Report
Teaser, summary, work performed and final results
Periodic Reporting for period 3 - Profile Infection (Unraveling changes in cellular gene expression during viral infection)
Teaser
The herpesvirus human cytomegalovirus (HCMV) infects the majority of the world\'s population, leading to severe diseases in millions of newborns and immunocompromised adults annually. During infection, HCMV extensively manipulates cellular gene expression to maintain conditions...
Summary
The herpesvirus human cytomegalovirus (HCMV) infects the majority of the world\'s population, leading to severe diseases in millions of newborns and immunocompromised adults annually. During infection, HCMV extensively manipulates cellular gene expression to maintain conditions favorable for efficient viral propagation. Identifying the pathways that the virus relies on or subverts is of great interest as they have the potential to provide new therapeutic windows and reveal novel principles in cell biology. Over the past years high-throughput analyses have profoundly broadened our understanding of the processes that occur during HCMV infection. However, much of previous analysis was focused on transcriptional changes at the lytic phase of infection leaving posttranscriptional regulation and the latent phase of the virus relatively less understood. Novel emerging technologies have the potential to extend our knowledge in areas that were heretofore unattainable.
The overall goal of our work is to decipher the multiple mechanisms by which HCMV modulates the host cell. For this, we use and develop multiple cutting-edge deep-sequencing technologies that will allow the analysis of novel aspects of host gene regulation during infection. Accordingly, the primary objectives of this research proposal are: 1) Deciphering posttranscriptional mechanisms that control cellular gene expression during infection; 2) Identifying and characterizing cellular protein diversification during infection; and 3) Uncovering the changes that occur in infected cells during latent infection.
The knowledge generated from these objectives will provide us with a clearer depiction of the changes that take place during HCMV infection, which in turn can facilitate the development of novel anti-viral strategies.
Work performed
Despite substantial variation in genome size, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent upon the protein synthetic machinery resident within their cellular hosts to translate viral mRNAs. Access to the translation apparatus, however, is patrolled by powerful host immune defenses programmed to restrict viral invaders. Our research focused on two major pathogens; a small RNA virus, Influenza A (IAV) and a large DNA virus, Human cytomegalovirus (HCMV).
Host shutoff is a common strategy used by many viruses to repress cellular mRNA translation, allowing efficient translation of viral mRNAs. In our work we used e RNA-seq, ribosome profiling and single molecule FISH to accurately quantify IAV induced host shutoff at both RNA and translation levels (Bercovich-Kinori et al. 2016). We reveal that IAV shutoff is mediated solely by interfering with cellular mRNA levels and we uncover that transcripts, coding for cell maintenance processes such as oxidative phosphorylation, were refractory to IAV shut-off affects. We further demonstrate that indeed continuous oxidative phosphorylation activity is important for IAV propagation. Our results thus challenge the notion of host shut-off being a blunt, indiscriminate instrument to halt host gene expression and reveal that shutoff could be selective, allowing maintenance of important housekeeping functions in infected cells. More broadly, this work lays the technological foundation for exploring host shutoff mechanisms during infection
A different tactic was observed in HCMV-infected cells, where host protein synthesis is not globally suppressed so it was assumed that host protein synthesis proceeded uninterrupted. Using ribosome profiling and RNA-seq we showed that HCMV dramatically re-shape the infected cell translation landscape. This translational reprogramming was dependent upon virally induced mTOR activation. Importantly, we show that interfering with the virus-induced activation of cellular mRNA translation can limit or enhance HCMV growth (Tirosh et al. 2015).
Although we obtained quantitative analysis of genes that are translationally regulated, our molecular understanding of this type of regulation is still at its infancy. In order to systematically identify cis-regulatory elements in mRNAs that affect posttranscriptional regulation, we mapped changes in mRNAs secondary structure along HCMV infection by utilizing in-vivo modification with dimethyl sulfate (DMS). DMS reacts with unpaired residues and the modifications are then mapped by deep sequencing. Our underlying assumption is that changes in translation should be mediated by changes in RNA binding protein interactions and that these interactions are likely to affect the DMS reactivity of the relevant mRNA region. Indeed, our measurements allowed us to identify hundreds of regions in human and viral mRNAs that show significant structural changes during viral infection. We are now using reporters, combinatorial mutagenesis and genome editing to elucidate the regulatory function and importance of these regions. Overall, this cutting edge approach allows us to identify new regulatory RNA elements and more broadly to shed light on how RNA structures help to regulate gene expression in mammalian cells.
Primary HCMV infection results in a lifelong infection due to HCMV ability to establish latent infection, with the characterized viral reservoir being hematopoietic progenitor cells (HPCs) and monocytes. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. A major limitation in studying latency is that natural latent cells are very rare and the experimental models utilize primary hematopoietic cells that are heterogenous, most likely consisting of several subpopulations differing in differentiation stage, thus may support non-uniform latent programs. In a manuscript we recent
Final results
We expect to accomplish all our original aims.
We plan to finish characterizing the cis regulatory elements we mapped using DMS sequencing and to extend the method to specifically map regulatory elements located in 5\' UTRs (manuscript just submitted for publication).
With regard to full characterization of the latency state. We will dissect changes in cellular transcripts during HCMV latency- in our scRNA-seq experiments we sequenced both infected and bystander cells (that were not infected) from the same culture. This allowed us to test the hypothesis that infection induces a host gene expression signature that distinguishes latent monocytes from their non-infected counterparts. Among the differentially expressed host genes we identified, several are coding for cell surface proteins. We will test if these proteins allow us to enrich for latent cells in experimental models of HCMV infection and further decipher whether these differences are due to cellular heterogeneity prior to infection, meaning these are cellular factors that affect infection efficiency or whether this differential expression is caused by changes that are being induced by the virus. Our ultimate goal is to identify cell surface markers that will allow us to enrich and characterize latent cells during natural latency.
We will elucidate the molecular determinants that govern the outcome of infection, lytic vs. latent - It is well established that differentiation state of hematopoietic cells affects the outcome of infection. HPCs and monocytes support latent infection whereas differentiation to dendritic cells or macrophages triggers virus reactivation and this reactivation is mediated by changes in chromatin modification. However, the prolonged and inefficient differentiation-dependent reactivation process makes it hard to identify the molecular mechanisms that drive this intricate process. In our scRNA-seq analysis we sequenced both latent and lytic cells. However, since our measurements were made at late time points post infection (when latency was established), these cell states were already very different and it was therefore impossible to learn about the initial determinants that governed these two outcomes. To tackle this issue, we plan to perform scRNA-seq analysis along early stages of infection, when latency state is starting to be established. We anticipate that these high temporal resolution measurements will allow us to better understand the dynamic that occurs in host and viral gene expression when latency or lytic infections are being established. Our hope is that these measurements, at single cell resolution, will allow us to pinpoint the determinants that govern these two discrete modes of infection. We believe that the combination of these complementary studies will help to uncover the complex interplay between virus and the host during latency and will pave the way to novel therapeutic strategies.