Our research group, led by Prof. Abel Viejo-Borbolla at the Institute of Virology, Hannover Medical School in Germany, focuses on immune- and neuromodulation by herpesviruses. Our latest study, ‘Viral modulation of type II interferon increases T cell adhesion and virus spread’ published in Nature Communications, reveals an unexpected finding: the glycoprotein C (gC) of varicella zoster virus (VZV) binds and modulates interferon gamma (IFN-γ) resulting in increased expression of intercellular adhesion molecule 1 (ICAM1) and ultimately enhancing T cell adhesion and infection.

Fig1:  The glycoprotein C (gC) of varicella zoster virus (VZV) modulates immune cells on two levels: Chemokines and interferon gamma. 

Our journey began with a simple question: Which cytokines could be bound by gC? I started this project during my bachelor’s thesis, supporting the at the time PhD student Víctor González-Motos. Using surface plasmon resonance, he found that gC bound both chemokines and interferons. My main role was to reproduce these results with a recombinant gC generated using a different expression system, and I fortunately succeeded.

After showing that gC belongs to a group of enhancing viral chemokine binding proteins (vCKBP) that increase chemokine-mediated immune cell migration¹, we wondered what was the functional relevance of its interaction with interferons. Since several studies showed that interferon inhibits VZV replication, we hypothesized that gC would block IFN function, similar to previously described viral interferon-binding proteins expressed by poxviruses. However, no such effect was observed, which was a bit frustrating at the time for our group.

Meanwhile, I left the group to obtain my Master of Science and later returned to pursue my PhD investigating the function of VZV gC in greater detail. After spending some time abroad during my PhD, I returned with a fresh perspective and decided to give this project another chance. Despite knowing about the gC-IFN interaction for over five years, no one had figured out its function. My doctoral advisor suggested to abandon this project due to repeated setbacks. I agreed to drop type I interferons but remained unconvinced about type II interferon, which had the strongest binding to gC. I persuaded him to perform one last experiment on gC and IFN-γ, focusing on an interferon-stimulated gene (ISG) related to cell migration, given gC's known modulation of chemokine activity.

Contrary to our expectations based on poxviral interferon-binding proteins, we found that adding gC together with IFN-γ increased ICAM1 expression on several cell types. This discovery led to deeper investigations into the gC–IFN-γ interaction, which became the main project of my doctoral thesis. RNAseq revealed that gC induced biased signaling on IFN-γ, modulating the expression of several genes involved in cell migration, such as chemokines and ICAM1, while leaving most ISGs unaffected. It's no wonder we hadn't seen any effects before. Sometimes, you need to look in the right place or use an unbiased approach to make great discoveries.

Fig2: (A) Surface plasmon resonance was performed to analyse potential interactions between glycoprotein C and cytokines. (B) Transcriptomics analysis showed that only a small subset of genes was differentially expressed upon co-stimulation with glycoprotein C and IFN-γ.

Since ICAM1 is crucial for cell adhesion and synapse formation, we tested our hypothesis that increased ICAM1 levels could enhance T cell adhesion and viral transmission from epithelial to immune cells. Our observations confirmed this, highlighting the critical role of gC in VZV's spread from epithelial cells to leukocytes, particularly T cells. This could contribute to VZV systemic dissemination during primary and secondary viremia, ultimately colonizing the skin and peripheral neurons where it establishes latency.

Fig3: (A) ICAM1 surface expression was measured by flow cytometry. The fold change by treatment with different domains of gC were calculated in the absence of and upon IFN-γ treatment. (B) Images of HaCaT cells infected with BAC-derived VZV that express either GFP instead of gC or a fusion product of gC-GFP (green). Adhered Jurkat T cells are shown in red. (C) The amount of infected PBMCs based on GFP-signal normalised to the transmission of the virus that lacks gC expression.

Just before my defence, a collaborator pointed out that the bacterial-produced IFN-γ we used lacked glycosylation, potentially important for the gC–IFN interaction. It felt like a punch in the face—how did we overlook this crucial detail? With my departure from the lab imminent, my doctoral advisor stepped in, conducting initial binding experiments with a mammalian-expressed interferon using surface plasmon resonance. Fortunately, we observed binding. Later, during the publication revision, my colleague Nina Plückebaum repeated some experiments with mammalian-expressed proteins in primary cells and confirmed our findings.

Our journey underscores the importance of perseverance, open-mindedness, and a willingness to revisit and re(de)fine hypotheses. Believing in your project, despite setbacks, can lead to significant scientific discoveries, as demonstrated by our identification of the first herpesviral IFN-binding protein and the first viral protein that enhances specific ISGs.

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¹ González-Motos V, Jürgens C, Ritter B, Kropp KA, Durán V, Larsen O, et al. (2017) Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration. PLoS Pathog 13(5): e1006346. https://doi.org/10.1371/journal.ppat.1006346

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Images modified from the original publication and my thesis.