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 #
5R01AI041927-15
Application #
8277298
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Lambros, Chris
Project Start
1997-08-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
15
Fiscal Year
2012
Total Cost
$364,467
Indirect Cost
$119,442
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
Jiang, Xiaohong; Sunkara, Naresh; Lu, Sangwei et al. (2014) Directing RNase P-mediated cleavage of target mRNAs by engineered external guide sequences in cultured cells. Methods Mol Biol 1103:45-56
Zeng, Wenbo; Vu, Gia-Phong; Bai, Yong et al. (2013) RNase P-associated external guide sequence effectively reduces the expression of human CC-chemokine receptor 5 and inhibits the infection of human immunodeficiency virus 1. Biomed Res Int 2013:509714
Xia, Chuan; Chen, Yuan-Chuan; Gong, Hao et al. (2013) Inhibition of hepatitis B virus gene expression and replication by ribonuclease P. Mol Ther 21:995-1003
Yang, Zhu; Mao, Guoliang; Liu, Yujun et al. (2013) Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay. Virol Sin 28:24-35
Zhang, Zhigang; Vu, Gia-Phong; Gong, Hao et al. (2013) Engineered external guide sequences are highly effective in inhibiting gene expression and replication of hepatitis B virus in cultured cells. PLoS One 8:e65268
Bai, Yong; Sunkara, Naresh; Liu, Fenyong (2012) Targeting mRNAs by engineered sequence-specific RNase P ribozymes. Methods Mol Biol 848:357-68
Rider, Paul J; Liu, Fenyong (2012) Crosstalk between HIV and hepatitis C virus during co-infection. BMC Med 10:32
Bai, Yong; Gong, Hao; Li, Hongjian et al. (2011) Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice. Proc Natl Acad Sci U S A 108:3222-7
Gong, Hao; Vu, Gia-Phong; Bai, Yong et al. (2011) A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors. PLoS Pathog 7:e1002120
Chen, Yanni; Shen, Ao; Rider, Paul J et al. (2011) A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication. FASEB J 25:4511-21

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