Hertel, L. | bioRxiv Science

bioRxiv2025

Increased Infectivity of Human Cytomegalovirus Strain TB40/E Conferred by Variants of the Envelope Glycoprotein UL128 and the Regulatory Protein IE2

Human cytomegalovirus (HCMV) infects various cell types in its human host, and this broad tropism plays a vital role in viral transmission, dissemination, and pathogenesis. HCMV strains differ in their ability to infect and replicate in different cell types, but the genetic determinants of cell tropism have only begun to be understood. A widely used HCMV strain, TB40/E, contains a mixture of genetically distinct virus variants. Only a few passages in ARPE-19 epithelial cells resulted in the selective enrichment of a substrain, termed TB40/EE, which infected epithelial cells more efficiently than the parental TB40/E and induced the formation of large multinucleated syncytia. Herein, we used sequence comparison and genetic engineering of a TB40/E-derived bacterial artificial chromosome clone, TB40-BAC4, to demonstrate that the high infectivity of TB40/EE and its ability to induce syncytia in epithelial cells depends on two single-nucleotide variants (SNVs) affecting the envelope glycoprotein UL128 and the major viral transactivator protein, IE2. While the intronic SNV in UL128 increased splicing of the UL128 transcript, it surprisingly decreased viral infectivity and replication in epithelial cells. The additional introduction of the IE2 SNV reversed this phenotype, increasing infectivity and syncytium formation. This SNV resulted in a D390H substitution and increased the levels of several early and late viral proteins, suggesting that it altered the ability of IE2 to activate viral genes. The same two SNVs increased the ability to infect THP-1-derived macrophages and JEG-3 trophoblast cells. These results demonstrate that HCMV cell tropism depends on both envelope glycoproteins and regulatory proteins. ImportanceDifferent genetic versions of human cytomegalovirus (HCMV) affect its ability to infect various human cell types. Here we focused on a commonly used strain, TB40/E, which contains a mix of virus variants. After growing it in epithelial cells, a specific variant called TB40/EE became dominant. This variant infected epithelial cells more effectively and caused the formation of large, fused cells (syncytia). In this study, we discovered that two small genetic changes were responsible for this behavior. One change affected a protein on the viral envelope (UL128) by altering how its RNA was processed. Surprisingly, this change reduced the viruss ability to spread, but a second change in a regulatory protein (IE2) reversed that effect. Together, these changes enhanced the viruss ability to infect not only epithelial cells but also macrophages and placental cells. This study highlights how small genetic tweaks can influence how HCMV targets different types of human cells.

microbiology↗

bioRxiv2018

Single-cell transcriptome analysis of CD34+ stem cell-derived myeloid cells identifies a CFU-GEMM-like population permissive to human cytomegalovirus infection.

Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34+ hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of susceptible and resistant cell types, and the cellular features characterizing permissive cells, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of [~] 7000 individual cells at day one post-infection using the 10X genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit - Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells susceptible to CMV replication, and provide a possible rationale for their preferential infection.\n\nAUTHOR SUMMARYMyeloid cells such as monocytes and dendritic cells are critical targets of CMV infection. To identify the cellular factors that confer susceptibility or resistance to infection, we profiled the transcriptomes of [~] 7,000 single cells from a population of semi-permissive myeloid cells infected with CMV. We found that viral RNAs are detectable in the majority of the cells, but that marked expression of CMV lytic genes occurs in only a small subset of cells transcriptionally related to a cluster of CFU-GEMM progenitors that express similar amounts of transcripts encoding interferon-related anti-viral factors as the rest of the population but higher levels of transcripts encoding proteins required for energy, RNA, and protein production. We thus conclude that the preferential infection of CFU-GEMM cells might be due to the pre-existing presence of an intracellular environment conducive to infection onset, rather than to the absence of anti-viral factors restricting viral entry or initial gene expression. Together, these findings uncover a new type of myeloid cells potentially permissive to CMV infection, expand our understanding of the cellular requirements for successful initiation of CMV infection, and provide new pro- and anti-viral gene candidates for future analyses and therapeutic interventions.

microbiology↗