Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins.
The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.
DeMasi
J
J
Program in Molecular Biology, Weill Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA.
Du
S
S
Lennon
D
D
Traktman
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015342:DNA Probes; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D016615:Telomere; D014616:Vaccinia virus / Q000235:genetics*; D014758:Viral Core Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D015342:DNA Probes; D004268:DNA-Binding Proteins; C109350:I1 protein, vaccinia virus; D014758:Viral Core Proteins; D014764:Viral Proteins