The N-end rule and retroviral infection: no effect on integrase

Guney Boso,Takafumi Tasaki,Nikunj V Somia,Yong Tae Kwon

doi:10.1186/1743-422x-10-233

Abstract

BackgroundIntegration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. Mature forms of integrase of several retroviruses (i.e. HIV-1 and MLV) bear conserved destabilizing N-terminal residues of the N-end rule pathway - a ubiquitin dependent proteolytic system in which the N-terminal residue of a protein determines its half life. Substrates of the N-end rule pathway are recognized by E3 ubiquitin ligases called N-recognins. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase. These findings have prompted us to investigate the involvement of the N-end rule pathway in the HIV-1 life cycle.ResultsThe infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted. HIV-1 integrase mutants of N-terminal amino acids (coding for stabilizing or destabilizing residues) were severely impaired in their infectivity in both human and mouse cells. Quantitative PCR analysis revealed that this inhibition was mainly caused by a defect in reverse transcription. The decreased infectivity was independent of the N-end rule since cells deficient in N-recognins were equally refractory to infection by the integrase mutants. MLV integrase mutants showed no difference in their infectivity or intravirion processing of integrase.ConclusionsThe N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase. The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.

Highlights

Integration of double stranded viral DNA is a key step in the retroviral life cycle
We have previously shown that concomitant impairment of three of the N-recognins that bind to destabilizing residues, UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase bearing a destabilizing residue [18]
Proteolytic cleavage of Gag/Pol generates destabilizing N-end rule residues We first examined whether the cleavage of the Gag Pol polyprotein by HIV-1 protease would generate mature proteins that expose destabilizing N-terminal residues of the N-end rule pathway

Summary

Introduction

Integration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase These findings have prompted us to investigate the involvement of the N-end rule pathway in the HIV-1 life cycle. First described by Alex Varshavsky and colleagues in 1986, this N-end rule is an ubiquitin dependent proteolytic system in which the identity of the N-terminal amino acid residue of a protein determines its half life in the cell [12] Even though it was originally discovered while expressing a bacterial protein in yeast cells the N-end rule pathway was subsequently found to be present in all organisms examined including mammals [13], plants [14], and bacteria [15]. With the discovery of several signaling components of the N-end rule pathway our understanding of this process has increased in the last decade

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