Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing

Abstract

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.

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

“…Thus, we wondered about the fate of infection in the remainder of the cells, which contain lower amounts of viral transcripts, and no detectable viral proteins. Intriguingly, a similar scenario was recently encountered following single-cell RNA sequencing of TB40/E-infected CD14+ and CD34 + cells [90]. Elevated levels of viral transcripts were observed in just ∼ 2% of monocytes, while the rest of the population, which contained lower amounts of a wide range of viral transcripts, were interpreted as potentially being latently infected.…”

“…This led us to speculate that the remaining CMV-transcript + cells in our population might be either latently infected or on a path toward latency. While this hypothesis requires additional testing, it remains a thrilling possibility, especially in view of recently presented evidence supporting the potential association of viral latency with quantitative rather than qualitative changes in viral gene expression [90]. Alternatively, it is of course possible for viral transcripts to simply be detected and eliminated by cellular defense mechanisms, producing an abortive infection.…”

“…CD34 + cells are extremely plastic, and can clearly give rise to clustered sub-populations of myeloid cells, some of which permissive to lytic infection. Being a minority in the population these clusters can easily escape detection and may introduce unwanted and unnoticed “lytic noise” [90] in studies of viral latency.…”

Single-cell RNA-sequencing of Herpes simplex virus 1-infected cells identifies NRF2 activation as an antiviral program

“…The characterization of cellular heterogeneity due to activation of different host pathways and the progression of viral infection is of great interest. Recent singlecell RNA-sequencing (scRNA-seq) efforts provided an unbiased characterization of virus-host interactions in individual cells which are masked at the population level [14][15][16][17][18][19][20][21][22]. However, deeper insights into unique molecular signatures and discovery of specific cell subsets could be obtained by increasing sequencing depth and the application of advanced analytical approaches to study viral infections.…”

EGR1 transcriptional control of human cytomegalovirus latency

“…CMV establishes latency in CD34+ hematopoietic progenitor cells (HPCs) and is carried through differentiation in cells of the myeloid lineage, including CD14+ monocytes (1). During latency in experimental models, CMV genes are expressed broadly but at very low levels (2, 3) and replication is restricted. Reactivation in immunodeficient individuals, such as stem cell or solid organ transplant recipients, is a major cause for morbidity and mortality (4–6).…”