BROMOVIRUS RNA REPLICATION AND TRANSCRIPTION REQUIRE COMPATIBILITY BETWEEN THE POLYMERASE- AND HELICASE-LIKE VIRAL-RNA SYNTHESIS PROTEINS

The positive-strand RNA bromoviruses encode two nonstructural proteins , 1a and 2a, involved in RNA-dependent RNA replication. These proteins have extensive sequence similarities with methyltransferase, helicase , and polymerase proteins of other plant and animal viruses. 1a and 2a can also form a complex in vitro. To explore whether 1a-2a interactio n is required for RNA replication in vivo, we reassorted the 1a and 2a genes from two different bromoviruses, brome mosaic virus (BMV) and c owpea chlorotic mottle virus (CCMV). 1a and 2a were expressed independ ently of viral replication by using RNA- or DNA-based transient expres sion, and their in vivo RNA replication activities were tested in prot oplasts with BMV and CCMV RNA3 templates. RNA-based transient expressi on confirmed prior indications that bromovirus RNA replication is more sensitive to reductions in 1a expression than to reductions in 2a exp ression. DNA-based expression of the homologous combinations of 1a and 2a supported high levels of RNA synthesis, but both 1a-2a heterologou s combinations exhibited RNA synthesis defects. The combination of CCM V 1a and BMV 2a did not support detectable synthesis of negative-stran d, positive-strand, or subgenomic RNA. The converse combination of BMV 1a and CCMV 2a was preferentially defective in positive-strand and su bgenomic RNA accumulation, showing that 1a-2a interaction is involved in these processes in ways distinct from negative-strand RNA synthesis , which was only slightly affected. These results indicate that at lea st some functions of 1a and 2a operate in a mutually dependent manner in vivo and that the mechanisms of positive- and negative-strand RNA s ynthesis are differentiated in part by features of such interactions.