Nucleotide Pools and the Replication of Sindbis Virus

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DESCRIPTION (provided by applicant): Close to 400 arthropod-borne viruses (arboviruses) have been identified, many of which cause serious human disease. Sindbis virus (SV), a mosquito-transmitted arbovirus, and the prototype virus of the family Togaviridae, genus aiphavirus, is the subject of this proposal. The intracellular rNTP's are the vital building blocks from which the SV RNA polymerase synthesizes the viral RNA. Yet, little is known concerning how the manipulation of the levels of theses substrates can affect the replication of SV, or for that matter of other cytoplasmic RNA viruses. Neither have there been any studies indicating how infection with an alphavirus affects the nucleotide pools in either a mosquito or vertebrate host cell. We have shown that pyrazofurin (PZF), a compound that prevents the synthesis of pyrimidine nucleotides inhibits the replication of SV in mosquito cells. Subsequently, we isolated a mutant of SV, SV VZF' which in contrast to the parental virus, is able to grow in PZF-treated mosquito cells; we then showed, as we expected, that mutations responsible for the resistance to PZF mapped to the sequence encoding nsP4 (the viral RNA polymerase). We shall compare the RNA polymerases encoded by SVPZF and by the parental virus, in order to test whether the former has a lower Km (higher affinity) for UTP and CTP. We also found that SVPZF has a second phenotype. It is restricted in BHK cells, but the restriction is relieved by adenosine. The restriction is associated with a decrease in the synthesis of subgenomic (SG) RNA. A single mutation is sufficient to cause the restriction phenotype, but this single mutation changes both an amino acid in nsP4 and the sequence of the SG promoter. Experiments are proposed to explain the restriction of SVPZF in BHK cells, and to evaluate the relative roles of the changes in nsP4 and the SG promoter in producing this phenotype. Some of these experiments will involve the use of double SG virus, and SV replicons. We shall also test whether the amino acid changes in nsP4, which we believe modify the affinity of the viral RDRP for UTP and CTP, affect the fidelity of the viral RDRP. Finally, we shall seek mutants of SV, isolated on the basis of their ability to grow in mosquito cells depleted only of CTP, or only of UTP, and compare their properties with those of SVPZF. The proposed studies will inform us for the first time as to how the size of the rNTP pools in the host cell can affect the activity of the viral RNA polymerase, and how a viral RNA polymerase can change so that it can function efficiently in the face of decreased levels of rNTP' s. These studies will also lead to a better understanding of the factors that regulate the synthesis of SG RNA. What we will learn will have practical application since alphavirus expression systems are being well developed in which foreign genes are expressed from the SG promoter. Our findings will also have application for the development of antiviral therapy.