Tempo-iOligo™was cited in Nature in a major infectious disease study that offers the first explanation as to why herpes simplex virus 1 (HSV-1) infection in the brain is very rare, despite the fact that most of us have been infected with the virus. The study used CRISPR screens to find new factors involved in controlling HSV-1 replication in the brain, and found that depletion of the transmembrane protein TMEFF1 in human neurons and in mice led to increased viral replication and neuronal death following HSV-1 infection.
Tempo’s iOligo cells are induced oligodendrocyte progenitors reprogrammed from human skin fibroblasts using Tempo’s proprietary reprogramming technology. While immortalized rodent cell lines are often used as models to understand human cell biology and pathophysiology, the evolutionary divergence between human and rodent cells, variations in signaling pathways, differences in gene expression signatures and anatomical differences make rodent cells less relevant compared to human iPSC-derived cell models. In this study, iOligo cells were used in a panel of CNS cell types to profile TMEFF1 gene expression.
Of all the known neurotropic viruses, i.e., viruses that can infect the CNS, herpes simplex virus 1 (HSV-1) is the most prevalent, with 60 – 80 % of the global population estimated to be sero-positive. HSV-1 typically infects its host via epithelial cells before spreading to sensory neurons. Infection is spread easily through oral contact, usually leading to cold sores and/or genital herpes.
In most cases, viral replication is brought under control by the host immune system and the virus exists in a latent form with occasional relapses or flare ups. However, in rare cases, HSV-1 can enter the CNS via peripheral nerves and cause a severe and potentially fatal infection in the brain known as herpes simplex encephalitis (HSE).
How do neurons fight off HSV-1 without damaging the CNS?
Previous research showed that loss-of-function mutations in several genes in the interferon (IFN) pathway – which is the major inducible innate anti-viral defense system – can increase an individual’s risk of developing HSE. More recently, disease-causing variants in several constitutive innate anti-viral factors have been discovered in patients with HSE (reviewed in 1). However, until recently, it was unknown why HSV-1 infection in the brain is so rare, despite the extremely high sero-prevalence of the virus, i.e., what is it that prevents HSV-1 from entering the CNS?
And how does the brain protect itself against HSV-1 without causing neuroinflammation, especially given the sensitivity of neurons to excessive inflammation and their limited self-renewal capacity? We know that the blood-brain barrier (BBB) protects the CNS by restricting the influx of immune cells and inflammatory mediators into the CNS, but the immune factors governing anti-viral responses in neurons remain elusive, and no neuron-specific mechanisms have been identified.
Genome-wide CRISPR screen reveals candidate HSV-1 restriction factors
In recent years, genome-wide CRISPR screens have revealed essential factors in host:virus interactions, including new host dependency factors and anti-viral restriction factors. To understand how neurons prevent HSV-1 from entering the CNS, researchers led by Professor Søren Riis Paludan at Aarhus University in Denmark conducted a genome-wide CRISPR screen to find new viral restriction factors in neuronal cells, whose depletion would lead to increased viral replication (2).
TMEFF1 prevents HSV-1 replication in neurons in vitro and in vivo
In total, 322 genes were identified as hits and the top-10 ranked genes included TMEFF1, NKX2-8 and CTXN1, all of which are known to be predominantly expressed in neuronal tissue. Deletion and overexpression experiments revealed the Type 1 transmembrane protein TMEFF1 to be the most potent of those three hits, on the basis that it was required and sufficient for HSV-1 restriction.
The researchers then deleted TMEFF1 in human neuron-like cells derived from human embryonic neuronal precursor cells (LUHMES) and in human neurons derived from human embryonic stem cells (hESCs). They found that CRISPR-Cas9-mediated knockout of TMEFF1 – similarly to knockout of the known HSV restriction factor SP100 – coincided with more efficient HSV-1 replication and an early increase in viral gene expression, as well as significantly increased cell death in the human neurons. In these experiments, TMEFF1 knockout had no effect on replication by two other neurotropic viruses: varicella zoster virus and poliovirus.
The critical role of TMEFF1 for HSV-1 replication was also evident in in vivo experiments conducted in TMEFF1 homozygous knockout mice, which exhibited lower survival rates, faster weight loss and increased symptom scores after HSV-1 infection compared to control mice. A neuron-specific role for TMEFF1 was further supported by immunohistochemistry and gene expression analysis using brainstem sections and cells isolated from mice. Findings from in vitro infection experiments conducted in various murine CNS cell types were also consistent with a neuron-specific role for TMEFF1 in mice, whereby deficiency coincided with increased viral replication in TMEFF1-deficient neurons but not in TMEFF1-deficient astrocytes or TMEFF1-deficient microglia. Viral replication outcomes in TMEFF1-deficient human astrocytes or TMEFF1-deficient human microglia were not reported in the current study.
