Periodic genome sequences facilitate packaging in a single-stranded DNA virus

ABSTRACT Icosahedral viruses organize and compact their genomes within volumetrically constrained capsids. Double-stranded (ds) DNA viral genomes form ordered spool-like structures when packaged into preformed capsids. By contrast, single-stranded (ss) RNA viral coat proteins recognize genomic sequences or structures, nucleating assembly around folded genomes. A similar mechanism may occur in some ssDNA systems; however, in parvo- and microviruses, the ss genome is concurrently synthesized and packaged into a preformed shell. In the øX174 X-ray virion structure, only ~12% of the genome is ordered. Consequently, the mechanism by which the genome is accommodated within the constrained capsid remains obscure. Sequence motifs within the øX174 genome produce a periodic segmentation pattern consistent with T = 1 icosahedral symmetry. To determine the function of these motifs, a subset within the first five packaged segments was altered. No regulatory elements or encoded amino acids were changed. The resulting mutant, øX DO5 , displayed a phenotype consistent with those of previously characterized packaging mutants. To further understand this phenomenon, the DNA replication-packaging pathway was characterized in cells co-infected with wild-type and øX DO5 . Each genome could both be separately tracked and distinguished throughout the pathway. Early, pre-packaging øX DO5 genome replication was comparable to the wild-type. However, øX DO5 genomes were severely diminished within virions, suggesting a defect in the transition from packaging intermediates to mature virions. IMPORTANCE Concurrent genome biosynthesis and packaging are specific to some families of single-stranded (ss) DNA icosahedral viruses. This evolutionary strategy combines elements found in both dsDNA and ssRNA systems. Like dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. However, in microviruses, such as øX174, these interactions do not facilitate capsid assembly around the genome. They occur after the ss genome enters the preformed procapsid. Sequence motifs within the øX174 genome produce a periodic segmentation pattern consistent with T = 1 icosahedral symmetry. The data provided herein demonstrate that altering these periodic motifs can lead to packaging defects, suggesting an additional level of selective pressure acting on ssDNA genomes.