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Immediately early 2 (IE-2) and DNA polymerase SiRNA as virus-specific antiviral against novel transplacental cytomegalovirus strain ALL-03 in vitro

Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, et al.

Infect Genet Evol, 2021 06;90:104783.
PMID: 33640483 DOI: 10.1016/j.meegid.2021.104783

OBJECTIVE: This study investigated the suitability of siRNA targeting specific genes that cause inhibition of virus replication in vitro especially for the virus that capable of crossing placenta and we employed a novel transplacental rat cytomegalovirus that mimics infection in human.
METHODS: Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells.
FINDINGS: Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300 pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P

Matched MeSH terms: Immediate-Early Proteins/genetics; Immediate-Early Proteins/pharmacology*
First report on interferon related developmental regulator-1 from Macrobrachium rosenbergii: bioinformatic analysis and gene expression

Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S

Fish Shellfish Immunol, 2012 May;32(5):929-33.
PMID: 22361112 DOI: 10.1016/j.fsi.2012.02.011

This study reports the first full length gene of interferon related developmental regulator-1 (designated as MrIRDR-1), identified from the transcriptome of Macrobrachium rosenbergii. The complete gene sequence of the MrIRDR-1 is 2459 base pair long with an open reading frame of 1308 base pairs and encoding a predicted protein of 436 amino acids with a calculated molecular mass of 48 kDa. The MrIRDR-1 protein contains a long interferon related developmental regulator super family domain between 30 and 330. The mRNA expressions of MrIRDR-1 in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) infected M. rosenbergii were examined using qRT-PCR. The MrIRDR-1 is highly expressed in hepatopancreas along with all other tissues (walking leg, gills, muscle, haemocyte, pleopods, brain, stomach, intestine and eye stalk). After IHHNV infection, the expression is highly upregulated in hepatopancreas. This result indicates an important role of MrIRDR-1 in prawn defense system.

Matched MeSH terms: Immediate-Early Proteins/genetics*; Immediate-Early Proteins/immunology*; Immediate-Early Proteins/chemistry
Development of a quantitative real-time RT-PCR for kinetic analysis of immediate-early transcripts of rat cytomegalovirus

Loh HS, Mohd-Azmi ML

Acta Virol., 2009;53(4):261-9.
PMID: 19941390

One-step real-time RT-PCR assay was developed for quantification of the immediate-early (IE), namely IE1 and IE2 transcripts of Rat cytomegalovirus (RCMV), strain ALL-03 in rat embryonic fibroblast cells (REF). This in-house SYBR Green I based RT-PCR was shown to have higher amplification efficiency and detection limit as compared to a commercially available real-time RT-PCR kit in quantifying these two transcripts. The quantification histogram revealed the divergence of transcription activities of the two IE genes. The IE1 transcript had a concentration peak at 7 hrs post infection (p.i.), whereas IE2 transcript at 20 hrs p.i. Regulation of IE expression is critical for determination, whether the infection is going to be abortive, lytic or latent. Therefore, this in-house developed quantitative RT-PCR assay offers an alternative for diagnosis and monitoring of the acute cytomegalovirus (CMV) infection directed at IE transcript detection.

Matched MeSH terms: Immediate-Early Proteins/genetics*; Immediate-Early Proteins/metabolism*
Cloning and localization of immediate early response 2 (ier2) gene in the brain of medaka

Moriya S, Chourasia D, Ng KW, Khel NB, Parhar IS

J. Chem. Neuroanat., 2016 11;77:24-29.
PMID: 27134039 DOI: 10.1016/j.jchemneu.2016.04.005

Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3'-, 5'- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.

Matched MeSH terms: Immediate-Early Proteins/biosynthesis; Immediate-Early Proteins/genetics*
Fulltext The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells

Ismail NA, Baines DL, Wilson SM

Eur J Pharmacol, 2014 Jun 05;732:32-42.
PMID: 24657276 DOI: 10.1016/j.ejphar.2014.03.005

Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na(+) channel subunits (α-, β- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser(221), -Ser(327) and -Thr(246). This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na(+) absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na(+) transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na(+) absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser(221)-phosphorylated, Ser(327)-phosphorylated and total Nedd4-2 without altering the abundance of Thr(246)-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, β- and γ-ENaC and had only a very small effect upon electrogenic Na(+) absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser(221)-, Ser(327)- and Thr(246)-phosphorylated and total Nedd4-2; increased the surface abundance of α-, β- and γ-ENaC and evoked a clear stimulation of Na(+) transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na(+) absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.

Matched MeSH terms: Immediate-Early Proteins/metabolism