Proteomic Analysis of Endemic Viral Infections in Neurons offers Insights into Neurodegenerative Diseases

Abstract

Endemic viral infections with low pathogenicity are often overlooked due to their mild symptoms, yet they can exert long-term effects on cellular function and contribute to disease pathogenesis. While viral infections have been implicated in neurodegenerative disorders, their impact on the neuronal proteome remains poorly understood. Here, we differentiated human induced pluripotent stem cells (KOLF2.1J) into mature neurons to investigate virus-induced proteomic changes following infection with five neurotropic endemic human viruses: Herpes simplex virus 1 (HSV-1), Human coronavirus 229E (HCoV-229E), Epstein-Barr virus (EBV), Varicella-Zoster virus (VZV), and Influenza A virus (H1N1). Given that these viruses can infect adults and have the potential to cross the placental barrier, their molecular impact on neurons may be relevant across the lifespan. Using mass spectrometry-based proteomics with a customized library for simultaneous detection of human and viral proteins, we confirmed successful infections and identified virus-specific proteomic signatures. Notably, virus-induced protein expression changes converged on key neuronal pathways, including those associated with neurodegeneration. Gene co-expression network analysis identified protein modules correlated with viral proteins. Pathway enrichment analysis of these modules revealed associations with the nervous system, including pathways linked to Alzheimer's and Parkinson's disease. Remarkably, several viral-induced proteomic alterations overlapped with changes observed in postmortem Alzheimer's patient brains, suggesting a mechanistic connection between viral exposure and neurodegenerative disease progression. These findings provide molecular insights into how common viral infections perturb neuronal homeostasis and may contribute to neurodegenerative pathology, highlighting the need to consider endemic viruses as potential environmental risk factors in neurological disorders.

Keywords: Alzheimer’s; KOLF2.1J; Neurodegeneration; Neuroinflammation; Neurons; Proteomics; Virus.

Figure 1.. Customized FASTA Database Reveals Virus-Specific Peptides in Infected Neuronal Cultures

(A) Schematic workflow of the study design. Human induced pluripotent stem cells (iPSCs, KOLF2.1J line) were differentiated into. After 26 days of induction, neurons were infected with various human viruses (HSV-1, HCoV-229E, EBV, VZV, and H1N1) at different concentrations. Samples were collected at 1, 2, and 5-days post-infection. Proteomic analyses using mass spectrometry were conducted to identify differentially expressed proteins, assess virus-specific effects, and investigate associated pathways. A Webapp was developed for data browsing (https://niacard.shinyapps.io/CARD_virus/). (B) Human and virus proteins were identified in neuronal cultures using mass spectrometry-based proteomics. (C) Detection and confirmation of virus-specific proteins in infected neuronal samples. Several proteins of interest showed alterations in expression across virus exposure, viral concentration, and exposure time

Figure 2.. Differential Expression Analysis of Common Genes in Viral Infections

(A) The dysregulated proteins identified in each comparison across different virus types and time points, as identified by gene count, providing an overview of the extent of protein changes. (B) A circular visualization displaying the relationships and overlaps between the dysregulated proteins across different viral infection conditions and time points, as noted by color. (C) A heatmap showing the changes in protein expression of dysregulated proteins across various virus types, highlighting specific patterns of protein alterations in response to each virus. (D) Pathway analysis of the dysregulated proteins shown in (C), revealing the pathways and processes that were significantly altered by viral infections in neuronal cultures.

Figure 3.. Pathways associated with neuron apoptosis and cognition

(A) The significant dysregulated proteins enriched in the negative regulation of neuron-apoptotic process (GO:0043524). Proteins were considered upregulated with log2 FC > 0.585 (adj. p-value < 0.05) and downregulated with log2 FC < −0.585 (adj. p-value < 0.05). (B) The significant dysregulated proteins enriched in the Cognition (GO:0050890). Proteins were considered upregulated with log2 FC > 0.585 (adj. p-value < 0.05) and downregulated with log2 FC < −0.585 (adj. p-value < 0.05). (C) The significant dysregulated proteins enriched in the anti neuron-apoptotic process (GO:0043524). Proteins were considered upregulated with log2 FC > 0.585 (adj. p-value < 0.05) and downregulated with log2 FC < −0.585 (adj. p-value < 0.05).

Figure 4.. WGCNA of Virus-Related Gene Modules

(A) Gene dendrogram obtained through average linkage hierarchical clustering, with the color row underneath the dendrogram showing the module assignment determined by the Dynamic Tree Cut method. The proteins are clustered in 18 modules based on protein abundance in virus infected neurons. (B) Module-trait association analysis, where each row corresponds to a gene module and each column corresponds to a trait. Each cell contains the corresponding p-value from the linear mixed-effects model, indicating the relationship between modules and traits. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (C) KEGG pathway analysis of the module 11 proteins, highlighting its association with nervous system function and NDDs. (D) Chord diagram depicting the module 11 proteins involved in seven neurological disorders identified through enrichment analysis. (E) Proteins expression of the module 11 in neurons across different time points and virus types post-infection. (F) Chord diagram depicting the module 11 proteins involved in ten nervous system pathways, as identified in the enrichment analysis.

Figure 5.. Correlation of Virus-Induced Protein Changes with Alzheimer’s Disease

(A) Heatmap displaying the correlation of altered proteins by viral exposure in neurons and their abundance alternation in the cerebrospinal fluid (CSF) and postmodern brain from AD patients compared to healthy controls. Proteins were considered upregulated with log2 FC > 0.585 (adj. p-value < 0.05) and downregulated with log2 FC < −0.585 (adj. p-value < 0.05). (B) Protein–protein interaction (PPI) network of shared dysregulation of proteins during viral infection and AD.