Francesca Calderazzo

Cell Cycle-dependent Metabolism of Pyrimidine Deoxynucleoside Triphosphates in CEM Cells

We incorporated 3H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G1 and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [3H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-3H]deoxycytidine into dCTP occurred almost exclusively in G1 cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [3H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-3H]Cytidine appeared in dCTP almost to the same extent in G1 as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G1, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631–637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation.

Pseudotyping of Moloney leukemia virus-based retroviral vectors with simian immunodeficiency virus envelope leads to targeted infection of human CD4+ lymphoid cells.

In view of our recent findings that a truncated form of the envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) was efficiently incorporated into MoMLV particles, we studied the generation of Moloney murine leukemia virus (MoMLV)/simian immunodeficiency virus (SIV) pseudotypes. Unlike HIV-1, both the wild-type SIV Env and a truncated form, which lacks most of the cytoplasmic domain of the transmembrane glycoprotein, were incorporated into MoMLV particles and generated infectious retroviral vectors which could transduce CD4+ sMAGI macaque cells. The infection depended on target cell CD4 expression, and was neutralized by both soluble CD4 and sera from SIV-infected macaques. We also observed pseudotype-mediated gene transfer of a green fluorescent protein marker into the CD4+ CEMX174 and C8166 lymphoid cell lines. More importantly, primary human lymphocytes were also successfully transduced ex vivo by MoMLV/SIV pseudotypes, albeit at lower efficiency, and gene transfer was specifically restricted to the CD4+ subset. These findings demonstrate that MoMLV/SIV pseudotypes can be used to transduce cells which are susceptible to SIV infection, and thus might be advantageously employed in animal models for direct in vivo delivery of gene therapy-based approaches.