Reverse genetic systems, used to rescue manipulated viral cDNAs into infectious RNA viruses, led to rapid advances in a large number of virus systems because they allow examination of specific mutations in the context of viral infection. The rotaviruses are a group of RNA genome viruses important in human disease for which no reverse genetic system exists. The goal of the experiments proposed here is development of a reverse genetic system for rotaviruses, and its application to questions that cannot be answered without reverse genetics. We have three specific aims: (1) In vitro approaches to reverse genetics for rotaviruses. We will examine methods for """"""""opening"""""""" core particles or virus-like particles, which result in intact particles that have replicase activity. These particles will be used to develop reverse genetic protocols. (2) In vivo approaches to reverse genetics for rotaviruses. Recently, a new model for mRNA segregation between translation and replication was presented that indicates replicated mRNAs never leave the viroplasm. We will investigate methods to introduce exogenous mRNAs into viroplasms, including (a) early introduction of exogenous RNAs into infected cells, (b) the use of tsE(1400) to manipulate the time of viroplasm formation, and (c) the isolation and manipulation of viroplasms to introduce exogenous RNA followed by reintroduction of viroplasms into cells. (3) Proof of principle application of the rotavirus reverse genetic system. We will convert a trypsin dependent virus (SA11 or RRV) to trypsin independence for plaque formation by rescuing segment 4 and VP4 from SA11-4F, a trypsin independent virus. In addition to electrophoretic mobility markers, we will introduce silent mutations into the coding region of SA11-4F VP4 for molecular confirmation of rescue. This gain of function format will allow direct selection of the desired engineered virus. These studies will make the power of reverse genetic analysis available for application to the rotaviruses for the first time. The current absence of reverse genetics renders many important problems impossible to solve. Reverse genetics will stimulate rapid advances in the rotaviruses. Rotaviruses are a significant cause of childhood disease for which no vaccine exists. Reverse genetic methods allow the examination of the effects of specific mutations in viral genes in the context of viral infection. Reverse genetic methods are not available for the rotaviruses. The ability to perform reverse genetics with rotaviruses will facilitate development of a vaccine or antivirals for this disease of childhood. ? ?
| Criglar, Jeanette; Greenberg, Harry B; Estes, Mary K et al. (2011) Reconciliation of rotavirus temperature-sensitive mutant collections and assignment of reassortment groups D, J, and K to genome segments. J Virol 85:5048-60 |
