Periodic Reporting for period 1 - VirIm (Dynamics and heterogeneity of early viral infection)
Okres sprawozdawczy: 2023-01-01 do 2025-06-30
Viruses are simple replicative units built from two components: a nucleic acid and a small number of associated proteins. Upon entering a cell, viruses co-opt the hosts machineries to copy their genetic material, while shutting down the hosts antiviral immune response. The success of viral infection differs dramatically from cell to cell through poorly understood mechanisms, resulting in heterogenous propagation through tissues, and ultimately, heterogeneous disease progression.
Although viruses have been studied for decades, the earliest steps of cellular infection have remained hidden, because only a few viral molecules are present at this stage, which presents formidable challenges for molecular analyses. Yet these initial events are critical for achieving a successful infection; transcription, translation and replication must be perfectly balanced to rapidly scale virus production before antiviral signalling pathways are activated. To overcome this barrier, we recently developed a first-in-kind imaging technology for simple positive-sense single stranded RNA (+ssRNA) viruses that transforms our ability to visualize early viral infection processes.
In this proposal, we will expand our single–molecule toolbox to gain molecular insights into early viral infection of the more complex group of negative-sense RNA (-ssRNA) viruses. We will focus on the respiratory syncytial virus (RSV), a -ssRNA virus which can be deadly in infants and vulnerable adults, and lacks effective treatments. Specifically, we will determine i) how viral transcription and replication are coordinated on single viral genomic RNA molecules to optimize early viral propagation, ii) what causes the early viral infection heterogeneity, and iii) how heterogeneity in early viral infection impacts infection outcome. Using our novel approaches, we will gain a deep understanding of viral biology, which will eventually inform therapeutic interventions.
Although viruses have been studied for decades, the earliest steps of cellular infection have remained hidden, because only a few viral molecules are present at this stage, which presents formidable challenges for molecular analyses. Yet these initial events are critical for achieving a successful infection; transcription, translation and replication must be perfectly balanced to rapidly scale virus production before antiviral signalling pathways are activated. To overcome this barrier, we recently developed a first-in-kind imaging technology for simple positive-sense single stranded RNA (+ssRNA) viruses that transforms our ability to visualize early viral infection processes.
In this proposal, we will expand our single–molecule toolbox to gain molecular insights into early viral infection of the more complex group of negative-sense RNA (-ssRNA) viruses. We will focus on the respiratory syncytial virus (RSV), a -ssRNA virus which can be deadly in infants and vulnerable adults, and lacks effective treatments. Specifically, we will determine i) how viral transcription and replication are coordinated on single viral genomic RNA molecules to optimize early viral propagation, ii) what causes the early viral infection heterogeneity, and iii) how heterogeneity in early viral infection impacts infection outcome. Using our novel approaches, we will gain a deep understanding of viral biology, which will eventually inform therapeutic interventions.
We have successfully developed a imaging system to visualize RSV infection with single vRNP resolution. Using the system, we have tracked RSV infection with high spatial and temporal resolution in single infected cells. We find that approximately half of infected cells fail to produce viral progeny, and identify formation of replication-competent inclusion bodies as a critical bottleneck towards successful infection. Excitingly, we show that virion heterogeneity underlies heterogeneity in infection success, and we identify viral protein concentrations inside virions as a key determinant of infection success
Our real-time single molecule imaging system to visualzie RSV infection is beyond the state of the art technically speaking. Moreover, we have characterized infection establishment, the earliest phase of infection, for the first time, providing important insights into how viruses colonize host cells, and what determines the outcome of infection.