Previous studies on the biological effects of the 2',3'-dideoxynucleosides (ddNs) have shown that while ddAdo is lethal to E. coli, ddThd has minimal effects on the growth of mammalian cell lines and that it inhibits retrovirus infection of some cell lines but not others. Previous studies have also shown that the 5'-triphosphate of ddThd, ddTTP, selectively inhibits cellular DNA polymerases beta and gamma and retroviral reverse transcriptases. Cellular DNA polymerase alpha is relatively resistant to ddTTP. We have extended these findings to show that the 5'-triphosphates of the other 3 ddNs (ddATP, ddCTP, and ddGTP) affect cellular DNA polymerases alpha, beta, and gamma in the same fashion as does ddTTP. We also show that all four ddNs in concentrations up to 100 microM have negligible effects on the growth of NIH Swiss 3T3 cells. These negligible effects may be due to inefficient intracellular phosphorylation of each nucleoside to the triphosphate. We have determined that, in several different cell lines, ddThd is phosphorylated only at a very slow rate to ddTTP, and in the one cell line tested (monkey CV-1 cells), ddAdo and ddGuo are also poorly phosphorylated. Both ddAdo and ddGuo, and probably ddThd, are converted by CV-1 cells to additional unknown compounds which may have biological activity. The four ddNs display effects of different magnitudes on certain virus infections. Although 30 microM ddThd inhibits herpes simplex I infection of CV-1 cells by 50%, 30 microM ddAdo has no effect. Infection of NIH Swiss 3T3 cells by 334C murine leukemia virus is inhibited 70-80% by ddAdo, ddCyd, and ddThd at 50 microM, but inhibition by 50 microM ddGuo is 100%.

Reverse transcriptase-polymerase chain reaction (RT-PCR) on 24 cerebrospinal fluid (CSF) specimens collected between February and August 1992 detected genome sequence of West Nile (WN) virus in 8 specimens and Japanese encephalitis (JE) virus in a single specimen. The results, combined with the data by IgM-ELISA on CSF indicated that a significant proportion of acute encephalitis cases in Karachi, Pakistan, were caused by WN virus infection, while JE virus caused a small fraction.

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.

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