p38 and OGT Sequestration into Viral Inclusion Bodies in Cells Infected with Human Respiratory Syncytial Virus Suppresses MK2 Activities and Stress Granule Assembly

Abstract

Respiratory syncytial virus (RSV) forms cytoplasmic inclusion bodies (IBs) that are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. The present study found that IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linked N-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the posttranslational addition of OGN to protein targets to regulate cellular processes, including signal transduction, transcription, translation, and the stress response. The virus-induced sequestration of p38-P in IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: oxidative stress resulting from arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection.

Respiratory Syncytial Virus: Infection, Detection, and New Options for Prevention and Treatment

“…According to this model, sequestration of O-linked N-acetylglucosamine transferase provides a mechanism for RSV to prevent stress granule assembly, since the modification of proteins with O-linked N-acetylglucosamine is an important early step in stress granule assembly (131). Sequestering host proteins in IBs to inhibit stress granule assembly is consistent with the negative correlation observed between the presence of large IBs and stress granule formation (128).…”

“…The host proteins that colocalize with IBs include a number of antiviral, chaperone, and signaling proteins such as MAVS (124), MDA5 (124), phosphorylated p38 (128), O-linked N-acetylglucosamine transferase (128), and HSP70 (129). The presence of antiviral and signaling proteins within IBs opens up the possibility that these proteins are specifically sequestered within the inclusions to prevent host cell responses that would be unfavorable to RSV infection.…”

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions

“…Marburg virus (MARV), another member of the filovirus family, recruits components of the endosomal complex required for transport (ESCRT) machinery to viral inclusions to enhance budding (80). The sequestration of cellular proteins within viral inclusions, including HuR and proteins involved in antiviral signaling, was also observed in respiratory syncytial virus (RSV) infection and dampened the antiviral host response (81)(82)(83). This supports the hypothesis that the sequestration of certain cellular proteins within viral inclusions promotes viral infection by disrupting antiviral signaling pathways.…”

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

“…This indicated that M and F may interact at or near the IBs and implicated these structures in the cascade of late-stage events that lead to virions. IBs are multifunctional bodies (45,46) and are believed to represent the sites of viral replication. Coexpression of N and P is sufficient to induce formation of IBs (47).…”