The evolution of genes within genes and the control of DNA replication in microviruses

Single-stranded DNA(ssDNA) viral life cycles must balance double-stranded DNA (dsDNA) and ssDNA biosynthesis. Previously published in vitro results suggest that microvirus C and host cell SSB proteins play antagonistic roles to achieve this balance. To investigate this in vivo, microvirus DNA replication was characterized in cells expressing cloned C or ssb genes, which would presumably alter the C:SSB protein ratios. Representatives of each microvirus clade (X174, G4, and α3) were used in these studies. α3 DNA replication was significantly more complex. Results suggested that the recognized α3 C gene (C_S: small) is one of two C genes. A larger 5′ extended gene could be translated from an upstream GTG start codon (C_B: big). Wild-type α3 acquired resistance to elevated SSB levels by mutations that exclusively frameshifted the C_B reading frame, whereas mutations in the origin of replication conferred resistance to elevated C protein levels. Expression of either the cloned C_B or C_S gene complemented am(C) mutants, demonstrating functional redundancy. When the C_S start codon was eliminated, strains were only viable if an additional amber mutation was placed in gene C and propagated in an informational suppressing host. Thus, C_B protein likely reaches toxic levels in the absence of C_S translation. This phenomenon may have driven the evolution of the C_S gene within the larger C _B gene and could constitute a unique mechanism of regulation. Furthermore, cross-complementation data suggested that interactions between the α3 C and other viral proteins have evolved enough specificity to biochemically isolate its DNA replication from G4 and X174.