Abstract
BackgroundHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV).ResultsBy cleavage activity assessing in vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS.ConclusionsThese results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.
Highlights
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals
We provide direct evidence that exogenous administration of chemically synthesized DNA-based External guide sequences (EGSs) is highly effective in inhibiting HCMV gene expression and growth in human cell culture and, demonstrate the feasibility of using DNA-based EGSs for the studies and treatment of infections caused by human viruses including HCMV
These features include that ? the nucleotides 3' and 5' adjacent to the site of cleavage are a guanosine and a pyrimidine, respectively, and ? a U is 8 nt downstream from this cleavage site. The interactions of these sequence elements with the EGS facilitate the formation of the mRNA-EGS complex into a ptRNA-like structure while those with ribonuclease P (RNase P) are critical for recognition and cleavage by the enzyme [22]
Summary
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. Human cytomegalovirus is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. This virus accounts for one of the most common opportunistic infections in AIDS patients (i.e., CMV retinitis). External guide sequences (EGSs) are antisense oligonucleotides that can be used in conjunction with ribonuclease P (RNase P) or the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) to diminish gene expression [6,7,8,9]. This enzyme catalyzes a hydrolysis reaction to remove the 5' leader sequence of tRNA precursors (ptRNA) by recognizing the common
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
