Human cytomegalovirus genetic determinants of viral tropism in epithelial cells and human leukocytes

Abstract

Human cytomegalovirus (HCMV) exhibits a broad cell tropism in vitro and in vivo. The ability of HCMV to replicate efficiently in different cell types is governed by specific envelope glycoproteins that mediate the binding to cellular receptors and consequently the virus entry into human cells. The infection of epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the host. Among human leukocytes, only cells of the myeloid lineage are permissive to HCMV and play a crucial role in both lytic and latent infection. By contrast, lymphoid cells, such as neutrophils, act as vehicle for HCMV dissemination in vivo although they do not support a lytic replicative cycle. HCMV strains differ in their ability to infect and replicate in various cell types, but the genetic basis of these differences has remained poorly investigated. The aim of this study was to characterize the genetic factors involved in the broad cell tropism of a specific HCMV strain, VR1814, and its ability to induce syncytium formation in epithelial cells and macrophages. By sequence comparison and genetic engineering of VR1814’s BAC clone, FIX, that displayed a strongly reduced infectivity in vitro, I identified specific variants of VR1814 in the envelope glycoproteins gB, UL128, and UL130 as major determinants of the increased tropism in epithelial cells and macrophages. I could show that an amino acid substitution in UL130 of FIX reduces the pentamer expression in viral particles, affecting both infectivity and syncytium formation in epithelial cells. Additionally, I demonstrated that a VR1814-specific mutation in US28 leading to a C-terminal truncation of the protein promotes IE gene expression in human macrophages, facilitating HCMV infection rather than latency in this cell type. This study also aimed to investigate HCMV trafficking in neutrophils, a process that occurs in vivo and may play a crucial role for viral dissemination in the human host. As VR1814 retained the capability to be transferred to human leukocytes, I established an in vitro system to study the transfer of cell-associated HCMV from infected endothelial cells to neutrophils. Heterogeneous viral material consisting of viral particles and dense bodies was found in large vesicles in neutrophils cytoplasm, suggesting that virus uptake from infected endothelial cells mainly occurred via endocytosis. In conclusion, this study identifies the viral genetic factors dictating HCMV tropism in epithelial cells and human leukocytes. Furthermore, the study highlights the importance of tools necessary for investigating the mechanisms and relevance of cell-cell fusion and leukocyte transfer in HCMV pathogenesis and dissemination.