Molluscum Contagiosum Virus Topoisomerase: Purification, Activities, and Response to Inhibitors

Abstract

Molluscum contagiosum virus (MCV), the only member of theMolluscipoxvirus genus, causes benign papules in healthy people but disfiguring lesions in immunocompromised patients. The sequence of MCV has been completed, revealing that MCV encodes a probable type I topoisomerase enzyme. All poxviruses sequenced to date also encode type I topoisomerases, and in the case of vaccinia virus the topoisomerase has been shown to be essential for replication. Thus, inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have cloned the gene for MCV topoisomerase, overexpressed and purified the protein, and begun to characterize its activities in vitro. Like other eukaryotic type I topoisomerases, MCV topoisomerase can relax both positive and negative supercoils. An analysis of the cleavage of plasmid and oligonucleotide substrates indicates that cleavage by MCV topoisomerase is favored just 3′ of the sequence 5′ (T/C)CCTT 3′, resulting in formation of a covalent bond to the 3′ T residue, as with other poxvirus topoisomerases. We identified solution conditions favorable for activity and measured the rate of formation and decay of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by coumermycin A1 (50% inhibitory concentration, 32 μM) but insensitive to five other previously reported topoisomerase inhibitors. This work provides the point of departure for studies of the mechanism of function of MCV topoisomerase and the development of medically useful inhibitors.

Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes

“…A network of contacts extending from −1 to the +9 region stabilize the catalytic domain in an active conformation (Perry et al, 2006). This is consistent with biochemical studies, which indicated that the topoisomerase when bound covers a considerably larger site than just the conserved 5′-(T/C)CCTT-3′ (Hwang, Burgin, and Bushman, 1999;Hwang et al, 2006;Hwang, Wang, and Bushman, 1998;Sekiguchi and Shuman, 1994). Comparison of the structure of the catalytic domain in the presence and absence of DNA indicates that DNA contacts stabilize a major conformational change which assembles the active site Perry et al, 2006).…”

“…Studies of DNA relaxation by poxvirus topoisomerases in vitro have often used the bacterial plasmid pUC19 as a substrate, and in the course of this work the topoisomerase recognition sites have been carefully quantified (Hwang, Wang, and Bushman, 1998;Shuman, 1991;Shuman and Prescott, 1990). To assess whether the PWM determined above is a reliable predictor of favored sites, we used it to predict sites in pUC19, then compared the calls to experimental results.…”

“…All poxviruses encode an unusual type IB topoisomerase, which is a member of a family that is now known to include enzymes from mimivirus and many bacteria (Benarroch et al, 2006;Gubser et al, 2004;Hwang, Wang, and Bushman, 1998;Krogh and Shuman, 2002;Shuman, 1998;Upton et al, 2003). At about 33 kDa in size, these are by far the smallest topoisomerases known.…”

“…All the poxvirus enzymes studied have been found to act at the core sequence 5′-(T/C)CCTT-3′ (base pairs +5 to +1; see Figure 1 for numbering of the DNA) together with optimal flanking sequences (Hwang, Burgin, and Bushman, 1999;Hwang, Wang, and Bushman, 1998;Shuman and Prescott, 1990;Tian et al, 2004). Purified enzymes have been studied from vaccinia, variola virus, molluscum contagiosum (MCV), leporipox (Palaniyar et al, 1996), and two entomopox viruses (MSV and AMV) (Bauer et al, 1977;Hwang, Rhodes, and Bushman, 2000;Hwang et al, 1999b;Hwang, Wang, and Bushman, 1998;Krogh et al, 1999;Perry et al, 2006;Petersen et al, 1997;Shuman, 1998). These studies have shown that phylogenetically diverse poxviruses encode enzymes with simliar specificities.…”

“…Previous studies have elucidated many key features of the DNA recognition sequence (Hwang, Burgin, and Bushman, 1999;Hwang, Wang, and Bushman, 1998;Shuman, 1991;Shuman and Prescott, 1990;Tian et al, 2004), but a complete functional characterization of the full site has not been reported. Such data is important both for clarifying the contributions of observed topoisomerase-DNA contacts and allowing analysis of topoisomerase sites in poxvirus genomes.…”