Transcriptional suppression of antiviral immune responses by HIV-1 Vpu
Final Report Abstract
The Human Immunodeficiency Virus 1 (HIV-1), a lentivirus, is the causative agent of the Acquired Immunodeficiency syndrome (AIDS). The proposed project focused on immune modulation by accessory proteins of HIV-1, at both the transcriptome and proteome level. In a first approach, it was shown that the viral accessory protein Vpu exerts broad immunosuppressive effects by inhibiting activation of the transcription factor NF-κB. Global transcriptional profiling of infected CD4 +T cells revealed that vpu-deficient HIV-1 strains induce stronger immune responses compared to the respective wild type viruses. Furthermore, gene set enrichment analyses in combination with cytokine arrays showed that Vpu suppresses the expression of NF-κB targets including interferons and lentiviral restriction factors. Of note, mutational analyses demonstrated that this immunosuppressive activity of Vpu is independent of its ability to counteract the restriction factor, innate sensor and NF-κB activator tetherin. However, Vpu-mediated inhibition of immune activation requires an arginine residue in the cytoplasmic domain that is critical for blocking NF-κB signaling downstream of tetherin. In a second approach, a Global Ubiquitination Pattern Analyses (GUPA) was performed by mass spectrometry, to detect changes of the ubiquitinated proteome of primary CD4+ T cells, infected with a primary HIV-1 isolate. To shed light on the role of HIV-1 accessory proteins, a diverse set of viral mutants was included in the study that do not express Vpu at all (vpu stop), or which have selectively lost their ability to suppress NF-κB activation (Vpu R50K). Additionally, an HIV-1 Vpu mutant that fails to recruit the E3 ubiquitin ligase complex (Vpu S58A/S62A) which is important for different Vpu functions such as CD4 degradation was also included. Last, a vpr-deficient HIV-1 mutant was included to identify potential targets of this accessory protein. By subsequently comparing the ubiquitinated proteome profiles of primary CD4+ T cells that are either uninfected (mock), infected with wt or different mutant viruses several potential novel Vpu and Vpr targets were identified. In-depth validation and mechanism of action studies are currently ongoing to follow up on these results. A third approach leveraged the Global Arrayed Protein Stability Analysis (GAPSA), which enables a targeted high-content analysis of protein levels, to identify degradation targets amongst the proximal interactome of the HIV-1 accessory protein Vpu, as defined by an innovative electron microscopy and proteomic approach. This combined approach identified a set of novel degradation targets of HIV-1 Vpu, including PREB/SEC12, RhoA, PDLIM1 and CIAPIN1, which might contribute to our understanding of how Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. In a last approach, it was shown that E3 ubiquitin-protein ligase Cullin 3 (Cul3) represents a host cell factor that regulates NF-κB activation, and thereby affects LTR-mediated HIV-1 gene expression in infected target cells. Thus, the results introduce Cul3 as novel important regulator of HIV-1 replication. Understanding how Cul3 restricts HIV-1 transcription could potentially lead the way towards new approaches for HIV/AIDS therapies and may provide novel strategies to clear the HIV-1 reservoir from infected individuals. Taken together, the results of the conducted investigations provide novel insights into the function of the HIV-1 accessory proteins Vpu and Vpr. Additionally, these results shed light on the intricate manipulation of the cellular transcriptome and proteome in HIV-1-infected infected target cells, and counteracting immune reactions, aiming to clear viral infection but also promote progression to AIDS. Potentially, results from these studies may also advance current therapeutic approaches, in which NF-κB is targeted to reactivate and eliminate HIV-1 from latent reservoirs in infected patients.
Publications
- (2019). HIV-1 Vpu is a potent transcriptional suppressor of NF- κB-elicited antiviral immune responses. eLIFE
Langer, Hammer, Hopfensperger, Klein, Hotter, De Jesus, Herbert, Pache, Smith, v. d. Merwe, Chanda, Fellay, Kirchhoff, Sauter
(See online at https://doi.org/10.7554/eLife.41930) - (2020). Sensor Sensibility- HIV-1 and the Innate Immune Response. Cells
Yin, Langer, Zhang, Herbert, Yoh, Koenig, Chanda
(See online at https://doi.org/10.3390/cells9010254) - (2020). The E3 ubiquitin-protein ligase Cullin 3 regulates HIV-1 transcription. Cells
Langer, Yin, Diaz, Portillo, Gordon, Rogers, Marlett, Krogan, Young, Pache, Chanda (
(See online at https://doi.org/10.3390/cells9092010) - (2021). A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor. PLOS Pathogens
Stoneham, Langer, De Jesus, Wozniak, Lapek, Deerinck, Thor, Pache, Chanda, Gonzalez, Ellisman, Guatelli
(See online at https://dx.doi.org/10.1371/journal.ppat.1009409)
