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

Background: 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. 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. Results: The 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. Conclusions: The 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.