Human cytomegalovirus (HCMV) causes one of the most common opportunistic infections in patients with AIDS. Disseminated HCMV infection in these patients is usually associated with gastroenteritis, pneumonia, and sight-threatening retinitis. The emergence of drug-resistant HCMV strains to currently available drugs (e.g. ganciclovir and foscarnet) has posed a need to develop new drugs and novel strategies to combat HCMV infections. This proposal represents our continued effort to develop ribonuclease P (RNase P) ribozyme as gene-targeting agents for therapy of infections by human viruses including HCMV. Recently, we have shown that RNase P ribozyme (M1GS RNA) can cleave the mRNAs coding for the HCMV essential protease (PR) and transcription regulator IE1 and IE2, and block viral gene expression and growth in cells. However, little is known about the mechanism of how RNase P ribozymes achieve efficient cleavage activity under physiological cellular environment. The specificity and antiviral activity of M1GSs in human cells that are clinically relevant to HCMV infections have not been studied. Furthermore, whether M1GS RNA is effective in down-regulating gene expression in animal models in vivo has not been reported. We propose in this research program to address these issues. In the initial part of the study, we will generate ribozyme variants that are highly active under physiological conditions. In vitro biochemical analyses will be carried out to characterize these ribozymes, and their interactions with cellular proteins will be investigated. Then, the engineered ribozymes will be expressed in human cells that are known to be infected by HCMV in vivo, and the antiviral activity of these ribozymes will be determined and their mechanisms in inhibiting viral replication will be investigated. Finally, using murine cytomegalovirus infection of mice as a model system, we will determine whether M1GS RNAs are effective in shutting down viral infection and preventing viral diseases in vivo in animals. These studies will generate novel ribozyme variants that are highly active in cells and can be used for gene-targeting applications. Moreover, these studies will provide insight into the mechanism of how M1GS ribozymes achieve efficient activity in physiological cellular conditions, and will reveal whether RNase P ribozymes are effective in downregulating gene expression in clinically-relevant human cells and in animals. Our proposed research will facilitate the development of M1GS ribozymes as gene-targeting therapeutic agents for treatment of infections by HCMV and other AIDS-associated human viruses.

Public Health Relevance

The objective of the proposed studies is to develop a new method to inhibit specific gene expression, which may be applied in both basic and clinical research, such as treatment of infection of human cytomegalovirus (HCMV), one of the most common opportunistic infections encountered in AIDS patients. This project aims to construct a macromolecule (RNA), termed RNase P ribozyme, which will be introduced into cells and will hydrolyze a specific RNA, e.g. HCMV messenger RNA and the genetic material of HIV which is made of RNA. Our study will facilitate the development of novel macromolecules that can be used as research tools and therapeutic agents for studies and treatment of infections of human viruses including HCMV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041927-14
Application #
8081099
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Lambros, Chris
Project Start
1997-08-01
Project End
2013-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
14
Fiscal Year
2011
Total Cost
$364,807
Indirect Cost
Name
University of California Berkeley
Department
Internal Medicine/Medicine
Type
Schools of Public Health
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Feng, Linyuan; Sheng, Jingxue; Vu, Gia-Phong et al. (2018) Human cytomegalovirus UL23 inhibits transcription of interferon-? stimulated genes and blocks antiviral interferon-? responses by interacting with human N-myc interactor protein. PLoS Pathog 14:e1006867
Li, Wei; Liu, Yujun; Wang, Yuanyuan et al. (2018) Engineered RNase P Ribozymes Effectively Inhibit the Infection of Murine Cytomegalovirus in Animals. Theranostics 8:5634-5644
Zhu, Dihan; Pan, Chaoyun; Sheng, Jingxue et al. (2018) Human cytomegalovirus reprogrammes haematopoietic progenitor cells into immunosuppressive monocytes to achieve latency. Nat Microbiol 3:503-513
Chen, Yuan-Chuan; Sheng, Jingxue; Trang, Phong et al. (2018) Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections. Viruses 10:
Li, Wei; Sheng, Jingxue; Xu, Mengqiong et al. (2017) Inhibition of Murine Cytomegalovirus Infection in Animals by RNase P-Associated External Guide Sequences. Mol Ther Nucleic Acids 9:322-332
Liu, Guoyu; Hai, Rong; Liu, Fenyong (2017) Detection of congenital cytomegalovirus in newborns using nucleic acid amplification techniques and its public health implications. Virol Sin 32:376-386
Sun, Xu; Chen, Weijie; He, Lingling et al. (2017) Inhibition of human cytomegalovirus immediate early gene expression and growth by a novel RNase P ribozyme variant. PLoS One 12:e0186791
Liu, Jin; Shao, Luyao; Trang, Phong et al. (2016) Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences. Sci Rep 6:27068
Lei, Lei; Wang, Wenbiao; Xia, Chuan et al. (2016) Salmonella Virulence Factor SsrAB Regulated Factor Modulates Inflammatory Responses by Enhancing the Activation of NF-?B Signaling Pathway. J Immunol 196:792-802
Pei, Zenglin; Jiang, Xiaohong; Yang, Zhu et al. (2015) Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection. PLoS One 10:e0129276

Showing the most recent 10 out of 44 publications