| Vaccinia virus, the prototypic member of the Poxvirus family, contains a 192-kb double-stranded DNA genome encoding approximately 200 proteins involved in DNA replication, gene expression, morphogenesis and virus/host interaction. The genome is a linear duplex with covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A/T-rich duplex stem that has twelve extrahelical bases, 10 on one strand and 2 on the other. The extrahelical bases are maintained during replication and are conserved in all poxviruses, suggesting that they play an important function in the viral life cycle. The telomeric hairpins form distinct complexes with proteins present in infected cells and in virions, and these interactions require the presence of extrahelical bases. The proteins responsible for complex formation with the hairpins were isolated and identified as vaccinia-encoded I1 and I6 proteins. Of the two, the I6 protein showed the greatest specificity and affinity for interaction with the viral hairpins. To gain insight into the role of I6 in vivo, clustered-charge-to-alanine mutagenesis and transient dominant selection were used to generate eight viruses bearing altered I6 alleles that might engender a temperature-sensitive (ts ) phenotype. All eight recombinant viruses (denoted as vI6-1, -2, -4, -5, -6, -9, -12 and -13) were isolated and were viable at 32°C, the permissive temperature. Of these, vI6-1, -2 and -13 formed small plaques and showed a decrease in viral yield at both permissive and non-permissive temperature. The I6-12 allele (tsI6-12) conferred a tight temperature-sensitive phenotype and was thus used to examine the stage(s) of the viral life cycle that were affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, concomitant with virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature virions, were observed. Instead, aberrant electron-dense virions and large crystalloids, most likely representing aggregated viral DNA, were seen. When purified, these aberrant tsI6-12 particles were found to have a normal protein content but to be devoid of viral DNA, indicative of a defect in DNA encapsidation. The I6-12 protein is stable during tsI6-12 infection, but the ability of this protein to bind viral hairpins is compromised, suggesting that the telomere-binding activity of I6 is essential for genome packaging. The tsI6-12 phenotype is virtually identical to that seen using vA32i-IPTG, a recombinant vaccinia virus in which the expression of A32 is repressed in the absence of IPTG. A32 is a putative ATPase with limited sequence homology to the DNA packaging proteins encoded by adenovirus and filamentous bacteriophages. A32 and I6-12 proteins were both found in purified tsI6-12 particles. However, in A32-deficient particles, no I6 protein was detected, implying that encapsidation of 16 is dependent on encapsidation of A32. Based on these data, we propose that I6 binds viral telomeres by virtue of extrahelical base recognition and directs the DNA into the virus particles through interactions with A32. |
