Generation of macrophage models to investigate the effect of host factors restricting HIV and HSV infection

Xiao, Qianhao (2021): Generation of macrophage models to investigate the effect of host factors restricting HIV and HSV infection. Dissertation, LMU München: Medizinische Fakultät

Abstract

Introduction Macrophages play a central role in the immune response to viral infection. While cellular responses are highly cell-type and pathogen-specific, little is known about innate mechanisms for sensing and restriction of viruses in macrophages and macrophage-like cells. MX2 and SAMHD1 were recently identified as antiviral cellular effectors targeting Human Immunodeficiency Virus Type 1 (HIV-1) and Herpes Simplex Virus Type 1 (HSV-1) in cell lines. Macrophages can be productively infected by HIV-1 and HSV-1 and constitute important cellular reservoirs in vivo, yet the roles of MX2 and SAMHD1 are only partially understood. Aims To fully understand cell type-specific host responses to these viruses, experimental model systems that allow screening approaches, genetic manipulation and physiologically relevant are needed. Here, we pursued the following aims: (i) Establish and validate trans-differentiated BLaER1 cells as a genetically amendable macrophage-like cell model for HIV-1 and HSV-1 infection, (ii) develop a rapid and efficient protocol to knockout genes in monocyte/macrophage primary cells, and (iii) characterize the antiviral potency of MX2 and SAMHD1 in both experimental models. Materials & methods Trans-differentiation and differentiation protocols as well as phenotypic and functional analyses of macrophages were established. Various approaches to study HIV-1 and HSV-1 infection by envelope pseudotyping, use of reporter viruses, virus-like-particles and specific drugs were optimized. Innate responses were monitored by PCR and immunoblotting. Nucleofection-mediated, CRISPR/Cas9-based gene editing in monocytes was developed and knockout efficiencies were validated genetically by immunodetection. Results (i) Trans-differentiated BLaER1 cells adopt a myeloid gene expression profile and display characteristics in response to HIV-1 or HSV-1 infection similar to primary macrophages. (ii) In comparison to siRNA-based approaches, CRISPR/Cas9-edited macrophages are viable, have a stable myeloid marker expression and targeted factors are efficiently depleted. (iii) Knockouts of MX2 or SAMHD1 result in enhanced HIV-1 and HSV-1 infection in trans-differentiated BLaER1 cells and primary macrophages, corroborating and expanding their role as restriction factors against these two human pathogens. (iv) A contamination with squirrel monkey retrovirus (SMRV) is discovered in BLaER1 cells. (v) BLaER1 cells can provide an easily accessible screening platform for cellular factors that are functionally relevant for virus infections. Conclusion Overall, these findings establish critical methodology for different types of experimental studies into virus-macrophage interactions. MX2 and SAMHD1 are broadly acting restriction factors that limit the infection of lentiviruses and herpesviruses in human macrophages.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 29. November 2021
• 1. Bericht­erstatter:in: Keppler, Oliver