Preferential incorporation of nucleoside analogs after template switching during human immunodeficiency virus reverse transcription

Abstract

We assessed the effects of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC) on reverse transcription in CD4-positive cells by isolating truncated human immunodeficiency virus (HIV) DNA fragments. Jurkat cells were treated with AZT (2 ,LM), ddl (200 ,uM), or 3TC (50 ,uM) The reverse transcriptase (RT) of the human immunodeficiency virus (HIV) is essential for the formation of the viral double-stranded DNA genome from its RNA templates (13,38). Blockage of this catalytic function leads to inhibition of viral replication (21-23, 37). Many nucleoside analogs, including 3'-azido-3'-deoxythymidine (AZT) (8, 23), 2',3'-dideoxyinosine (ddl) (20-22, 33, 39), and the racemic mixture of 2',3'-dideoxy-3'-thiacytidine (BCH-189) plus both the (+) and (-) enantiomeric forms of this compound (3TC) (6,7,34,36) can effectively obstruct HIV replication in tissue culture and presumably in HIV-infected subjects.RNA-dependent DNA polymerization begins with the formation of a transcription complex of RT (p66/pSi) and tRNALYS3, coupled to the primer-binding sequence (PBS) (4, 24). This step is thought to occur in the cytoplasm after viral entry. The first template switch can be defined as the completion of synthesis of (-) strong-stop HIV DNA accompanied by RNase H digestion of the U5/repeat (R) region of the long terminal repeat (LTR), allowing for annealing to a second RNA template and synthesis of near-full-length (-) DNA (9,12,19,29). Incorporation of triphosphorylated 3'-hydroxyl-deficient nucleoside analogs by RT is believed to irreversibly terminate the elongating DNA strand during reverse transcription. These analogs have higher affinity for RT than for host DNA polymerase activities and therefore possess preferential inhibitory activity for the former enzyme (14,28,37 (514) 340- 7502.The mechanisms which underlie nucleoside analog antiviral activity are still poorly understood. The triphosphate (TP) form of these analogs is thought to be responsible for proviral DNA chain termination. Although AZT-TP and ddITP can chain terminate elongating DNA strands synthesized from RNA or DNA templates (22, 27, 37), these effects have only been demonstrated in vitro with nonviral synthetic templates, usually poly(rA) oligo(dT). Large decreases of HIV proviral DNA in the presence of AZT have been noted (35), but only one study has identified AZT-chain-terminated HIV DNA fragments in CD4-positive cells (40). We have isolated and characterized truncated HIV DNA in HIV-exposed Jurkat cells that were pretreated with 3TC, AZT, or ddl, through extractions of low-molecular-weight (LMW) DNA (15) and quantitative PCR amplifications. We found that HIV DNA fragments isolated from drug-treated cells were efficiently truncated after the synthesis of (-) strong-stop DNA. Little or no further chain termination by nucleoside analogs was observed over the subsequent synthesis of 8,500 nucleotides (nt) following the first template switch.We utilized an endogenous HIV type 1 (HIV-...

DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity

“…AZT chain-terminated DNA is incapable of promoting further elongation, because of the absence of a 3' hydroxyl group (Zack et al, 1992). We have also shown, by quantitative PCR amplification, that AZT can chain-terminate elongating viral DNA in activated peripheral blood lymphocytes, quiescent brain macrophages (Geleziunas et al, 1993) and Jurkat cells (Arts & Wainberg 1994). We found that chain termination by AZT in viral particles occurred only after the first template switch, which is consistent with the mode of action of nucleoside analogues in HIV-infected cells (Arts & Wainberg, 1994).…”

“…We have also shown, by quantitative PCR amplification, that AZT can chain-terminate elongating viral DNA in activated peripheral blood lymphocytes, quiescent brain macrophages (Geleziunas et al, 1993) and Jurkat cells (Arts & Wainberg 1994). We found that chain termination by AZT in viral particles occurred only after the first template switch, which is consistent with the mode of action of nucleoside analogues in HIV-infected cells (Arts & Wainberg, 1994). However, virus generated in the presence of AZT was as infectious as wild-type for H9 cells, suggesting that HIV genome-derived DNA is not involved in infection.…”

“…We found that HIV RNA was at least 1000 times more abundant in virions than DNA, and that virtually no viral DNA was present in protease-defective viral particles. Recently, we have used the same method of quantitative PCR amplification to demonstrate chain termination by AZT during HIV infection of activated peripheral blood lymphocytes, brain macrophages (Geleziunas et al, 1993) and Jurkat cells (Arts & Wainberg, 1994).…”

“…between 107 and one and then amplified by PCR for use as a quantification control for DNA amplifications. HIV RNA containing the LTR was obtained by in vitro transcription from a HIV RNA expression plasmid (Arts & Wainberg, 1994). Levels of RNA transcripts were quantified by spectrophotometry.…”

Transient Selection of A Hepatitis B Virus Polymerase Gene Mutant Associated With A Decreased Replication Capacity and Famciclovir Resistance

“…One problem that may be frequently encountered during prolonged administration of nucleoside analogs is the selection of HBV resistant strains with mutation in the viral polymerase as already described with antiviral therapy of HIV infection. 1,13 Sequence alignment and comparison with the crystal structure of the HIV reverse transcriptase have shown that most of the mutations associated with resistance to famciclovir or lamivudine reside in domains B and C of the HBV polymerase. [14][15][16] Resistant viruses, with mutations in the catalytic site (domain C) of the viral polymerase (M539V and M539I), similar to that observed in HIV resistant strains to Abbreviations: HBV, hepatitis B virus; PCR, polymerase chain reaction; ALT, alanine aminotranferase; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; tid, thrice daily.…”