RNA encapsidation assay.

Analysis of viral RNA encapsidation assay provides a rapid means of assaying which of the progeny RNA are competent for packaging into stable mature virions. Generally, a parallel analysis of total RNA and RNA obtained from purified virions is advisable for accurate interpretation of the results. In this, we describe a series of in vivo assays in which viral RNA encapsidation can be verified. These include whole plants inoculated either mechanically or by Agroinfiltration and protoplasts. The encapsidation assay described here is for an extensively studied plant RNA virus, brome mosaic virus, and can be reliably applied to other viral systems as well as with appropriate buffers. In principle, the encapsidation assay requires purification of virions from either symptomatic leaves or transfected plant protoplasts followed by RNA isolation. The procedure involves grinding the infected tissue in an appropriate buffer followed by a low speed centrifugation step to remove the cell debris. The supernatant is then emulsified with an organic solvent such as chloroform to remove chlorophyll and cellular material. After a low seed centrifugation, the supernatant is subjected to high speed centrifugation to concentrate the virus as a pellet. Depending on the purity required, the partially purified virus preparation is further subjected to sucrose density gradient centrifugation. Following purification of virions, encapsidated RNA is isolated using standard phenol-chloroform extraction procedure. An important step in the encapsidation assay is the comparative analysis of total and virion RNA preparations by Northern hybridization. This would allow the investigator to compare the number of progeny RNA components synthesized during replication vs. encapsidation. Northern blots are normally hybridized with radioactively labeled RNA probes (riboprobes) for specific and sensitive detection of desired RNA species.