PHOTOINACTIVATION (365 NM) OF VACCINIA AND HERPES SIMPLEX VIRUSES INDUCED BY A NEW BUILT-IN DNA PHOTOSENSITIZER: 4-THIOTHYMIDINE

The thymidine analogue 4-thiothymidine (s⁴T) strongly absorbs light at wavelengths in the UVA range (Λ_max 335 nm) and we have examined the photoinactivation of vaccinia and herpes simplex viruses grown in the presence of this nucleoside. The cells used in this study (Vero, mouse 1D-TK⁺) were able to grow at the same rate when cultured in the presence of 2 mM s⁴T or 2 mM thymidine, albeit at a slower rate than control cells. Consistent with this finding, viruses grown in the presence of1–4 mM s⁴T were obtained in reduced yield but retained full infectivity. Both viruses were specifically inactivated by irradiation with 365 nm light and their photosensitivity, as measured by the initial slope of the inactivation curve, increased in parallel with the concentration of s⁴T added to the culture medium. More than 90% of vaccinia virus grown in the presence of 4 mM s⁴T was inactivated. Organomercurial agarose chromatography of sheared DNA isolated from vaccinia virus grown in the presence of 2 mM s⁴T showed that approximately 2.5% of DNA fragments were specifically retained, as compared to 0.2% for control DNA. This value corresponds to at least one s4T residue incorporated per 30 000 nucleotides of vaccinia virus DNA. In fact, it is likely that this ratio is actually approximately 10 times higher because of the incomplete retention of control thiolated oligodeoxynucleotides. The incorporation of s⁴T into vaccinia virus DNA was required for photoinactivation as (1) the expression of a viral or cellular thymidine kinase was required to confer photosensitivity, and (2) virus plaque reduction assays revealed that maximal photosensitivity coincided with the first rounds of viral DNA replication. The photo-inactivated virus was unable to induce detectable synthesis of several early proteins after infection of cells. These data show that s⁴T is incorporated into the DNA of vaccinia virus grown in the presence of the analogue and then behaves as a built-in UVA light photosensitizer.