Abstract
BackgroundHuman immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication.ResultsA proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages.ConclusionOur current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.
Highlights
Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane
Because Gag phosphorylation is important for its functional role, we focused on human protein kinases as potential Gag regulators
When a relative light unit per cutoff (RLU/Co) ratio of ≥ 3.0 was used as the threshold, we found that 22 host kinases could selectively interact with HIV-1 Gag and were identified as primary kinase candidates for the phosphorylation of HIV-1 Gag (Figure 1B)
Summary
Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. Concomitant with, or after these viral particles pinch off and are released from the host cell via budding, the virusencoded protease becomes activated and cleaves Gag into its functional subdomains, matrix (MA, p17), capsid (CA, p24), and nucleocapsid (NC, p7), as well as several shorter segments: SP1 (spacer peptide 1), SP2, and p6. This proteolytic maturation in tandem with the incorporation of viral enzymes and accessory proteins into virions results in the acquisition of HIV-1 infectivity [5,6,7,8]
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