Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells

Maeda, Fumio; Arii, Jun; Hirohata, Yoshitaka; Maruzuru, Yuhei; Koyanagi, Naoto; Kawaguchi, Yasushi

doi:10.1128/jvi.00271-17

Cited by 20 publications

(28 citation statements)

References 79 publications

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“…Membrane compartments are affected during HSV-1 infection as well. Examples include the concentration of the endoplasmic reticulum (ER) around the nucleus [9], the alteration of mitochondrial dynamics [10], and the fragmentation of the Golgi apparatus [11].…”

Section: Introductionmentioning

confidence: 99%

Spatiotemporal analysis of host cell modification during herpes simplex virus 1 replication

Scherer

Manton

Soh

et al. 2019

Preprint

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Herpesviruses are large and complex viruses, and have a long evolutionary history with their host species. One poorly understood, but important, area in virus-host interaction is the extensive remodelling of intracellular organelles and morphology that occurs during active viral replication. We have constructed a recombinant reporter virus that allowed us to classify four distinct stages in the infection cycle of herpes simplex virus 1. Single-cell analysis of the impact of the viral load on replication dynamics demonstrates the utility of this timestamping method for tracking the infection cycle. High-resolution microscopy analysis of cellular structures in live and fixed cells in concert with our dual-fluorescent reporter virus have enabled us to generate a detailed overview over the spatial and temporal organisation of the cytoskeleton and organelles during viral replication. This provides the first systems-level analysis of the morphological changes involved in viral replication.Scherer et al.2 / 24

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Section: Introductionmentioning

confidence: 99%

Spatiotemporal analysis of host cell modification during herpes simplex virus 1 replication

Scherer

Manton

Soh

et al. 2019

Preprint

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“…Furthermore, an ER-associated ubiquitin E3 ligase was found to bind to pUL50 and promote UL50-mediated UBE1L degradation. Recently, HSV-1 pUL34, a homolog of HCMV pUL50, was shown to remodel the global ER architecture, including ER compression around the nuclear membrane during virus infection, which may lead to the recruitment of nuclear egress regulators to the nuclear membrane (57). Therefore, it is also an intriguing scenario that the pUL50-triggered remodeling of the ER architecture is responsible for the exposure of UBE1L to ER-associated ubiquitin E3 ligases, such as RNF170.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

Lee

Kim

et al. 2018

J Virol

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Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation. Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

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“…Several viruses are known to affect ER organization. For example, herpes simplex virus 1 (HSV-1) infection results in compression of ER around the nuclear membrane, most likely to facilitate nuclear egress of HSV-1 nucleocapsids (51). SFV infection also affected the late endosomal and lysosomal markers.…”

Section: Discussionmentioning

confidence: 99%

Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

Pietilä

Hemert

Ahola

2018

J Virol

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Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER) and late endosome markers enriched in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the co-fractionation of the RC-carrying plasma membrane and ER suggests that SFV may recruit ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs. Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA and replication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes enriched with the replication complexes demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have here developed a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

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Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells

Cited by 20 publications

References 79 publications

Spatiotemporal analysis of host cell modification during herpes simplex virus 1 replication

Spatiotemporal analysis of host cell modification during herpes simplex virus 1 replication

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

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