The objective of this research is to understand what determines whether a viral infection is lytic or persistent. The mammalian reoviruses have been used as a model system to explore the molecular determinants of viral replication in the cell and viral pathogenesis in the host. Reoviruses are typically cytolytic, but can establish persistent infection in L cells if high-passage (HP) stocks are used to initiate infection. A model has been proposed which suggests that specific gene segments play important roles in reovirus persistence. The L2 gene is important for the generation of mutations during high passage, the S4 gene is important for initiation of persistence, and the S1 gene is important for maintenance of the persistent state. These genes each encode capsid proteins with well-characterized functions. In order to study the mechanism of persistent reovirus infection in cell culture, we will determine the S4 and S1 nucleotide sequences of viruses isolated from independent persistently infected L-cell cultures initiated with HP stocks of wild-type reovirus strain type 3 Dearing (T3D). Changes in the deduced amino acid sequences of the proteins encoded by these genes will be compared to the corresponding T3D sequences in order to develop a model to explain how mutations in S4 and S1 can affect the outcome of viral infection. In addition, we have found that an ammonium chloride (AC) sensitive step in reovirus replication is subject to change during reovirus persistence. Ac acts to inhibit the intralysosomal digestion of virions following receptor-mediated endocytosis. Viruses isolated from persistently infected cultures are resistant to growth inhibition in cells treated with AC, suggesting that modifications of the intralysosomal digestion of virions can regulate the lytic potential of the virus and lead to the establishment of persistent infection. We will conduct experiments to better understand the nature of the AC-sensitive step in reovirus replication by using other reagents which inhibit the acidification of endosomes and lysosomes, and we will determine the genetic basis of AC-resistance through the use of reassortant viruses. Finally, we have found the CNS clearance of viruses isolated from persistently infected cultures is delayed in comparison to T3D, suggesting that there is an in vivo correlate to in vitro persistence. We will investigate the pathology produced by CNS infection with viruses isolated from persistently infected cultures, and we will determine the genetic basis of delayed clearance from the CNS by using reassortant viruses. Furthermore, we will serially passage viruses isolated from persistently infected cultures in the newborn mouse brain in order to determine whether viruses which manifest delayed CNS clearance can be adapted to establish persistence in the animal. Our approach, which utilizes molecular studies of genes known to be altered in reovirus persistence in addition to studies of the pathogenesis of reoviruses isolated from persistently infected cultures, will allow us to gain insight into the molecular basis of viral persistence and to explore the mechanism of viral-mediated cellular injury.
| Danthi, Pranav; Guglielmi, Kristen M; Kirchner, Eva et al. (2010) From touchdown to transcription: the reovirus cell entry pathway. Curr Top Microbiol Immunol 343:91-119 |
