Mutational analysis of complementary-sense genes of African cassava mosaic virus DNA A

We have investigated the ability of African cassava mosaic virus DNA A mutants, containing disrupted complementary-sense genes, to infect Nicotiana benthamiana and to replicate in Nicotiana tabacum protoplasts. Three overlapping open reading frames (ORFs) with the capacity to encode proteins with an Mr greater than 10K (AC1, AC2 and AC3) are highly conserved between geminiviruses that infect dicotyledonous plants and one (AC4) is less well conserved. Of these, only AC1 is a prerequisite for DNA replication; disruption of this ORF rendered the DNA noninfectious in plants and prevented DNA replication in protoplasts. Disruption of ORF AC2 prevented plant infection but mutants were capable of autonomous replication and replicated DNA B in trans in protoplasts to produce DNA forms that comigrated with wild-type virus DNAs. The AC2 mutant phenotype suggests that the product of this ORF is involved in virus spread within the plant. Mutants in which ORF AC3 had been disrupted retained the ability to replicate and to infect plants systemically although symptom development was delayed and attenuated, and mutant DNA accumulated to much lower levels (10 to 20%) in comparison with wild-type infection. Typical geminate virus particles were observed in extracts of plants infected with ORF AC3 mutants indicating that this gene is not essential for coat protein synthesis or virus assembly but possibly acts by modulating virus levels in infected tissues. Disruption of ORF AC4 had no effect on infectivity or symptom development suggesting that this ORF is maintained only because it overlaps the highly conserved ORF AC1.