scholarly journals Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Anthony Khong ◽  
Craig H. Kerr ◽  
Clarence H. L. Yeung ◽  
Kathleen Keatings ◽  
Arabinda Nayak ◽  
...  
Keyword(s):  
Virus Infection ◽  
Stress Granule ◽  
Granule Formation ◽  
Viral Translation ◽  
Content Type ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Viral Yield ◽  
Infected Cells ◽  
Paralysis Virus

ABSTRACT Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication. IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.

scholarly journals The Leader Protein of Theiler's Virus Prevents the Activation of PKR

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Fabian Borghese ◽  
Frédéric Sorgeloos ◽  
Teresa Cesaro ◽  
Thomas Michiels
Keyword(s):  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Granule Formation ◽  
Double Stranded Rna ◽  
Eif2α Phosphorylation ◽  
Leader Protein ◽  
Infected Cells ◽  
L Proteins

ABSTRACT Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

scholarly journals Stable Formation of Compositionally Unique Stress Granules in Virus-Infected Cells

2009 ◽  
Vol 84 (7) ◽  
pp. 3654-3665 ◽  
Author(s):  
Joanna Piotrowska ◽  
Spencer J. Hansen ◽  
Nogi Park ◽  
Katarzyna Jamka ◽  
Peter Sarnow ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Viral Infections ◽  
De Novo ◽  
Stress Granules ◽  
Stress Granule ◽  
Initiation Factor ◽  
Granule Formation ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACT Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.

scholarly journals The 5′ Untranslated Region of a Novel Infectious Molecular Clone of the Dicistrovirus Cricket Paralysis Virus Modulates Infection

2015 ◽  
Vol 89 (11) ◽  
pp. 5919-5934 ◽  
Author(s):  
Craig H. Kerr ◽  
Qing S. Wang ◽  
Kathleen Keatings ◽  
Anthony Khong ◽  
Douglas Allan ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Molecular Mechanisms ◽  
Insect Cells ◽  
Infectious Clone ◽  
Virus Infectivity ◽  
Viral Translation ◽  
Content Type ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Paralysis Virus

ABSTRACTDicistroviridaeare a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species,Cricket paralysis virus(CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection ofin vitro-transcribed RNA from the CrPV clone intoDrosophilaSchneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adultDrosophilaflies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5′ untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells.IMPORTANCEDicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistrovirus, cricket paralysis virus (CrPV). We show that transfection of the CrPV clone RNA intoDrosophilacells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects and expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistrovirus life cycle and host-virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5′ untranslated region of the CrPV clone increased viral translation in reporter constructs but decreased virus infectivity, thus revealing a balance that interplays between viral translation and replication.

scholarly journals Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Keisuke Nakagawa ◽  
Krishna Narayanan ◽  
Masami Wada ◽  
Shinji Makino
Keyword(s):  
Middle East ◽  
Virus Replication ◽  
Stress Responses ◽  
Middle East Respiratory Syndrome ◽  
Stress Granule ◽  
Accessory Protein ◽  
Viral Mrnas ◽  
Translation Arrest ◽  
Viral Translation ◽  
Infected Cells

ABSTRACTStress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. The MERS-CoV 4a accessory protein is known to inhibit SG formation in cells in which it was expressed by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-Δp4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-Δp4-induced SG formation. MERS-CoV-Δp4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells, and inhibition of SG formation by small interfering RNA-mediated depletion of the SG components promoted MERS-CoV-Δp4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-Δp4 replication was not due to either the induction of type I and type III interferons or the accumulation of viral mRNAs in the SGs. Rather, it was due to the inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2α phosphorylation and likely by the confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of the 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication.IMPORTANCEMiddle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that the MERS-CoV 4a accessory protein, which was reported to block SG formation in cells in which it was expressed, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this report is the first to show the biological significance of SG in CoV replication and provides insight into the interplay between MERS-CoV and antiviral stress responses.

scholarly journals Ubiquitination is essential for recovery of cellular activities after heat shock

Science ◽  
2021 ◽  
Vol 372 (6549) ◽  
pp. eabc3593
Author(s):  
Brian A. Maxwell ◽  
Youngdae Gwon ◽  
Ashutosh Mishra ◽  
Junmin Peng ◽  
Haruko Nakamura ◽  
...  
Keyword(s):  
Heat Stress ◽  
Cultured Cells ◽  
Nucleocytoplasmic Transport ◽  
Stress Granule ◽  
Eukaryotic Cells ◽  
Granule Formation ◽  
Cellular Processes ◽  
Specific Proteins ◽  
Global Increase ◽  
As Stress

Eukaryotic cells respond to stress through adaptive programs that include reversible shutdown of key cellular processes, the formation of stress granules, and a global increase in ubiquitination. The primary function of this ubiquitination is thought to be for tagging damaged or misfolded proteins for degradation. Here, working in mammalian cultured cells, we found that different stresses elicited distinct ubiquitination patterns. For heat stress, ubiquitination targeted specific proteins associated with cellular activities that are down-regulated during stress, including nucleocytoplasmic transport and translation, as well as stress granule constituents. Ubiquitination was not required for the shutdown of these processes or for stress granule formation but was essential for the resumption of cellular activities and for stress granule disassembly. Thus, stress-induced ubiquitination primes the cell for recovery after heat stress.

scholarly journals Dynamic Oscillation of Translation and Stress Granule Formation Mark the Cellular Response to Virus Infection

2012 ◽  
Vol 12 (1) ◽  
pp. 71-85 ◽  
Author(s):  
Alessia Ruggieri ◽  
Eva Dazert ◽  
Philippe Metz ◽  
Sarah Hofmann ◽  
Jan-Philip Bergeest ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cellular Response ◽  
Stress Granule ◽  
Granule Formation

scholarly journals Why Cells and Viruses Cannot Survive without an ESCRT

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 483
Author(s):  
Arianna Calistri ◽  
Alberto Reale ◽  
Giorgio Palù ◽  
Cristina Parolin
Keyword(s):  
Endosomal Sorting ◽  
Cellular Processes ◽  
Escrt Machinery ◽  
Cellular Environment ◽  
Intracellular Parasites ◽  
Cell Life ◽  
Infected Cells ◽  
Intracellular Organelles ◽  
Simplex Virus ◽  
Hsv 1

Intracellular organelles enwrapped in membranes along with a complex network of vesicles trafficking in, out and inside the cellular environment are one of the main features of eukaryotic cells. Given their central role in cell life, compartmentalization and mechanisms allowing their maintenance despite continuous crosstalk among different organelles have been deeply investigated over the past years. Here, we review the multiple functions exerted by the endosomal sorting complex required for transport (ESCRT) machinery in driving membrane remodeling and fission, as well as in repairing physiological and pathological membrane damages. In this way, ESCRT machinery enables different fundamental cellular processes, such as cell cytokinesis, biogenesis of organelles and vesicles, maintenance of nuclear–cytoplasmic compartmentalization, endolysosomal activity. Furthermore, we discuss some examples of how viruses, as obligate intracellular parasites, have evolved to hijack the ESCRT machinery or part of it to execute/optimize their replication cycle/infection. A special emphasis is given to the herpes simplex virus type 1 (HSV-1) interaction with the ESCRT proteins, considering the peculiarities of this interplay and the need for HSV-1 to cross both the nuclear-cytoplasmic and the cytoplasmic-extracellular environment compartmentalization to egress from infected cells.

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