Exceptional Flexibility in the Sequence Requirements for Coronavirus Small Envelope Protein Function

ABSTRACT The small envelope protein (E) plays a role of central importance in the assembly of coronaviruses. This was initially established by studies demonstrating that cellular expression of only E protein and the membrane protein (M) was necessary and sufficient for the generation and release of virus-like particles. To investigate the role of E protein in the whole virus, we previously generated E gene mutants of mouse hepatitis virus (MHV) that were defective in viral growth and produced aberrantly assembled virions. Surprisingly, however, we were also able to isolate a viable MHV mutant (ΔE) in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, were deleted. We have now constructed an E knockout mutant that confirms that the highly defective phenotype of the ΔE mutant is due to loss of the E gene. Additionally, we have created substitution mutants in which the MHV E gene was replaced by heterologous E genes from viruses spanning all three groups of the coronavirus family. Group 2 and 3 E proteins were readily exchangeable for that of MHV. However, the E protein of a group 1 coronavirus, transmissible gastroenteritis virus, became functional in MHV only after acquisition of particular mutations. Our results show that proteins encompassing a remarkably diverse range of primary amino acid sequences can provide E protein function in MHV. These findings suggest that E protein facilitates viral assembly in a manner that does not require E protein to make sequence-specific contacts with M protein.

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Evolved Variants of the Membrane Protein Can Partially Replace the Envelope Protein in Murine Coronavirus Assembly

Journal of Virology ◽

10.1128/jvi.01850-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12872-12885 ◽

Cited By ~ 21

Author(s):

Lili Kuo ◽

Paul S. Masters

Keyword(s):

Membrane Protein ◽

Protein Function ◽

Hepatitis Virus ◽

M Protein ◽

Wild Type Virus ◽

M Gene ◽

E Gene ◽

E Protein ◽

M Proteins ◽

New Gene

ABSTRACT The coronavirus small envelope (E) protein plays a crucial, but poorly defined, role in the assembly of virions. To investigate E protein function, we previously generated E gene point mutants of mouse hepatitis virus (MHV) that were defective in growth and assembled virions with anomalous morphologies. We subsequently constructed an E gene deletion (ΔE) mutant that was only minimally viable. The ΔE virus formed tiny plaques and reached optimal infectious titers many orders of magnitude below those of wild-type virus. We have now characterized highly aberrant viral transcription patterns that developed in some stocks of the ΔE mutant. Extensive analysis of three independent stocks revealed that, in each, a faster-growing virus harboring a genomic duplication had been selected. Remarkably, the net result of each duplication was the creation of a variant version of the membrane protein (M) gene that was situated upstream of the native copy of the M gene. Each different variant M gene encoded an expressed protein (M*) containing a truncated endodomain. Reconstruction of one variant M gene in a ΔE background showed that expression of the M* protein markedly enhanced the growth of the ΔE mutant and that the M* protein was incorporated into assembled virions. These findings suggest that M* proteins were repeatedly selected as surrogates for the E protein and that one role of E is to mediate interactions between transmembrane domains of M protein monomers. Our results provide a demonstration of the capability of coronaviruses to evolve new gene functions through recombination.

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The Small Envelope Protein E Is Not Essential for Murine Coronavirus Replication

Journal of Virology ◽

10.1128/jvi.77.8.4597-4608.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4597-4608 ◽

Cited By ~ 118

Author(s):

Lili Kuo ◽

Paul S. Masters

Keyword(s):

Envelope Protein ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Selection System ◽

Rna Recombination ◽

Wild Type ◽

Phenotypic Changes ◽

E Gene ◽

E Protein ◽

Virion Envelope

ABSTRACT The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (ΔE) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The ΔE mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the ΔE mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.

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Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

Journal of Virology ◽

10.1128/jvi.72.10.7885-7894.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 7885-7894 ◽

Cited By ~ 117

Author(s):

Françoise Fischer ◽

Carola F. Stegen ◽

Paul S. Masters ◽

William A. Samsonoff

Keyword(s):

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Wild Type ◽

E Gene ◽

E Protein ◽

Wild Type Phenotype ◽

Necessary And Sufficient ◽

Targeted Recombination

ABSTRACT Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

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A general mechanism for drug promiscuity: Studies with amiodarone and other antiarrhythmics

The Journal of General Physiology ◽

10.1085/jgp.201511470 ◽

2015 ◽

Vol 146 (6) ◽

pp. 463-475 ◽

Cited By ~ 18

Author(s):

Radda Rusinova ◽

Roger E. Koeppe ◽

Olaf S. Andersen

Keyword(s):

Membrane Proteins ◽

Lipid Bilayer ◽

Protein Function ◽

Single Channel ◽

General Mechanism ◽

Antiarrhythmic Agents ◽

Diverse Range ◽

Lipid Mixtures ◽

Membrane Protein Function

Amiodarone is a widely prescribed antiarrhythmic drug used to treat the most prevalent type of arrhythmia, atrial fibrillation (AF). At therapeutic concentrations, amiodarone alters the function of many diverse membrane proteins, which results in complex therapeutic and toxicity profiles. Other antiarrhythmics, such as dronedarone, similarly alter the function of multiple membrane proteins, suggesting that a multipronged mechanism may be beneficial for treating AF, but raising questions about how these antiarrhythmics regulate a diverse range of membrane proteins at similar concentrations. One possible mechanism is that these molecules regulate membrane protein function by altering the common environment provided by the host lipid bilayer. We took advantage of the gramicidin (gA) channels’ sensitivity to changes in bilayer properties to determine whether commonly used antiarrhythmics—amiodarone, dronedarone, propranolol, and pindolol, whose pharmacological modes of action range from multi-target to specific—perturb lipid bilayer properties at therapeutic concentrations. Using a gA-based fluorescence assay, we found that amiodarone and dronedarone are potent bilayer modifiers at therapeutic concentrations; propranolol alters bilayer properties only at supratherapeutic concentration, and pindolol has little effect. Using single-channel electrophysiology, we found that amiodarone and dronedarone, but not propranolol or pindolol, increase bilayer elasticity. The overlap between therapeutic and bilayer-altering concentrations, which is observed also using plasma membrane–like lipid mixtures, underscores the need to explore the role of the bilayer in therapeutic as well as toxic effects of antiarrhythmic agents.

