Targeted Modulation of Host Immune Proteins by Human Cytomegalovirus
Open Access
- Author:
- Sandhu, Praneet Kaur
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 15, 2020
- Committee Members:
- Nicholas Jay Buchkovich, Dissertation Advisor/Co-Advisor
Nicholas Jay Buchkovich, Committee Chair/Co-Chair
Todd Schell, Committee Member
Jianming Hu, Committee Member
Neil David Christensen, Outside Member
Ralph Lauren Keil, Program Head/Chair - Keywords:
- Human cytomegalovirus
MHC class II
Virology
MARCH1
Immune evasion
Immune modulation - Abstract:
- Human cytomegalovirus is a ubiquitous pathogen in the human population that can cause severe health consequences in immunocompromised patients and neonates. The virus modulates host immunity to facilitate viral replication within humans. This includes attenuation of innate immune activation within infected cells and dampening of the adaptive immune responses. Thus, elucidation of how the virus alters the host immune system is key to developing remedial strategies for HCMV infections. CD4+ T lymphocytes are adaptive immune cells that are important for controlling viral infections within the host. The activation of CD4+ T cells occurs when they recognize antigenic peptides displayed on immune proteins called major histocompatibility complex class II (MHC class II). Thus, viruses hinder CD4+ T cell activation by interfering with MHC class II antigen presentation. MHC class II is constitutively expressed in specialized, antigen-presenting cells (APCs), which include cells of the myeloid lineage. Myeloid cells play an important role in the HCMV lifecycle in vivo. However, the regulation of endogenous MHC class II in myeloid cells during HCMV infection is not well-understood. We investigated the impact of HCMV infection on MHC class II in Kasumi-3 cells, a myeloid cell line that endogenously expresses MHC class II. We found that HCMV decreases the synthesis of MHC class II by inhibiting transcription of MHC class II and its master regulator class II transactivator (CIITA). This mechanism of MHC class II regulation was found to be independent of the immunomodulatory unique short (US) region of the HCMV genome and previously reported viral genes involved in MHC class II regulation. Importantly, the reduction in MHC class II synthesis required the expression of the immediate early proteins of the virus. Thus, we found that HCMV decreases endogenous CIITA and MHC class II expression in infected myeloid cells. Cells encode innate immune sensors to detect presence of viral ligands. This includes sensing of viral nucleic acids within the cytoplasm, which stimulates innate immune responses. Consequently, viruses block the activation of the cytoplasmic innate sensors to prevent immune activation. We found that HCMV induces the expression of MARCH1, an E3 ubiquitin ligase that targets membrane proteins for ubiquitination and lysosomal degradation, in non-expressing fibroblasts. This induction of MARCH1 in fibroblasts is remarkable because MARCH1 expression is limited to APCs to regulate immune proteins specifically expressed within these cells. However, we observed that MARCH1 is highly expressed during the late stages of HCMV infection and localizes to the Golgi in the cytoplasmic viral assembly compartment (cVAC), the site of viral maturation. We identified stimulator of interferon genes (STING), a cytoplasmic DNA sensor, as the target of the Golgi-localized MARCH1 in HCMV-infected fibroblasts. In support of this, we saw an increase in STING expression and its associated antiviral gene transcription upon short, hairpin RNA (shRNA)-mediated MARCH1 knockdown. Consequently, there was reduced cytoplasmic viral activity and infectious virus production upon loss of MARCH1 during HCMV infections. Thus, HCMV induces MARCH1 to target the antiviral STING protein to reduce innate immune signaling and promote viral replication. This dissertation highlights how HCMV effectively modulates the host immune response. The cessation of endogenous MHC class II synthesis upon HCMV infection reduces the expression of MHC class II, a T cell stimulating protein, and ablates the activation of the adaptive immune CD4+ T cells. Additionally, HCMV induces MARCH1 to target the immune protein STING and dampen the innate immune response in infected cells. Thus, HCMV alters the expression of cellular proteins to make the host immune environment favorable for the viral lifecycle.