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The herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutant, ts1222, has a defect within the gene specifying the small subunit of ribonucleotide reductase. Sequence determination of the lesion revealed that the mutant DNA had a single base pair deletion at the 3' end of the gene. The mutation altered the translational reading frame such that the codons of all but one of the last 15 amino acids of the protein were changed and the termination codon removed. Although ts1222 did not induce detectable amounts of enzyme activity at both 31 degrees and 39.5 degrees, it replicated as well as wild-type virus at 31 degrees in exponentially growing tissue culture cells under one step growth conditions. At 39.5 degrees, however, ts1222 behaved as a ts mutant. These findings suggest that at low temperatures the virus-coded enzyme is dispensable for virus growth in actively dividing tissue culture cells but at high temperatures the enzyme is essential for virus replication. Under these conditions altered properties of the host cell contribute to the ts phenotype of the mutant. In the presence of hydroxyurea, which inactivates both the cellular and virus ribonucleotide reductases, growth of the mutant at 31 degrees was inhibited more than wild-type virus replication. Growth of the mutant at the permissive temperature was also sensitive to high concentrations of thymidine whereas wild-type virus multiplication was resistant to the nucleoside. It is therefore likely that ts1222 is dependent on the cellular ribonucleotide reductase for growth at this temperature. In serum-starved cells, growth of the mutant virus at 31 degrees was severely impaired. Thus, like thymidine kinase, the HSV-coded ribonucleotide reductase is required for virus multiplication in resting tissue culture cells.

2-Hydroxypropyl-beta-cyclodextrin (HPβCD) has gained recent attention as a potential therapeutic intervention in the treatment of the rare autosomal-recessive, neurodegenerative lysosomal storage disorder Niemann-Pick Disease Type C1 (NPC1). Notably, HPβCD formulations are not comprised of a single molecular species, but instead are complex mixtures of species with differing degrees of hydroxypropylation of the cyclodextrin ring. The degree of substitution is a critical aspect of the complex mixture as it influences binding to other molecules and thus could potentially modulate biological effects. VTS-270 (Kleptose HPB) and Trappsol® Cyclo™ are HPβCD products under investigation as novel treatments for NPC1. The purpose of the present work is to compare these two different products; analyses were based on ion distribution and abundance profiles using mass spectrometry methodology as a means for assessing key molecular distinctions between products. The method incorporated electrospray ionization and analysis with a linear low-field ion mobility quadrupole time-of-flight instrument. We observed that the number of hydroxypropyl groups (the degrees of substitution) are substantially different between the two products and greater in Trappsol Cyclo than in VTS-270. The principal ions of both samples are ammonium adducts. Isotope clusters for each of the major ions show doubly charged homodimers of the ammonium adducts. In addition, both products show doubly charged homodimers from adduction of both a proton and ammonium. Doubly charged heterodimers are also present, but are more intense in Trappsol Cyclo than in VTS-270. Based on the analytical differences observed between VTS-270 and Trappsol Cyclo with respect to the degree of substitution, the composition and fingerprint of the complex mixture, and the impurity profiles, these products cannot be considered to be the same; the potential biological and clinical implications of these differences are not presently known.

An assay for the presence of functional large (RR1) and small (RR2) subunits of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been developed. The system utilizes two temperature-sensitive mutants, ts1207, which has a lesion in RR1, and ts1222, which has a lesion in RR2. In cells infected with ts1207 at 39.5 degrees C, the defective RR1 is unable to associate with RR2 to form an active enzyme, and, as a result, a pool of functional RR2 and defective RR1 accumulates. Evidence presented in this paper suggest that cells infected with ts1222 at either 31 degrees C or 39.5 degrees C accumulate a pool of functional RR1, but do not contain detectable RR2. Virus-specific ribonucleotide reductase activity was produced in cells coinfected with both mutants at 39.5 degrees C, each virus contributing one functional subunit to the holoenzyme. No enzyme activity was detected in cells infected with each mutant alone at this temperature. When partially purified extracts of cells infected with ts1207 at the nonpermissive temperature were mixed with those from ts1222-infected cells, a fully functional enzyme was also formed. These results demonstrate that HSV-1 ribonucleotide reductase activity can be reconstituted both in vivo and in vitro from the nondefective subunits produced by ts1222 and ts1207.

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