HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system

doi:10.1016/S1357-2725(99)00097-7

The International Journal of Biochemistry & Cell Biology

Volume 31, Issue 12, December 1999, Pages 1443-1452

https://doi.org/10.1016/S1357-2725(99)00097-7 Get rights and content

Abstract

Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

Introduction

Genome replication is one of the key steps in the life cycle of human immunodeficiency virus type 1 (HIV-1). The HIV-1 reverse transcriptase (RT), which plays a crucial role in HIV-1 genome replication in infected cells [1], is a heterodimer of 66 (p66) and 51 (p51) kDa polypeptides [2], [3] that are identical except for C-terminal truncation in the smaller subunit. The RT catalyzes several reactions: RNA-dependent DNA synthesis, hydrolysis of RNA (ribonuclease H) and DNA-dependent DNA synthesis [4], [5]. One potential mechanism through which these reactions may be regulated is covalent post-translational modification(s) of the RT, although no such modification(s) have yet been reported.

Protein phosphorylation is among the best characterized post-translational modification and it regulates enzyme activities and signal transduction in eukaryotes [6]. Many transcription factors, including the ligand-dependent steroid/thyroid receptor family, are phosphoproteins and are regulated through the action of protein kinases [7], [8], [9]. Further, several proteins involved in DNA/RNA metabolism are subject to and regulated by phosphorylation. These include DNA polymerases [10], [11], [12], [13], topoisomerases I [14] and II [15], and RNA polymerases I [16] and II [17]. Progression through the cell cycle depends on the phosphorylation and dephosphorylation of the cyclins and the kinases they in turn regulate [18]. In addition, many viral proteins such as adenovirus DNA polymerase [19] and the β-subunit of avian sarcoma virus DNA polymerase [20] are phosphoproteins.

In the present study, the phosphorylation potential of HIV-1 RT was determined in vivo and in vitro. The results show that bacterially expressed HIV-1 RT is an in vitro substrate for several purified protein kinases. In vivo phosphorylation of HIV-1 RT is also examined and discussed.

Section snippets

Materials and methods

[³²P]Orthophosphate (carrier- and HCl-free, 9000 Ci/mmol) and anti-HIV-1 RT monoclonal antibody (diluted ascites fluid) were from NEN-Dupont (Cat. No. NEA-9304]. [γ-³²P]ATP (4500 Ci/mmol) was from ICN. protein kinase C (PKC) from rat brain, protein G-agarose beads, PDBu and H-7 were from Calbiochem. Cell culture media were from GIBCO-BRL, and phosphate free Grace's medium was custom made by Sigma. Okadaic acid was from LC Laboratories. The catalytic subunit of bovine heart protein kinase A

Results

The primary structure of HIV-1 RT includes a number of potential phosphorylation sites (Table 1). For example, nine matches are found to the CK II consensus phosphorylation sequence (Ser/Thr–X–X–Glu/Asp) [21]; for example at the sites ³Ser–Pro–Ile–Glu⁶ and ³Thr–Glu–Met–Glu⁴² . In addition, 21 matches exist to the PKC consensus sequence (Ser/Thr–X–Lys/Arg, Lys/Arg–X–X–Ser/Thr and Lys/Arg–X–Ser/Thr [21]); for example ⁴⁶Lys–Ile–Ser⁴⁸ and ⁵⁵⁰Lys–Leu–Val–Ser⁵⁵³. Bacterially expressed

Discussion

In this study the phosphorylation of HIV-1 RT was investigated using purified kinases in vitro and using two different in vivo cell culture systems. Reversible phosphorylation is an important post-translational modification that regulates the activity of numerous proteins, including DNA and RNA polymerases. HIV-1 RT is a nucleic acid-binding enzyme, which is crucial for the replication of the HIV virus. RT is the target for a number of anti-AIDS drugs. The HIV-1 RT amino acid sequence includes

Acknowledgements

We thank Drs. Bill Beard, Miles Cloyd, Pierre Boulanger and Chris Delves for helpful discussions and advice. We also thank Dr. Hong Guo for purified kinase samples. We wish also to thank Dr. Ella Englander for helpful discussions and for initiating some of the preliminary experiments. This work was supported, in part, by NSF Grant DMB9019882 to Z.D. and by NIH Grant DK41058 to E.B.T., and by a Grant from the UTMB Retroviral program to S.H.W. Some of the studies described herein were performed

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^☆: This work is dedicated to the memory of the late Dr. Amalendra Kumar.

¹: Present address and reprint request: School of Biomedical Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, UK. Tel.: +44-334-467-282; fax: +44-1334-462-995.

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HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system☆

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

FEBS Lett.

J. Biol. Chem.

Curr. Opin. Cell Biol.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Virology

Methods Enzymol.

Protein Expr. Purif.

J. Biol. Chem.

J. Biol. Chem.

Nature

J. Virol.

J. Acq. Immun. Def. Synd.

Nature

Endocrinol. Rev.

J. Biol. Chem.