could be abolished by addition of the zinc and copper chelator o-phenanthroline, which inhibited zinc-containing purified poly(A) polymerase and destroyed the poly(A) polymerase containing nuclear matrix structure, resulting in a solubilization of the enzyme.
Insights
HIV-1 infection increases poly(A) polymerase activity by modifying the enzyme, not by increasing its quantity. This posttranslational modification, likely phosphorylation, affects nuclear poly(A) polymerase crucial for RNA processing.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Polyadenylation is critical for mRNA stability and translation.
- Human immunodeficiency virus-1 (HIV-1) infection alters cellular processes.
- Nuclear poly(A) polymerase and endoribonuclease IV regulate poly(A) tail dynamics.
Purpose of the Study:
- To investigate the effect of HIV-1 infection on poly(A) polymerase and endoribonuclease IV activity.
- To elucidate the mechanism behind the observed changes in enzyme activity.
- To understand the role of posttranslational modification in regulating poly(A) polymerase function during viral infection.
Main Methods:
- Infection of Molt-3 cells with HIV-1.
- Assays for poly(A) polymerase and endoribonuclease IV activity.
- In vitro phosphorylation studies using purified kinases and poly(A) polymerase.
- Phosphoamino acid analysis.
- Nuclear matrix isolation and enzyme binding studies.
- Effect of chelators on enzyme activity and nuclear matrix structure.
Main Results:
- HIV-1 infection rapidly increased poly(A) polymerase activity and decreased endoribonuclease IV activity.
- Increased poly(A) polymerase activity resulted from posttranslational modification, likely phosphorylation by protein kinase NI or protein kinase C.
- Phosphorylation occurred on serine and threonine residues.
- Poly(A) polymerase and endoribonuclease IV are nuclear matrix-bound.
- O-phenanthroline abolished the phosphorylation-induced enhancement of activity, inhibited purified enzyme, and disrupted the nuclear matrix structure.
Conclusions:
- HIV-1 infection modulates poly(A) polymerase activity through phosphorylation, impacting mRNA processing.
- Nuclear matrix association and zinc-dependent activity are crucial for poly(A) polymerase function.
- These findings highlight a novel mechanism of viral regulation of host cell machinery.