We study processes that lead to replication of RNA viruses and their interactions with the host. In one project, we concentrate on the study of poliovirus translation, RNA replication, and genome packaging. We have focused on a ribonucleoprotein complex that forms at the 5'-end of the viral RNA. This complex is composed of the first 100 nucleotides of the genomic RNA, a viral-encoded protein, and a ribosomal-associated cellular factor. The structural details and specific function of the complex have yet to be defined. Mutations that disrupt the formation of this complex reduce the synthesis of positive strand viral RNA, and also greatly affect translation. To analyze the function of this complex in vivo, we have developed a system in which to study poliovirus replication. Microinjection of viral RNA into Xenopus laevis oocytes initiates a complete and authentic viral replication cycle, which yields a high level of infectious viruses, but only if polioviral RNA is co-injected with factors present in HeLa cells. Two additional mammalian cell factors are required for viral replication in oocytes: one involved in initiation of translation and the other in RNA synthesis. Thus, microinjection in oocytes can be used, essentially, as an in vitro system in which to identify and further analyze the function of viral and cellular factors and biochemically dissect mechanisms of poliovirus synthesis.

More recently, we have also started to examine the role of genetic diversity on a virus population and its consequences for virus fitness, adaptation and pathogenesis. The quasispecies hypothesis postulates that genomic variation in RNA virus populations enhances fitness by facilitating adaptation to changing environments encountered during infection. To test this theory, we examined whether limiting genomic diversity affects viral fitness and pathogenesis. A variant of the poliovirus RNA-dependent RNA polymerase (3D pol ) that enhances replication fidelity reduces diversity within the virus population. The high fidelity mutant replicates at wildtype levels but is unable to adapt to adverse growth conditions. In infected animals, the reduced genomic diversity led to restricted tissue tropism and an attenuated pathogenic phenotype. Importantly, expanding the quasispecies of the high fidelity mutant by chemical mutagenesis prior to infection restored tissue tropism and pathogenesis to wildtype levels. This study provides direct evidence for the quasispecies theory and establishes a compelling link between mutation rate, population fitness and pathogenesis.

Selected References

Gitlin, L., Karelsky, S., and Andino, R. (2002) Short interfering RNA confers intracellular antiviral immunity in human cells. Nature, 418: 430-434.

Vignuzzi M, Stone JK, Andino R. (2005) Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications. Virus Res, 107: 173-81.

Gitlin, L., Stone, J.K., and Andino, R. ( 2005) Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches. J Virol, 79: 1027-35.

Web Site: http://www.ucsf.edu/andino