Characterizing the virus microenvironment during herpesvirus infection
During viral infection, multicellular organisms utilize cell-to-cell communication to mount a preemptive antiviral defense. The ways in which host cells perform this communication via interferon and cytokine signaling are well studied and considered to be advantageous to the host. However, accumulating evidence also points to the ability of viruses to exploit host multicellular communication to promote infection. The balance and complexity of these rivaling communication events remain unclear. To begin to understand this, our lab has recently adapted a cellular microenvironment labeling platform and applied it to study the virus microenvironment (VME). Using this fluorescent labeling platform, cell populations are separated based on their proximity to infection, allowing us to specifically capture cell populations corresponding to infected cells, uninfected proximal cells, and uninfected distal cells. We have applied this platform to investigate the VME of the β-herpesvirus human cytomegalovirus (HCMV). Surprisingly, in contrast to what we predicted based on a canonical understanding of interferon signaling, proximal cells were less resistant to secondary infection by either HCMV or herpes simplex virus type 1 (HSV-1) than their distal counterparts. Proteomic analyses revealed differences in interferon-stimulated gene (ISG) and cell cycle-related protein abundances in proximal versus distal cells. Cell cycle profiling confirmed an increase in the proportion of mitotic cells in the proximal population. I hypothesize that cell cycle dysregulation of nearby cells represents a viral strategy to enhance the spread of infection. Therefore, in my first aim, I will determine the mechanism by which HCMV dysregulates cell cycle control in proximal cells. Based on our detection of selected HCMV proteins in proximal cells, I hypothesize that this mechanism involves the delivery of these proteins from infected to proximal cells, thereby promoting cell cycle entry and subsequent mitotic arrest. I will next investigate how mitotic dysregulation of proximal cells promotes viral spread. I hypothesize that this effect is driven largely by host-mediated innate immune suppression during mitosis. For my second aim, I propose to develop a temporally controllable VME labeling platform with broader applicability to viruses that replicate more rapidly than HCMV. Currently, this application is precluded by slow labelling kinetics. To do this, I will optimize the rate of proximal cell labeling through genetic manipulation of the labeling protein domain that mediates its cellular uptake. Next, I will integrate an inducible protein secretion system to allow fine temporal control over the onset of labeling. Finally, I will apply my improved labeling platform to compare the VMEs of α-, β-, and γ-herpesviruses—HSV-1, HCMV, and Kaposi’s sarcoma-associated herpesvirus (KSHV), respectively. Altogether, my thesis research will aim to expand the scope of our understanding about how viruses modulate cellular communication to evade host defenses and facilitate virus spread.
Myhrvold & te Velthuis Labs
Knot a problem: targeting secondary structure with Cas13
RNA’s unique chemical makeup lends it an enormous diversity of functions far beyond simply transducing genetic information. Its 2’ OH group makes it far more reactive than DNA and permits it to form complex secondary structures that frequently dictate its function. However, the dynamic and ephemeral nature of these structures means they are difficult to study and perturb, and there is a lack of adequate tools for doing so. Cas13 is an RNA-targeting CRISPR enzyme that has emerged as an extremely powerful tool for studying RNA. It is highly sensitive and sequence-specific, and these traits have been leveraged to build robust nucleic acid detection platforms. Cas13 holds promise for applications far beyond detection, such as transcriptome engineering, RNA editing, targeted antivirals, and RNA imaging. However, it has two fundamental limitations that substantially limit its potential: it cannot target structured RNA, and it exhibits nonspecific RNA cleavage when activated. My project will aim to address these weaknesses. I will conduct a comprehensive study to describe the rules governing secondary structure targeting by Cas13 using multiplexed, high-throughput Cas13 detection screens with structured targets. Next I will fuse Cas13 to a viral RNA helicase to i) give it the ability to unwind double-stranded RNA and ii) reduce off-target RNA cleavage by shortening the duration of Cas13 activation.
Functional ecology and evolution of marine macroalgae microbiomes
Marine algae are a diverse group of organisms that serve crucial roles in global carbon fixation and as a food source. Recently, the Donia Lab reported the first case of an obligate bacterial endosymbiont of marine macroalgae. Candidatus Endobryopsis kahalalidefaciens (cEK) produces a library of defensive toxins, the kahalalides, that protects its algal host, Bryopsis sp. from predation; this system highlights one example of how the microbiome plays a crucial role in macroalgae host physiology and ecology. Studying the regulation and stability of this symbiosis will shed light onto the functional interactions between microbes and algal host. First, I will establish the Bryopsis-cEK system, as a novel macroalgal laboratory model. Using this model, I will develop perturbation assays that mimic and vary real life conditions, including abiotic environmental factors (light/dark cycles, nutrients, and temperature), competition dynamics, encounters with pathogens, and predator-prey interactions. By measuring the outcomes of these variables on host ecology, fitness, and transcriptional activity, in addition to symbiont load, transcriptional activity, and defensive molecule output, I aim to identify environmental signals that mediate the symbiosis and the molecular mechanisms by which they act. In search of similar functional symbioses, I will also perform the first survey of marine bacterial symbiont–macroalgal host interactions over geographical space. By using comprehensive metagenomic, metatranscriptomic, metabolomic, and phylogenetic analyses on over 300 environmental algae samples, I aim to identify functionally relevant, novel bacterial symbionts of marine macroalgae. Based on a preliminary analysis of our dataset, at least one case of a new algal symbiosis has been identified, which I will further investigate the molecular bases of and their effects on bacterial-host ecology and coevolution. The development of ecologically relevant laboratory assays, in addition to the comprehensive survey of macroalgal microbiomes performed here, will provide the basis for future studies investigating microbiome-host evolution and a quantitative standard with which to characterize marine algal symbioses.