Chemical methylation of RNA and DNA viral genomes as a probe of in situ structure.
We used [methyl-(3)H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for phiX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [methyl-(3)H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, phiX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation.
D000225:Adenine / Q000378:metabolism; D000260:Adenoviruses, Human / Q000235:genetics; D001358:Avian Sarcoma Viruses / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D005814:Genes, Viral; D007304:Insect Viruses / Q000235:genetics; D012089:Mammalian orthoreovirus 3 / Q000235:genetics; D008745:Methylation; D009029:Mosaic Viruses / Q000235:genetics; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D013463:Sulfuric Acid Esters; D014616:Vaccinia virus / Q000235:genetics
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D013463:Sulfuric Acid Esters; D012313:RNA; D000225:Adenine; C007482:dimethyl sulfate