Watson A, Wilburn W, Ho HT, Datema R; International Conference on AIDS.
Int Conf AIDS. 1991 Jun 16-21; 7: 98 (abstract no. W.A.1027).
Bristol Myers Squibb, Seattle, WA, USA
OBJECTIVE: Human Immunodeficiency virus (HIV-1) infection of resting, quiescent CD4 lymphocytes can lead to production of unintegrated, proviral DNA which becomes integrated into the host genome to produce infectious virions after mitotic stimulation. In vivo, quiescent PBL predominate and represent a frequent potential target for HIV infection. Inhibition of HIV infection by nucleosides requires conversion of these compounds to triphosphates by cellular kinases. We show here that AZT and D4T, whose phosphorylation is dependent upon thymidine kinase, are not phosphorylated in quiescent PBL. On the other hand, phosphorylation of the ddI analogs FddI and FddA is observed in both quiescent and PHA stimulated PBL. The overall objective of these studies is to correlate cellular nucleoside activation levels to the inhibition of HIV DNA synthesis. To accomplish this a drug evaluation assay using PCR has also been developed. METHODS: Activation of nucleosides by PBL is measured by SAX HPLC of a 60% methanol cell extract using conventional procedures. PCR Assay: Circulating PBL from normal donors are depleted of B cells and monocytes and infected with HIV-1 (LAV strain) in the presence or absence of nucleosides. Following 16hr to 24hr incubation, DNA preparations are made. HIV DNA is detected using SK38 and SK39 primers to amplify a 115 nucleotide fragment of HIV gag. Identity of the amplified fragment is confirmed using an end-labelled gag-specific probe. RESULTS: Quiescent PBL are able to phosphorylate both FddI and FddA to their triphosphates. The thymidine kinase-dependent nucleosides, AZT and D4T, are not phosphorylated in resting cells but are in PHA activated PBL. PCR assays of resting PBL exposed to HIV in the presence or absence of drugs showed that new HIV DNA synthesis is frequently masked by contaminant HIV DNA found in viral stocks. Extensive DNAse pre-treatment of viral stocks was found essential. The PCR studies correlating cellular nucleoside activation levels to the inhibition of HIV DNA synthesis are ongoing. SUMMARY: Activation of cell cycle independent nucleosides (such ad ddI and its analog FddI) in resting cells may affect viral DNA synthesis, and account for the observations that ddI and AZT have similar antiviral potencies in vivo despite the significantly higher activity of AZT in vitro when assayed with replicating cells.
Publication Types:
Keywords:
- Acquired Immunodeficiency Syndrome
- Anti-HIV Agents
- CD4-Positive T-Lymphocytes
- DNA
- DNA, Viral
- Didanosine
- HIV
- HIV Infections
- HIV Seropositivity
- HIV-1
- Humans
- In Vitro
- Nucleosides
- Polymerase Chain Reaction
- Stavudine
- Zidovudine
- reverse transcriptase, Human immunodeficiency virus 1
Other ID:
UI: 102192219
From Meeting Abstracts