Fig 3 -

(A) Hcmv-miR-UL148D targets ERN1 through 3’UTR binding: (A) HEK293T cells were transfected with hcmv-miR-UL148D mimic or hcmv-miR-UL148D mimic and inhibitor together. RNA was isolated and the ectopic expression level of hcmv-miR-UL148D was measured through qRT-PCR, the miRNA levels were normalized to 5s rRNA in the corresponding samples. Result represents the mean ± SEM (n = 3 independent experiments) ****, p(B) hcmv-miR-UL148D downregulates the ERN1 mRNA expression. HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor followed by staurosporine treatment. RNA was isolated and expression of ERN1 was quantified and compared with respect to untreated cells. GAPDH was used as a housekeeping control and data plotted as a measure of relative expression. Results represent the mean ± SEM (n = 3 independent experiments) **p(C) Cartoonistic representation of pEZX-MT06-3’UTRWT/DEL dual-luciferase reporter vector, highlighting the binding site of hcmv-miR-UL148D. (D) hcmv-miR-UL148D targets the ERN1 3’UTR. HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor along with either pEZX-MT06-3’UTRWT-ERN1 or pEZX-MT06-3’UTRDEL-ERN1 luciferase reporter construct. Luciferase activity was measured 24 hr post transfection. Unlike the wild-type, the luciferase activity of the mutant pEZX-MT06-3’UTRDEL-ERN1 was not inhibited by hcmv-miR-UL148D.</p

hcmv-miR-UL148D inhibits IRE1α protein level in staurosporine treated HEK293T cells.

(A) The IRE1α protein levels were analyzed in different groups of cells transfected with hcmv-miR-UL148D, hcmv-miR-UL148D and its inhibitor followed by Staurosporine treatment. The IRE1α protein and the β-actin levels were examined through western blot. The bands of the blots have been cropped with no further manipulation. (B) Relative IRE1α protein levels in different groups of cells were quantified through ImageJ software. The IRE1α levels were normalized to β-actin and plotted as fold change with respect to control. Results represent the mean ± SEM (n = 3 independent experiments) **p<0.01; ***p<0.001.</p

Human Cytomegalovirus miR-UL70-3p Downregulates the H2O2-Induced Apoptosis by Targeting the Modulator of Apoptosis-1 (MOAP1)

Human Cytomegalovirus (HCMV) is a prototypic beta herpesvirus, causing persistent infections in humans. There are medications that are used to treat the symptoms; however, there is no cure yet. Thus, understanding the molecular mechanisms of HCMV replication and its persistence may reveal new prevention strategies. HCMV evasive strategies on the antiviral responses of the human host largely rely on its significant portion of genome. Numerous studies have highlighted the importance of miRNA-mediated regulation of apoptosis, which is an innate immune mechanism that eradicates virus-infected cells. In this study, we explore the antiapoptotic role of hcmv-miR-UL70-3p in HEK293T cells. We establish that hcmv-miR-UL70-3p targets the proapoptotic gene Modulator of Apoptosis-1 (MOAP1) through interaction with its 3&rsquo;UTR region of mRNA. The ectopic expression of hcmv-miR-UL70-3p mimic significantly downregulates the H2O2-induced apoptosis through the translational repression of MOAP1. Silencing of MOAP1 through siRNA also inhibits the H2O2-induced apoptosis, which further supports the hcmv-miR-UL70-3p mediated antiapoptotic effect by regulating MOAP1 expression. These results uncover a role for hcmv-miR-UL70-3p and its target MOAP1 in regulating apoptosis