Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins

Schmitt, Janika; Poole, Emma; Groves, Ian; Owen, David J; Graham, Stephen C; Sinclair, John; Kelly, Bernard T

doi:10.1038/s44319-024-00063-3

Cited by 2 publications

References 64 publications

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The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

Freeman,

Dooley,

Beucler

et al. 2024

Preprint

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Human cytomegalovirus (HCMV) is a β-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gαq-dependent PLC-β signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5′ UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

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The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

Freeman,

Dooley,

Beucler

et al. 2024

Preprint

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Virus-Specific Nanobody-Chimeras Degrade the Human Cytomegalovirus US28 Protein in CD34+ Cells

Poole,

Schmitt,

Graham

et al. 2024

Pathogens

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After primary infection, human cytomegalovirus (HCMV) establishes lifelong persistence, underpinned by latent carriage of the virus with spontaneous reactivation events. In the immune-competent, primary infection or reactivation from latency rarely causes disease. However, HCMV can cause significant disease in immune-compromised individuals such as immune-suppressed transplant patients. Latency, where the viral genome is carried in the absence of the production of infectious virions, can be established in undifferentiated cells of the myeloid lineage. A number of stimuli can cause virus reactivation from latency to occur, beginning with the induction of viral immediate-early (IE) lytic gene expression. The suppression of viral IE gene expression to establish and maintain latent infection is known to result from a balance of viral and cellular factors. One key viral factor involved in this is the G protein-coupled receptor US28. Recently, we have shown that US28 is targeted for degradation by a modified nanobody (PCTD-Vun100bv) based on the novel PACTAC (PCSK9-antibody clearance-targeting chimeras) approach for targeted protein degradation. Furthermore, we have shown that this PCTD-Vun100bv-induced degradation of US28 results in IE gene expression in experimentally latently infected CD14+ monocytes. However, HCMV also establishes latency in CD34+ bone marrow cells, the progenitors of CD14+ cells. Here, we show that PCTD-Vun100bv also causes US28 degradation in these CD34+ primary cells, again resulting in the induction of viral IE gene expression. Additionally, we show that PCTD-Vun100bv can target US28 in naturally latently infected CD14+ monocytes from an HCMV-seropositive donor, allowing these latently infected cells to be killed by HCMV-specific cytotoxic T cells from that same donor. These observations support the view that targeting US28 for degradation during natural latency could be a tractable ‘shock-and-kill’ strategy to target the latent HCMV reservoir in myeloid cells.

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Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins

Cited by 2 publications

References 64 publications

The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

Virus-Specific Nanobody-Chimeras Degrade the Human Cytomegalovirus US28 Protein in CD34+ Cells

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