The MAPK/ERK Pathway and the Role of DUSP1 in JCPyV Infection of Primary Astrocytes

Abstract

JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.

PI3K/AKT/mTOR Signaling Pathway Is Required for JCPyV Infection in Primary Astrocytes

“…All cell types were treated w U0126 for 1 h, treated with PMA for 5 min, and ERK phosphorylation was nearly redu to comparable levels to cells treated with U0126 alone (Figure 1C). Together, these suggest that although elevated levels of ERK phosphorylation promote JCPyV infec [66], inhibition of ERK phosphorylation does not decrease JCPyV infection in prim astrocytes when compared to cells immortalized with SV40 T Ag. Thus, these data gest that the mechanisms of MAPK cell signaling activation utilized in JCPyV infectio NHAs differs from those in SVGAs and NHA-Ts.…”

“…First, background fluorescence from the 800 nm channel (wells that only received 2 • antibody) were subtracted to the 800 nm channel, in which the protein of interest was being quantified. Next, the ratio was determined using this new value (protein of interest), normalized to the 700 nm channel (CellTag) or the overall number of cells in each well [61,65,66].…”

“…RNA-seq data is available under the accession number: GSE183322. The read counts were generated as previously described [66] and analyzed using RStudio (version 1.2.1335, Boston, MA, USA) and R/edgeR (version 3.30.3) http://bioconductor.org/packages/release/bioc/html/edgeR.html) (accessed on 1 June 2021) [67]. Genes expressed were mapped to the PI3K/AKT signaling pathway from KEGG (https://www.kegg.jp) (accessed on 1 June 2021) [68][69][70] by obtaining the gene symbols and then converting them to Ensembl Gene IDs using EnsemblBioMart (Ensembl version 103, https://www.ensembl.org) (accessed on 1 June 2021) [71].…”

“…The Ensembl Gene IDs were matched to the RNA-seq data to acquire the genes expressed in the PI3K/AKT signaling pathway. The list of expressed PI3K/AKT genes was then merged with the results of the R/edgeR analysis [66] and subdivided based on an unadjusted p value of less than 0.10. Venn diagrams showing the overlap between the differentially expressed genes for each cell type and timepoint were created using the R package, eulerr (version 6.1.0, https://cran.r-project.org/web/packages/eulerr/) (accessed on 1 June 2021) [72].…”

“…In addition to the PI3K/AKT/mTOR signaling pathway implicated in JCPyV infection [48,50,59], the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway is required for JCPyV infection [61,73,74]. This pathway is temporally regulated, specifically being phosphorylated upon JCPyV infection, and infection of immortalized cells is significantly reduced when cells are treated with ERK siRNAs or inhibitors [61,66,73,74]. The MAPK/ERK pathway overlaps with the PI3K/AKT/mTOR pathway and both pathways have important roles in cell survival and differentiation [39,75].…”