Understanding how viruses manipulate ubiquitin ligases to avoid innate immunity.

摘要

Soon after viruses enter the cell, they encounter host innate and adaptive immune responses that must be evaded. Restriction factors are proteins from the host innate immune response that impair the establishment of viral infection.;Primate lentiviruses encode "accessory" proteins, which are not required for viral replication but essential to counteract host restriction factors. The primate lentivirus accessory proteins are Nef, Vif, Vpu, Vpr, and Vpx. The main mechanism used by these proteins to counteract restriction factors is the manipulation of the ubiquitin proteasome system. While Vpu usurps Cul1, Vpr and Vpx hijack Cul4A by associating with DCAF1.;Through interaction with DCAF1, Vpx degrades SAMHD1, a protein that impairs viral reverse transcription in myeloid cell lineages. Vpr, on the other hand, induces poly-ubiquitination of Mus81 to activate the endonuclease complex SLX4com. SLX4com activation was suggested to induce cell cycle arrest in G2/M. SIVagm Vpr is homologous to HIV-1 Vpr in sequence and structure, but these two proteins have important functional differences. Unlike HIV-1 Vpr, SIVagm Vpr degrades SAMHD1 and induces G2 arrest but in a species-specific manner. In this work, we generated chimeric proteins between HIV-1 Vpr and SIVagm Vpr, in order to understand the structure-function relationships in these proteins. We showed a de novo ability to arrest cell cycle in human cells when the C-terminus of HIV-1 Vpr was grafted onto SIVagm Vpr. Using point mutants of HIV-1 Vpr, we were able to uncouple degradation of Mus81 from cell cycle arrest, suggesting that these two functions are independent.;To study the amino acid residues of DCAF1 that are responsible for interactions with Vpr and Vpx, we generated a large array of point mutants. We demonstrated that Vpr and Vpx interact with DCAF1 using a similar region on DCAF1, but establishing interactions with different residues.;We then used an inhibitor of the Neddylation pathway (MLN4924) to examine the role of CRUL in the abilities of accessory proteins to target host cell proteins. Our results demonstrate that inhibition of CRUL with MLN4924 did not affect downregulation of tetherin, suggesting that the trans-golgi entrapment is responsible for the observed effect.