TMEFF1 blocks HSV-1 entry to the CNS, independently of the interferon pathway
Gene expression profiling of several cell types found in the CNS brain revealed that TMEFF1 is predominantly expressed in human and murine neurons, with weaker expression in human and murine astrocytes, human and murine microglia, and human oligodendrocytes (sourced as iPSC-derived Oligodendrocyte Progenitor Cells from Tempo Bioscience).
Mechanistic studies to shed light on how TMEFF1 restricts HSV-1 from entering the CNS included gene expression analyses, in vitro viral entry experiments in HEK293 cells using wild-type and entry-deficient HSV-1 mutants, transmission electron microscopy (TEM) to visualize viral capsids inside HEK293 cells, and fluorescence microscopy to track GDP-expressing HSV-1 at different time-points post-infection. Co-immunoprecipitations were carried out to look for proteins interacting with TMEFF1. Structural analysis was carried out to investigate how certain proteins interacted with TMEFF1 using recombinantly expressed truncated versions of those proteins and AlphaFold was used to predict interaction surfaces.
Based on the findings of those studies, the researchers conclude that TMEFF1 works through a constitutive mechanism that is independent of Type 1 IFN, since TMEFF1 expression was not altered in the presence of cytokines, and TMEFF1 did not impact IFN1 activity.
Incubation of HEK293 cells with wild-type HSV-1 or HSV-1 viral-entry mutants – namely a viral glycoprotein D mutant and a viral glycoprotein B mutant which are impaired in cell binding and virus-cell fusion, respectively – indicated that TMEFF1 does not block viral attachment to cells but that TMEFF1 overexpression led to decreased viral DNA levels in HEK293 cytoplasm and nuclear fractions as well as reduced viral capsid protein V5 in cytoplasmic lysates. These findings were supported by TEM and viral titers.
Viral tracking experiments revealed that TMEFF1 knockout reduced the level of HSV-1 entry in hESC and LUHMES-derived neurons; this was supported by early localization of viral capsids at the nuclear rim in wild-type cells but not in TMEFF1-deficient cells. Co-immunoprecipitation experiments identified non-muscle myosin (NMHC) heavy chains IIA and IIB (which are involved in HSV-1 entry via viral glycoprotein B (gB) as interaction partners of TMEFF1, and nectin-1, which is a receptor for HSV-1 binding through viral glycoprotein D (gD) also co-precipitated with TMEFF1. The authors also show that HSV-1 progeny yield was markedly reduced in cells in which NMHC-IIA, NMHC-IIB and NECTIN1 were knocked out, further suggesting the importance of their interaction with TMEFF1 for viral entry, at least in the cell types explored in this study. The results of structural characterizations to determine the regions of TMEFF1 important for those interactions are described in detail in the recent article (2).
In sum, the study is the first to report and characterize a neuron-specific restriction factor for HSV-1, and the finding that TMEFF1 uses a dual mechanism to control HSV-1 entry highlights a unique mechanism for the human CNS in preventing HSV-1 entry.
Human astrocytes, a type of glial cell.
We at Tempo are intrigued as to why such an important mechanism has evolved to be dependent upon neurons only, which make up approximately 10 % of the cells in the brain, while the glial cells that make up the remaining 90 % seem redundant in this process, at least in mice. We look forward to studies that further explore this mechanism for HSV-1, which may eventually also shed light on how other important viruses, e.g., SARS-CoV-2, enter the human brain.
Stay tuned for more citation alerts from Tempo Bioscience!
References:
- Paludan SR, Pradeu T, Masters SL, Mogensen TH. Constitutive immune mechanisms: mediators of host defense and immune regulation. Nat Rev Immunol. 2021 Mar;21(3):137-150. doi: 10.1038/s41577-020-0391-5.
- Dai Y, Idorn M, Serrero MC, et al. TMEFF1 is a neuron-specific restriction factor for herpes simplex virus. Nature. 2024 Aug;632(8024):383-389. doi: 10.1038/s41586-024-07670-z.
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Culturing and Characterising Organoids – What Do We Need to Know? (Tempo Bioscience, 2024)
Karen O’Hanlon Cohrt is an independent Science Writer with a PhD in biotechnology from Maynooth University, Ireland (2011). After her PhD, Karen relocated to Denmark where she held postdoctoral positions in mycology and later in human cell cycle regulation, before moving to the world of drug discovery. Karen has been a full-time science writer since 2017, and has since then held numerous contract roles in science communication and editing spanning diverse topics including diagnostics, molecular biology, and gene therapy. Her broad research background provides the technical know-how to support scientists in diverse areas, and this in combination with her passion for learning helps her to keep abreast of exciting research developments as they unfold. Karen is currently based in Ireland, and you can follow her on Linkedin here.
