Roy C. French

Molecular studies of brome mosaic virus using infectious transcripts from cloned cDNA.

Publisher Summary Development of a convenient and powerful recombinant DNA technology has led to considerable interest among RNA virologists in directly or indirectly infectious cDNA clones of RNA viruses. Such clones allow unambiguous, stable isolation of a genetically well-defined virus type from heterogeneous and potentially unstable virus populations and provide a reliable means of separating different physical components of a divided RNA genome. Most importantly, such clones provide a practical means of introducing designed alterations into RNA genomes, including the alterations that would be conditionally or unconditionally lethal in the virus itself. This ability can be used as the basis for a wide range of informative molecular genetic studies, previously not accessible to RNA virology. At the date of this writing, all RNA viruses for which infection has been successfully expressed from cloned cDNA encapsidate single-stranded messenger-sense RNAs, which are directly infectious in the absence of any viral proteins. Negative- and double-stranded RNA viruses, which require presynthesized viral proteins, as well as RNA for infectivity, may demand somewhat sophisticated strategies for expression from cloned cDNA.

Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo.

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.