Active cAMP-dependent Protein Kinase Incorporated within Highly Purified HIV-1 Particles Is Required for Viral Infectivity and Interacts with Viral Capsid Protein

Christine Cartier,Bénédicte Hemonnot,Bernard Gay,Martine Bardy,Céline Sanchiz,Christian Devaux,Laurence Briant

doi:10.1074/jbc.m301257200

Abstract

Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

Highlights

Protein phosphorylation is one of the primary processes by which external physiological stimuli influence intracellular events in eucaryotic cells
CAMP-dependent Protein Kinase Catalytic Subunit Is Incorporated within Highly Purified human immunodeficiency virus type 1 (HIV-1) Viral Particles—We and others have previously reported that several serine-threonine and tyrosine kinases are packaged within HIV-1 particles
The presence of a protein with a molecular weight of 40,000 was revealed by the mean of a serum specific for the catalytic subunit of cAMP-dependent protein kinase (C-PKA) (Fig. 1A, lane 4), suggesting that C-PKA is incorporated within purified HIV-1 particles

Summary

Introduction

Protein phosphorylation is one of the primary processes by which external physiological stimuli influence intracellular events in eucaryotic cells. Rhabdoviridae [9], Hepadnaviridae [10], Retroviridae including Rauscher murine leukemia virus [11], and RNA tumor viruses [12] were found to incorporate host cell protein kinases within their membrane or inside the viral core Even though their precise contribution has not yet been identified, there is ample evidence to suggest that virus-associated protein kinases are crucial for viral infectivity, and their possible function includes both the regulation of the viral nucleic acid replication and transcription and the modification of virus structural proteins that leads to either uncoating or encapsidation of viral nucleic acids. Additional phosphorylation on serine residues was reported and was proposed to promote membrane dissociation of the reverse transcription complex from the cell membrane at the site of entry, allowing its nuclear translocation [16, 19] Such modifications were found to occur in preintegration complexes isolated from target cells and in native virions, suggesting that host cell serine-threonine kinases might possibly be incorporated within HIV particles [16]. Despite the fact that the precise function of these modification remains unknown, by similarity to other viruses (i.e. poliovirus and herpes virus), it can be hypothesized that phosphorylation of HIV-1 capsid protein probably generates some repulsive forces among proteinprotein interactions that participate in viral core destabilization and viral particle uncoating

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Results

Conclusion