The ubiquitin-specific peptidase USP15 regulates human papillomavirus type 16 E6 protein stability.

Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages.

Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.

Low-level HIV replication in mixed glial cultures is associated with alterations in the processing of p55(Gag).

We report a novel long-lived infection model in human mixed glial cultures (microglia) whereby cells harbor replication-competent HIV-1 for up to 2.5 months after infection; a model that potentially mimics latency within the central nervous system (CNS). Infection of mixed glial cultures in the presence of serum, cytokines, and growth factors (activating conditions) resulted in a robust productive infection of microglial cells as previously described for purified microglia. In contrast, similar mixed glial cells cultured in serum-free medium without cytokines or growth factors (mirroring a nonactivated CNS) supported HIV-1 entry, reverse transcription, integration, and transcription, yet released little or no infectious virus. We found instead that nonactivated mixed glial cells expressed almost 10-fold less Gag protein, but more importantly, analysis of the intracellular Gag products in quiescent cells showed an aberrant p55/p24 Gag processing phenotype that appeared to be due to the premature activity of the viral protease. These results suggest that the cellular environment in nonactivated microglia cells in these mixed glial cultures is not conducive to proper Gag processing and virus release. This long-lived infection model will be useful in identifying factors that are key for viral maturation in cells of the macrophage lineage.