Analysis of a Non-coding RNA in Ectromelia Virus

Emily Berger, Vassar College ’10 and Prof. David Esteban

Ectromelia virus (ECTV) is an Orthopoxvirus and the causative agent of mousepox. Other members of the poxvirus family include Variola virus, which causes smallpox in humans, Vaccinia virus, which was used in the smallpox vaccine of the 19th and 20th centuries, and Cowpox virus, which was used in the development of the first known vaccine in 1796. Its close similarities to the unavailable Variola virus make ECTV a desirable model for the study of poxviruses.

The genome of ECTV encodes a recently identified non-coding RNA. That is, the gene is transcribed into a hairpin RNA, but is not translated into protein. The purpose of this study was to analyze the gene to determine its function and expression patterns in viral infection and replication. To do this, a knockout mutant virus was created, replacing the stem-loop structure of the RNA with a red fluorescent protein. BSC-1 cell cultures were infected with wildtype ECTV also containing a red fluorescent protein, as well as the knockout mutant ECTV. As viral infection spreads in a confluent cell culture, infected cells release viral progeny, which subsequently infect neighboring cells. The size of these circular zones of infected cells, referred to as plaques, is used to assay the replication of a virus. Fluorescence microscopy was used to characterize the morphology of plaques from both viral infections. Mutant virus lacking the hairpin RNA was seen to be defective in replication or transmission, resulting in smaller plaques. PCR is currently being performed on viral DNA from the mutant ECTV infections. This DNA will be sequenced to confirm the identity of the virus sample as the mutant genotype. Reverse transcriptase PCR (RT-PCR) was used to determine the presence of a transcript of the gene of interest at specific time points during the course of infection. The size of DNA fragments detected from samples isolated at various times during infection can be used to identify the RNA as an early or late transcript, meaning the gene is expressed by the virus either early or late in the infection cycle. The knowledge gained by determining the function of the non-coding RNA in the Ectromelia genome may be applicable to related viruses, giving us a greater understanding of the infection and replication cycles of poxviruses in general.