Dataset Information

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03).

ABSTRACT:

Background

Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy.

Methods

The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.

Results

There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses.

Conclusions

In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.

SUBMITTER: Balakrishnan KN

PROVIDER: S-EPMC7590257 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03).

Balakrishnan Krishnan Nair KN Abdullah Ashwaq Ahmed AA Bala Jamilu Abubakar JA Jesse Faez Firdaus Abdullah FFA Abdullah Che Azurahanim Che CAC Noordin Mustapha Mohamed MM Mohd-Azmi Mohd Lila ML

Virology journal 20201027 1

<h4>Background</h4>Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and ...[more]

PMID: 33109247

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Project description:Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is the major transactivator for viral gene expression and is required for lytic replication. In addition to transcriptional activation, IE2 is known to mediate transcriptional repression of promoters, including the major immediate-early (MIE) promoter and a bidirectional promoter within the lytic origin of replication (oriLyt). The activity of IE2 is modulated by another viral protein, UL84. UL84 is multifunctional and is proposed to act as the origin-binding protein (OBP) during lytic replication. UL84 specifically interacts with IE2 to relieve IE2-mediated repression at the MIE and oriLyt promoters. Originally, UL84 was thought to be indispensable for viral replication, but recent work demonstrated that some strains of HCMV (TB40E and TR) can replicate independently of UL84. This peculiarity is due to a single amino acid change of IE2 (UL122 H388D). Here, we identified that a UL84-dependent (AD169) Δ84 viral mutant had distinct IE2 localization and was unable to synthesize DNA. We also demonstrated that a TB40E Δ84 IE2 D388H mutant containing the reversed IE2 amino acid switch adopted the phenotype of AD169 Δ84. Further functional experiments, including chromatin-immunoprecipitation sequencing (ChIP-seq), suggest distinct protein interactions and transactivation function at oriLyt between strains. Together, these data further highlight the complexity of initiation of HCMV viral DNA replication. IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals and is also the leading viral cause of congenital birth defects. After initial infection, HCMV establishes a lifelong latent infection with periodic reactivation and lytic replication. During lytic DNA synthesis, IE2 and UL84 have been regarded as essential factors required for initiation of viral DNA replication. However, previous reports identified that some isolates of HCMV can replicate in a UL84-independent manner due to a single amino acid change in IE2 (H388D). These UL84-independent strains are an important consideration, as they may have implications for HCMV disease and research. This has prompted renewed interest into the functional roles of IE2 and UL84. The work presented here focuses on the described functions of UL84 and ascertains if those required functions are fulfilled by IE2 in UL84-independent strains.

Project description:The 86-kDa major immediate-early protein, IEP86 (IE2, IE2(579aa), or ppUL122a), from the Towne and AD169 strains of human cytomegalovirus show four amino acid variations, namely, R68Q, K455E, T541A, and seven consecutive serines beginning at position 258 in Towne and eight serines in AD169. A commonly utilized IEP86 cDNA expression clone (herein called the original cDNA) (E. Baracchini, E. Glezer, K. Fish, R. M. Stenberg, J. A. Nelson, and P. Ghazal, Virology 188:518-529, 1992) shows the Towne R68 and seven serines but contains the AD169 E455 and A541 plus two amino acid mutations, M242I and A463T. In transcriptional activation analyses using several promoters, the IEP86 produced by the original cDNA was 40 to 60% less active than wild-type (WT) Towne IEP86, whereas AD169 IEP86 was two to three times more active than WT Towne IEP86. To determine which amino acid variations or mutations accounted for the differences in transcriptional activation, they were individually tested in the WT Towne IEP86 background. K455E, M242I, and the eighth serine had little effect on transcriptional activation or sumoylation when inserted into the Towne background. T541A significantly increased transcriptional activation on all promoters tested and showed increased sumoylation; T541A is the primary reason that WT AD169 IEP86 has increased activity over WT Towne IEP86. The increased sumoylation seen with T541A was quantitatively reduced to WT Towne levels when the K455E alteration was present, suggesting that K455 may be a sumoylation site or that E455 may cause alterations in the IEP86 structure which affect overall sumoylation. A463T was very deleterious to transcriptional activation and caused reduced sumoylation. The A436T mutation in the original cDNA is partially compensated by the presence of the T541A variation. Phosphopeptide mapping suggests that a threonine at 463 or 541 does not introduce a phosphorylation site. However, the A463T mutation does affect phosphorylation at a distant site, suggesting that it alters the conformation of the protein. Promoter-specific effects were noted with some of the amino acid variations, particularly T541A. Structural modeling is presented which suggests how A463T and T541A alter the functional structure of WT Towne IEP86. A hydrophobic core containing A463 is predicted to be responsible for the functional integrity of the carboxy-terminal region of IEP86 between amino acids 344 and 579.

Project description:The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

Dataset Information

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03).

Background

Methods

Results

Conclusions

Publications

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03).

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