HIV-1 infection is blocked at an early stage in cells devoid of mitochondrial DNA.

Human immunodeficiency virus type I (HIV-1) exploits various host cellular pathways for efficient infection. Here we report that the absence of mitochondrial DNA (mtDNA) in ρ(0) cells markedly attenuates HIV-1 infection. Importantly, reduced infection efficiency in ρ(0) cells is not simply the result of impaired oxidative phosphorylation (OXPHOS) because pharmacological OXPHOS inhibition did not inhibit HIV-1 infection.

A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.

Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3'-deoxy-3'-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence.

Stable complexes formed by HIV-1 reverse transcriptase at distinct positions on the primer-template controlled by binding deoxynucleoside triphosphates or foscarnet.

Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analog of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide.

Chain-terminating dinucleoside tetraphosphates are substrates for DNA polymerization by human immunodeficiency virus type 1 reverse transcriptase with increased activity against thymidine analogue-resistant mutants.

Nucleoside reverse transcriptase inhibitors are an important class of drugs for treatment of human immunodeficiency virus type 1 (HIV-1) infection. Resistance to these drugs is often the result of mutations that increase the transfer of chain-terminating nucleotides from blocked DNA termini to a nucleoside triphosphate acceptor, resulting in the generation of an unblocked DNA chain and synthesis of a dinucleoside polyphosphate containing the chain-terminating deoxynucleoside triphosphate analogue. We have synthesized and purified several dinucleoside tetraphosphates (ddAp4ddA, ddCp4ddC, ddGp4ddG, ddTp4ddT, Ap4ddG, 2'(3')-O-(N-methylanthraniloyl)-Ap4ddG, and AppNHppddG) and show that these compounds can serve as substrates for DNA chain elongation and termination resulting in inhibition of DNA synthesis.

Effects of primer-template sequence on ATP-dependent removal of chain-terminating nucleotide analogues by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP.

Relationship between 3'-azido-3'-deoxythymidine resistance and primer unblocking activity in foscarnet-resistant mutants of human immunodeficiency virus type 1 reverse transcriptase.

Phosphonoformate (foscarnet) is a pyrophosphate (PP(i)) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP(i) binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP(i) or a nucleotide such as ATP) which also interacts with the PP(i) binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3'-azido-3'-deoxythymidine (AZT).

Effects of dipeptide insertions between codons 69 and 70 of human immunodeficiency virus type 1 reverse transcriptase on primer unblocking, deoxynucleoside triphosphate inhibition, and DNA chain elongation.

Finger insertion mutations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (T69S mutations followed by various dipeptide insertions) have a multinucleoside resistance phenotype that can be explained by decreased sensitivity to deoxynucleoside triphosphate (dNTP) inhibition of the nucleotide-dependent unblocking activity of RT. We show that RTs with SG or AG (but not SS) insertions have three- to fourfold-increased unblocking activity and that all three finger insertion mutations have threefold-decreased sensitivity to dNTP inhibition. The additional presence of M41L and T215Y mutations increased unblocking activity for all three insertions, greatly reduced the sensitivity to dNTP inhibition, and resulted in defects in in vitro DNA chain elongation.

Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase.

Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3'-azido-3'-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that pi-pi interactions between RT and an aromatic structure may be facilitated by these mutations.