Biology subjects

Bossert, M.

Publications and source records attributed to Bossert, M..

3 recordsLinked to original sources

bioRxiv2026

Identification of Viral and Cellular Proteins in Proximity to the HSV-2 pUL16 Tegument Protein in Infected Cells

Orthologs of the herpes simplex virus (HSV) pUL16 tegument protein are conserved throughout the Orthoherpesviridae family. During HSV infection, pUL16 functions in the nuclear egress of nascent nucleocapsids from the nucleus to the cytoplasm, prevents the docking of nascent cytoplasmic nucleocapsids to nuclear pore complexes, promotes the final envelopment of cytoplasmic nucleocapsids, and enhances cell-to-cell spread of virus infection. How pUL16 performs these diverse functions is poorly understood. To gain further insight into the mechanisms by which pUL16 mediates its activities, we utilized a BioID approach to identify cellular and viral proteins in proximity to pUL16 during the infection of human keratinocytes. By comparing proteins in proximity to pUL16 during infection with proteins in proximity to its well-known virus-encoded binding partner, pUL21, we provide new insight into the activities of pUL16 that likely occur in complex with pUL21 and those that are independent of pUL21. A key function of pUL21 is to deliver protein phosphatase 1 (PP1) to viral and cellular substrates to mediate their dephosphorylation. Intriguingly, the findings presented suggest that pUL16 interactions with pUL21 may regulate the isoform of PP1 that is bound to pUL21 and thereby regulate the specificity of substrate dephosphorylation. ImportanceHSV-1 and HSV-2 are important human pathogens that currently infect roughly 3.8 billion and 520 million people, respectively. These viruses cause lifelong, recurrent, infections and cause a variety of diseases including vesicular lesions of the oral and genital mucosa, corneal blindness, meningitis, encephalitis and devastating neonatal infections. The HSV pUL16 tegument protein performs a number of critical functions for the virus that influence virion assembly and the spread of infection between cells. In this study we have identified cellular and viral proteins that are proximal to pUL16 during infection of human keratinocytes, providing new insight into the mechanisms used by pUL16 to perform its activities.

microbiology↗

bioRxiv2025

Proximity Labelling Identifies Proteins Associated with HSV-2 pUL21 at Early and Late Times After Infection

pUL21 is a conserved and multifunctional alphaherpesvirus tegument protein that is critical for both early and late stages in the herpes simplex virus type 2 (HSV-2) replication cycle; however, how pUL21 participates in these activities is poorly understood. To help elucidate the role of pUL21 in these various activities, we used a proximity-dependent biotin identification (BioID) approach by constructing an HSV-2 strain encoding pUL21 fused to the non-specific biotin ligase, miniTurbo (pUL21mT). Cells infected with this strain were treated with exogenous biotin at early and late times post-infection and biotinylated proteins were affinity-purified and identified by mass spectrometry. This approach enabled the identification of many viral and cellular proteins in proximity to pUL21mT at late times after infection, including those involved in cell adhesion and junction organization, as well as components of the spliceosome and the nuclear envelope. We also utilized this system to identify proteins in proximity to tegument-delivered pUL21mT immediately following viral entry, thereby providing a comprehensive profile of pUL21 proximal interactors at both early and late stages of infection. These proximal interactions were further validated by pUL21 affinity purification experiments, which confirmed that many viral and cellular proteins identified by BioID associate with pUL21. These findings provide insights into the role of HSV-2 pUL21 during infection and highlight BioID as a powerful tool for investigating virus-host interactions at multiple stages of infection.

microbiology↗

bioRxiv2022

The Herpes Simplex Virus Tegument Protein pUL21 is Required for Viral Genome Retention Within Capsids

During virion morphogenesis herpes simplex virus nucleocapsids transit from the nucleoplasm to the cytoplasm, through a process called nuclear egress, where the final stages of virion assembly occur. Coupled to nuclear egress is a poorly understood quality-control mechanism that preferentially selects genome-containing C-capsids, rather than A- and B-capsids that lack genomes, for transit to the cytoplasm. We and others have reported that cells infected with HSV strains deleted for the tegument protein pUL21 accumulate both empty A-capsids and C-capsids in the cytoplasm of infected cells. Quantitative microscopy experiments indicated that C-capsids were preferentially selected for envelopment at the inner nuclear membrane and that nuclear integrity remained intact in cells infected with pUL21 mutants, prompting alternative explanations for the accumulation of A-capsids in the cytoplasm. More A-capsids were also found in the nuclei of cells infected with pUL21 mutants compared to their wild type (WT) counterparts, suggesting pUL21 might be required for optimal genome packaging or genome retention within capsids. In support of this, more viral genomes were prematurely released into the cytoplasm during pUL21 mutant infection compared to WT infection and led to enhanced activation of cellular cytoplasmic DNA sensors. Mass spectrometry and western blot analysis of WT and pUL21 mutant capsids revealed an increased association of the known pUL21 binding protein, pUL16, with pUL21 mutant capsids, suggesting that premature and/or enhanced association of pUL16 with capsids might result in capsid destabilization. Further supporting this idea, deletion of pUL16 from a pUL21 mutant strain rescued genome retention within capsids. Taken together, these findings suggest that pUL21 regulates pUL16 addition to nuclear capsids and that premature, and/or, over-addition of pUL16 impairs HSV genome retention within capsids. ImportancepUL21 is a conserved, multifunctional alphaherpesvirus protein involved in cell-to-cell spread of infection, transport of capsids along microtubules, and regulation of the phosphorylation status of both viral and host proteins. pUL21 thereby controls diverse processes such as nuclear egress of capsids and cellular lipid trafficking. This study provides additional insight into HSV-1 and HSV-2 pUL21 activities and suggests that, by binding pUL16, pUL21 prevents pUL16 from interacting prematurely with nuclear capsids that would otherwise lead to capsid destabilization and premature ejection of viral genomes. Thus, prevention of pUL21 interaction with pUL16 may prove to be a useful strategy for interfering with virion assembly.

microbiology↗