The overall goal of this study is to determine whether RNA virus infection and its associated diseases can be prevented and/or terminated by the presence of virus-specific ribozymes. Viral diseases exact an enormous worldwide toll in morbidity and, increasingly, in mortality. However, the list of effective antiviral treatments remains distressingly short. One problem which has contributed to this is specificity; most substances which exert an antiviral effect are toxic also to the host cells in which the virus resides. Thus the development of a general method to prevent and/or treat viral diseases without harm to the host would be of great benefit. Specificity can be achieved by targeting the nucleic acid sequence of the virus: the experiments proposed herein study the feasibility of using ribozymes for this purpose. A ribozyme is an RNA molecule which cleaves RNA in a sequence-specific manner; ribozymes can be designed to cleave at a specific site in essentially any chosen RNA. Therefore by employing these molecules to cleave virus RNAs, while avoiding host sequences, we can potentially overcome the problem of specificity; furthermore the present burgeoning sequence information not only about many viruses, but also about the human genome, can only aid us in the search for specificity. The effects of ribozymes will be studied in a model system using lymphocytic choriomeningitis virus (LCMV). The advantages of this system are severalfold. (1) LCMV is an RNA virus with no DNA stage; all stages of the viral lifecycle are, therefore, vulnerable to ribozyme attack. Hopefully, results obtained from the study of this agent may be successfully extrapolated to other RNA viruses. (2) The complete sequence of the virus genome is known, allowing specific ribozymes to be readily designed. (3) The interactions between the virus and its natural host, the mouse, have been extensively studied and documented; we thus have access to an excellent animal system in which to study in vivo the effects of ribozymes on viral pathogenicity. For example, LCMV can establish both acute and persistent infections: thus the effects of ribozymes on both types of virus infection can be studied. Furthermore, the diseases associated with LCMV infection are well-characterized, and therefore the therapeutic effects of ribozymes can be readily determined. The effects of ribozymes will be studied at three levels. (i) In vitro. What are the criteria for maximal ribozyme effectiveness? Can a system be designed to simultaneously deliver ribozymes of multiple specificities? (ii) In cell culture. Several expression systems will be evaluated. Are ribozymes selected for their efficacy in vitro able to markedly depress viral replication/maturation in cell culture? If so, what is the mechanism underlying this reduction in virus? (iii) In vivo. Ribozymes will be expressed in all mouse tissues, or in selected cell types. What effects do such ribozymes have, on both viral titers, and on virus-induced diseases? The effects of ribozymes upon the virus also will be studied. When a virus grows in the presence of antiviral ribozymes, do ribozyme-resistant viruses emerge? If so, what is the mechanism? Do these resistant viruses have altered pathogenicity? How can the production of resistance be minimized?
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