Human parvovirus B19 has been epidemiologically identified as the etiologic agent of transient aplastic crisis in hemoglobinopathic patients, i.e. patients with sickle cell diseases or thalassemias, childhood exanthem (fifth disease), polyarthralgia syndrome in adults, and possibly hydrops fetalis. The genome of the B19 virus consists of about 5.4 kb of single-stranded DNA, which encodes at least two capsid proteins of about 84,000 and 58,000 daltons. Although the virus appears to replicate by the mechanism called """"""""self-primed DNA replication"""""""" as proposed for other parvoviruses, details of the DNA replication in general remain undefined. Furthermore, little is known about the regulation of DNA replication in the B19 virus.
The aim of this proposal is to study the mechanism and the regulation of B19 virus DNA replication using virus-infected human bone marrow tissue culture as the experimental model system. To achieve this goal, 1) a detailed time course of the appearance of the viral transcripts and proteins in reference to DNA synthesis will be studied initially by use of gel electrophoreses and blot-analyses. 2) Inhibitors of protein and RNA synthesis will be applied at various times, post infection. Cessation or delay of DNA synthesis caused by the interruption of protein and/or RNA synthesis during the infection should indicate the presence of critical regulatory component(s) for the DNA replication. The inhibited products will be identified from the profile of proteins/RNA deduced from specific aim #1. 3) An in vitro DNA replication system for the B19 virus will be developed. This system will offer an opportunity too fractionate and identify components necessary for B19 viral DNA replication. The in vitro studies will be carried out with both normal and hemoglobinopathic bone marrow cells in order to detect possible difference in their synthetic activities. 4) An attempt will be made to infect beta-thalassemic mice with the B19 virus in order to create a mouse model for aplastic crisis. This mouse model will be used to study pathophysiological features of the disorder. A long term objective of these studies is the development of recombinant parvovirus(es) (either adeno-associated viruses or the B19 virus) containing normal gene(s) for hemoglobin, which may be useful for gene therapy of hemoglobinopathies.
|Ohi, S; Kim, B C (1996) Synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors. J Pharm Sci 85:274-81|