Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.The assembly of adenovirus (Ad) particles has been studied using a large number of viral temperature-sensitive mutants, blocked at different stages of assembly at the restrictive temperature, and by pulse-chase kinetic analyses (7,8,10). The results from these studies suggest that Ad DNA is inserted into preformed empty capsids late in the viral life cycle. Whether the viral genome enters the prohead in association with viral core proteins, or as a separate entity, is unclear. The exact structure of the viral DNA before and during entry also is not known. Ad DNA packaging occurs in a polar fashion from the left end of the genome. Polarity of adenoviral DNA packaging was initially demonstrated by the finding that left end sequences were overrepresented within incomplete viral particles containing DNA of subgenomic length (6, 43). The left ends of Ad type 3 (Ad3), Ad5, and Ad16, representatives of Ad subgroups B and C, harbor conserved packaging signals (15,19,33,36). These conserved cis-acting packaging domains suggest a similar mechanism of selective and polar DNA packaging for all Ad subgroups.Ad5 DNA encapsidation is dependent on cis-acting sequences located on the left end of the genome between nucleotides (nt) 194 and 380 (15, 16, 22, 39). The left end of the Ad5 genome is depicted in Fig. 1A. Through deletion and linker scanning mutagenesis, seven repeats have been identified within this...

Epigenetic information is encoded in the mammalian genome in the form of cytosines methylated at the 5 position. Cytosine methylation has multiple biological effects, but our understanding of these effects has lagged because extant methods for mapping methylation sites genome-wide have severe shortcomings. For instance, the gold standard bisulfite sequencing approach suffers from the use of harsh reaction conditions resulting in DNA cleavage and incomplete conversion of unmethylated cytosine to uracil. We report here on a new photochemical method in which a DNA (cytosine-5)-methyltransferase can be used to covalently attach reactive functionalities which upon irradiation at ∼350 nm initiate photoinduced intramolecular reactions that convert modified C to T analogues. We synthesized a model compound, a cinnamyl ether-containing cytidine derivative, and demonstrated its conversion to a thymidine analogue using mild conditions and a DNA-compatible wavelength (∼350 nm), enabled by the use of a triplet sensitizer, thioxanthone. Transfer of a cinnamyl ether or comparable reactive functionality from an AdoMet analog to cytosine followed by the use of this photoconversion method would require only small amounts of DNA and allow complete methylation profiling on both long and short read sequencing platforms.

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