Periodic Reporting for period 2 - DissectCMV (Creating a comprehensive functional map of the viral and host factors in HCMV infection)
Période du rapport: 2021-12-01 au 2023-05-31
Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus, present in the majority of human population. Although asymptomatic in most healthy adults, HCMV is an opportunistic pathogen in immunocompromised adults, as well as a significant cause of congenital disease. Like all herpesviruses, HCMV can establish latent infection that persists for the lifetime of the host.
Currently, there is no vaccine against HCMV and no antivirals that target the latent phase of infection. Alleviating, or even preventing, the medical consequences of this widespread virus requires a deeper understanding of HCMV biology.
HCMV genome is 236kb of double-stranded DNA, the largest genome of a virus known to infect humans and contains hundreds of proteins, the functions of most of these proteins remains unknown.
HCMV infects a wide range of cell types 2, but with variable infection outcomes. Cells from the myeloid lineage are thought to play a critical role in HCMV latency and reactivation. Hematopoietic stem cells (HSCs) and blood monocytes are the main cells in which HCMV latency has been characterized. While macrophages and dendritic cells, which are terminally differentiated, were shown to be permissive for productive HCMV infection, yet the molecular basis for the different infection outcome between monocytes and their differentiated counterparts is not understood.
In DissectCMV, we seek to bridge the above-described central knowledge gaps by developing novel technology that will allow us to probe viral and cellular gene functions during HCMV infection and to dissect the molecular factors that govern infection outcome (latent vs. lytic)
We belive the insights gained will advance HCMV research and indicate new potential therapeutic interventions. More generally, we hope the tools we will develop will provide a paradigm for studying complex host-pathogen interactions.
Currently, there is no vaccine against HCMV and no antivirals that target the latent phase of infection. Alleviating, or even preventing, the medical consequences of this widespread virus requires a deeper understanding of HCMV biology.
HCMV genome is 236kb of double-stranded DNA, the largest genome of a virus known to infect humans and contains hundreds of proteins, the functions of most of these proteins remains unknown.
HCMV infects a wide range of cell types 2, but with variable infection outcomes. Cells from the myeloid lineage are thought to play a critical role in HCMV latency and reactivation. Hematopoietic stem cells (HSCs) and blood monocytes are the main cells in which HCMV latency has been characterized. While macrophages and dendritic cells, which are terminally differentiated, were shown to be permissive for productive HCMV infection, yet the molecular basis for the different infection outcome between monocytes and their differentiated counterparts is not understood.
In DissectCMV, we seek to bridge the above-described central knowledge gaps by developing novel technology that will allow us to probe viral and cellular gene functions during HCMV infection and to dissect the molecular factors that govern infection outcome (latent vs. lytic)
We belive the insights gained will advance HCMV research and indicate new potential therapeutic interventions. More generally, we hope the tools we will develop will provide a paradigm for studying complex host-pathogen interactions.
Macrophages are known targets of HCMV and considered to be permissive for productive infection, while monocytes, their precursors, are latently infected. In our ongoing work we revealed that infection of macrophages is more complex than previously appreciated and can result in either productive or non-productive infection. By analyzing the progression of HCMV infection in monocytes and macrophages using single cell transcriptomics, we uncover that a major factor dictating productive infection is the level of viral gene expression, and specifically the expression of the major immediate early proteins, IE1 and IE2, at early stages of infection. On the cellular side, we reveal that the cell intrinsic levels of interferon stimulated genes (ISGs), but not their induction, is a main determinant of infection outcome and that intrinsic ISG levels are downregulated with monocyte differentiation, partially explaining why macrophages are more susceptible to productive HCMV infection. We further show that, compared to monocytes, non-productive macrophages maintain higher levels of viral transcripts and are able to reactivate, raising the possibility that they may serve as latency reservoirs. Overall, by harnessing the tractable system of monocyte differentiation and using single cell transcriptomicwe decipher some of the underlying principles that control HCMV infection outcome.
HCMV as a slow an intricate life cycle involving many cellular compartments and pathways, but not much is unknown about the roles cellular genes play in HCMV infection. CRISPR/Cas9 technology have accelerated the discovery gene functions. However, in the context of infection these screens predominantly reveal hits that support the early steps of viral replication, specifically entry. In order to expand the range of discovery to later stages of viral infection, we developed a new platform for performing CRISPR screens in which the sgRNA is encoded in the viral genome instead of the host genome. This way, changes in sgRNA abundance report directly on its effect on viral propagation and not on the cell fate. We used this platform, which we name Virus Encoded Knock-Out System (VEKOS), to screen for cellular genes that play a role in HCMV infection, and identify dozens of new restriction and dependency factors. Moreover, screening with VEKOS allows us to isolate the specific stage of viral replication cycle affected by the sgRNA, producing a high-resolution view on cellular gene effects on post-entry stages of infection. We are now validating the results of this screen and planning follow-up studies to some of the genes we identified.
HCMV as a slow an intricate life cycle involving many cellular compartments and pathways, but not much is unknown about the roles cellular genes play in HCMV infection. CRISPR/Cas9 technology have accelerated the discovery gene functions. However, in the context of infection these screens predominantly reveal hits that support the early steps of viral replication, specifically entry. In order to expand the range of discovery to later stages of viral infection, we developed a new platform for performing CRISPR screens in which the sgRNA is encoded in the viral genome instead of the host genome. This way, changes in sgRNA abundance report directly on its effect on viral propagation and not on the cell fate. We used this platform, which we name Virus Encoded Knock-Out System (VEKOS), to screen for cellular genes that play a role in HCMV infection, and identify dozens of new restriction and dependency factors. Moreover, screening with VEKOS allows us to isolate the specific stage of viral replication cycle affected by the sgRNA, producing a high-resolution view on cellular gene effects on post-entry stages of infection. We are now validating the results of this screen and planning follow-up studies to some of the genes we identified.
The goal of this project is to gain a broad and in-depth view of the components essential for HCMV and to understand better the components that regulate infection outcome. My lab has strong expertise in applying high-throughput approaches and in the frame of this project we consolidate our expertise to attain a multifaceted understanding of HCMV functional components. The merging of the knowledge gained from our work will provide a more comprehensive picture of the determinants that play a role during HCMV infection in both lytic and latent infection and should help to expand our therapeutic options.