SummaryA subset of viral genes is required for the long-term latent infection of hematopoietic cells by human cytomegalovirus (HCMV). Here, we show that a latency-associated gene product (LUNA) promotes the disruption of cellular PML bodies during latency. Mutation and inhibitor studies reveal that LUNA encodes a deSUMOylase activity responsible for this disruption. Specifically, LUNA encodes a conserved Asp-Cys-Gly motif common to all deSUMOylases. Importantly, mutation of the putative catalytic cysteine is sufficient to reverse LUNA-mediated PML dispersal and markedly reduces the efficiency of viral reactivation. The depletion of PML from cells is sufficient to rescue the reactivation of the LUNA-deficient viruses, arguing that targeting PML is an important biological role of LUNA. Finally, we demonstrate that reactivation of naturally latent HCMV is blocked by deSUMOylase inhibitors. Thus, latent HCMV primes the cellular environment for efficient reactivation via the activity of a virally encoded deSUMOylase.
Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data.
To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high-throughput strategy to screen a GlaxoSmithKline Published Kinase Inhibitor Set. This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen, we identified compounds within chemotypes that impede HCMV protein production and identified kinase proteins associated with inhibition of HCMV protein production that are potential novel anti-HCMV targets. We focused our study on a top 'hit' in our screen, SB-734117, which we found inhibits productive replication of several HCMV strains. Kinase selectivity data indicated that SB-734117 exhibited polypharmacology and was an inhibitor of several proteins from the AGC and CMCG kinase groups. Using Western blotting, we found that SB-734711 inhibited accumulation of HCMV immediate-early proteins, phosphorylation of cellular proteins involved in immediate-early protein production (cAMP response element-binding protein and histone H3) and histone H3 lysine 36 trimethylation (H3K36me3). Therefore, we identified SB-734117 as a novel anti-HCMV compound and found that inhibition of AGC and CMCG kinase proteins during productive HCMV replication was associated with inhibition of viral protein production and prevented post-translational modification of cellular factors associated with viral protein production.
The emergence of SARS-CoV-2 variants has exacerbated the COVID-19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N-terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine-tune spike; this may provide a mechanism for SARS-CoV-2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immunedriven antigenic variation and ongoing adaptation to a new host.
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