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Exploring the role of identity fusion and group identification in predicting parochialism amongst Indonesian Islamic groups

10.31234/osf.io/e8ytr ◽

2019 ◽

Author(s):

Christopher Michael Kavanagh ◽

Susilo Wibisono ◽

Rohan Kapitány ◽

Whinda Yustisia ◽

Idhamsyah Eka Putra ◽

...

Keyword(s):

Group Identification ◽

Control Sample ◽

Theoretical Models ◽

Religious Groups ◽

Diverse Range ◽

Beliefs And Practices ◽

Identity Fusion ◽

Beliefs And Practice ◽

Fusion Theory

Indonesia is the most populous Islamic country and as such is host to a diverse range of Islamic beliefs and practices. Here we examine how the diversity of beliefs and practices among Indonesian Muslims relates to group bonding and parochialism. In particular, we examine the predictive power of two distinct types of group alignment, group identification and identity fusion, among individuals from three Sunni politico-religious groups - a fundamentalist group (PKS), a moderate group (NU), and a control sample of politically unaffiliated citizens. Fundamentalists were more fused to targets than moderates or citizens, but contrary to fusion theory, we found across all groups, that group identification (not fusion) better predicted parochialism, including willingness to carry out extreme pro-group actions. We discuss how religious beliefs and practice impact parochial attitudes, as well as the implications for theoretical models linking fusion to extreme behaviour.

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PPM1G promotes the progression of hepatocellular carcinoma via phosphorylation regulation of alternative splicing protein SRSF3

Cell Death and Disease ◽

10.1038/s41419-021-04013-y ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Dawei Chen ◽

Zhenguo Zhao ◽

Lu Chen ◽

Qinghua Li ◽

Jixue Zou ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Alternative Splicing ◽

Protein Function ◽

Vital Role ◽

Normal Tissues ◽

Mechanistic Analysis ◽

Highly Correlated ◽

Splicing Patterns

AbstractEmerging evidence has demonstrated that alternative splicing has a vital role in regulating protein function, but how alternative splicing factors can be regulated remains unclear. We showed that the PPM1G, a protein phosphatase, regulated the phosphorylation of SRSF3 in hepatocellular carcinoma (HCC) and contributed to the proliferation, invasion, and metastasis of HCC. PPM1G was highly expressed in HCC tissues compared to adjacent normal tissues, and higher levels of PPM1G were observed in adverse staged HCCs. The higher levels of PPM1G were highly correlated with poor prognosis, which was further validated in the TCGA cohort. The knockdown of PPM1G inhibited the cell growth and invasion of HCC cell lines. Further studies showed that the knockdown of PPM1G inhibited tumor growth in vivo. The mechanistic analysis showed that the PPM1G interacted with proteins related to alternative splicing, including SRSF3. Overexpression of PPM1G promoted the dephosphorylation of SRSF3 and changed the alternative splicing patterns of genes related to the cell cycle, the transcriptional regulation in HCC cells. In addition, we also demonstrated that the promoter of PPM1G was activated by multiple transcription factors and co-activators, including MYC/MAX and EP300, MED1, and ELF1. Our study highlighted the essential role of PPM1G in HCC and shed new light on unveiling the regulation of alternative splicing in malignant transformation.

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New Insights on the Role of TRP Channels in Calcium Signalling and Immunomodulation: Review of Pathways and Implications for Clinical Practice

Clinical Reviews in Allergy & Immunology ◽

10.1007/s12016-020-08824-3 ◽

2021 ◽

Author(s):

Saied Froghi ◽

Charlotte R. Grant ◽

Radhika Tandon ◽

Alberto Quaglia ◽

Brian Davidson ◽

...

Keyword(s):

Immune System ◽

Calcium Release ◽

Transient Receptor Potential ◽

Trp Channels ◽

Calcium Influx ◽

Calcium Signalling ◽

Chronic Fatigue ◽

Diverse Range ◽

Immune System Activation

AbstractCalcium is the most abundant mineral in the human body and is central to many physiological processes, including immune system activation and maintenance. Studies continue to reveal the intricacies of calcium signalling within the immune system. Perhaps the most well-understood mechanism of calcium influx into cells is store-operated calcium entry (SOCE), which occurs via calcium release-activated channels (CRACs). SOCE is central to the activation of immune system cells; however, more recent studies have demonstrated the crucial role of other calcium channels, including transient receptor potential (TRP) channels. In this review, we describe the expression and function of TRP channels within the immune system and outline associations with murine models of disease and human conditions. Therefore, highlighting the importance of TRP channels in disease and reviewing potential. The TRP channel family is significant, and its members have a continually growing number of cellular processes. Within the immune system, TRP channels are involved in a diverse range of functions including T and B cell receptor signalling and activation, antigen presentation by dendritic cells, neutrophil and macrophage bactericidal activity, and mast cell degranulation. Not surprisingly, these channels have been linked to many pathological conditions such as inflammatory bowel disease, chronic fatigue syndrome and myalgic encephalomyelitis, atherosclerosis, hypertension and atopy.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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