Induction of apoptosis by a nonnucleoside human immunodeficiency virus type 1 reverse transcriptase inhibitor.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase (RT) by both nucleoside and nonnucleoside RT inhibitors profoundly inhibits virus replication. Nucleoside RT inhibitors are known to be toxic, but there is little information regarding the toxicities of nonnucleoside RT inhibitors (NNRTI). We demonstrate that efavirenz (an NNRTI) induces caspase- and mitochondrion-dependent apoptosis of Jurkat T cells and human peripheral blood mononuclear cells. The clinical relevance of these observations is not yet clear.

Induction of cell death in human immunodeficiency virus-infected macrophages and resting memory CD4 T cells by TRAIL/Apo2l.

Because the persistence of human immunodeficiency virus (HIV) in cellular reservoirs presents an obstacle to viral eradication, we evaluated whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induces apoptosis in such reservoirs. Lymphocytes and monocyte-derived macrophages (MDM) from uninfected donors do not die following treatment with either leucine zipper human TRAIL (LZhuTRAIL) or agonistic anti-TRAIL receptor antibodies. By contrast, such treatment induces apoptosis of in vitro HIV-infected MDM as well as peripheral blood lymphocytes from HIV-infected patients, including CD4(+) CD45RO(+) HLA-DR(-) lymphocytes. In addition, LZhuTRAIL-treated cells produce less viral RNA and p24 antigen than untreated controls. Whereas untreated cultures produce large amounts of HIV RNA and p24 antigen, of seven treated CD4(+) CD45RO(+) HLA-DR(-) cell cultures, viral RNA production was undetectable in all, p24 antigen was undetectable in six, and proviral DNA was undetectable in four. These data demonstrate that TRAIL induces death of cells from HIV-infected patients, including cell types which harbor latent HIV reservoirs.

Effect of cessation of highly active antiretroviral therapy during a discordant response: implications for scheduled therapeutic interruptions.

Although treatment with combination antiretroviral therapy leads to a reduction in the level of plasma viremia and an improvement in CD4 T cell count for most patients, for a minority of patients, an improvement in CD4 T cell count occurs despite the failure of treatment to suppress viral replication. Recent reports suggest that these discordant improvements in CD4 T cell count may last for months to years and are associated with improved clinical outcomes. In a retrospective observational study, we evaluated the effect of therapy cessation on 8 patients with discordant immunologic responses to therapy and found that improved CD4 T cell responses are dependent upon ongoing drug pressure. If antiretroviral agents that are likely to resuppress the virus are not available, we suggest that patients continue the therapy associated with immunologic improvement to maximize the clinical benefit of the discordant response.

Mechanisms of HIV-associated lymphocyte apoptosis.

Infection with the human immunodeficiency virus (HIV) is associated with a progressive decrease in CD4 T-cell number and a consequent impairment in host immune defenses. Analysis of T cells from patients infected with HIV, or of T cells infected in vitro with HIV, demonstrates a significant fraction of both infected and uninfected cells dying by apoptosis. The many mechanisms that contribute to HIV-associated lymphocyte apoptosis include chronic immunologic activation; gp120/160 ligation of the CD4 receptor; enhanced production of cytotoxic ligands or viral proteins by monocytes, macrophages, B cells, and CD8 T cells from HIV-infected patients that kill uninfected CD4 T cells; and direct infection of target cells by HIV, resulting in apoptosis. Although HIV infection results in T-cell apoptosis, under some circumstances HIV infection of resting T cells or macrophages does not result in apoptosis; this may be a critical step in the development of viral reservoirs. Recent therapies for HIV effectively reduce lymphoid and peripheral T-cell apoptosis, reduce viral replication, and enhance cellular immune competence; however, they do not alter viral reservoirs. Further understanding the regulation of apoptosis in HIV disease is required to develop novel immune-based therapies aimed at modifying HIV-induced apoptosis to the benefit of patients infected with HIV.

Mutations in the HIV type 1 integrase of patients receiving long-term dideoxynucleoside therapy do not confer resistance to zidovudine.

Metabolites of AZT can inhibit HIV-1 integrase in vitro (Mazumder A, et al., Proc Natl Acad Sci USA 1994;91:5771-5775). To determine if long-term dideoxynucleoside therapy can lead to the emergence of HIV-1 AZT-resistant variants containing mutations in the integrase, we have sequenced the proviral DNA encoding the HIV-1 integrase of nine HIV-1-infected patients at different time points during treatment. Four of the nine patients developed mutations during the course of treatment. Although most mutations occurred at nonconserved amino acids, one patient developed a mutation at codon (R166T), a residue that is conserved among all integrases from known HIV-1 isolates. This mutation was introduced in the recombinant HIV-1 integrase protein to determine if it could confer resistance to AZT in vitro. We show that the R166T integrase mutant is still proficient at carrying 3'-processing and 3' end-joining but that the enzyme is not resistant to AZT-TP. Our results suggest that it is unlikely that integrase inhibition contributes to the antiviral activity of AZT.

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff.

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.

Identification of a nucleotide binding site in HIV-1 integrase.

HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3'-azido-3'-deoxythymidine (AZT) analog 3',5-diazido-2', 3'-dideoxyuridine 5'-monophosphate (5N3-AZTMP) and proteolytic mapping identified the amino acid 153-167 region of integrase as the site of photocrosslinking. Docking of 5N3-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.

Trapping of mammalian topoisomerase I and recombinations induced by damaged DNA containing nicks or gaps. Importance of DNA end phosphorylation and camptothecin effects.

We used purified mammalian topoisomerases I (top1) and oligonucleotides containing a unique top1 cleavage site to study top1-mediated cleavage and recombination in the presence of nicks and short gaps mimicking DNA damage. In general, top1 cleavage was not induced opposite to the nicks, and nicks upstream from the top1 cleavage site suppressed top1 activity. Irreversible top1 cleavage complexes ("suicide products" or "aborted complexes") were produced in DNA containing nicks or short gaps just opposite to the normal top1 cleavage site. Camptothecin enhanced the formation of the aborted top1 complexes only for nicks downstream from the cleavage site. These aborted (suicide) complexes can mediate DNA recombination and promote illegitimate recombination by catalyzing the ligation of nonhomologous DNA fragments (acceptors). We report for the first time that top1-mediated recombination is greatly enhanced by the presence of a phosphate at the 5' terminus of the top1 aborted complex (donor DNA). By contrast, phosphorylation of the 3' terminus of the gap did not affect recombination. At concentrations that strongly enhanced inhibition of intramolecular religation, resulting in an increase of top1 cleavable complexes, camptothecin did not reduce recombination (intermolecular religation). Nicks or gaps with 5'-phosphate termini would be expected to be produced directly by ionizing radiations or by processing of abasic sites and DNA lesions induced by carcinogens or drugs used in cancer chemotherapy. Thus, these results further demonstrate that DNA damage can efficiently trap top1-cleavable complexes and enhance top1-mediated DNA recombination.

Polyomavirus large T-antigen binds the "pRb related' protein p130 through sequences in conserved region 2.

The transforming potential of DNA tumor viruses derives mainly from the ability of their encoded oncogene products to interact with cellular proteins. Many of these viral oncoproteins share regions of sequence similarity, designated conserved region 1 and 2, which have been implicated in complex formation with pRb, the product of the retinoblastoma tumor suppressor gene, and related p107 and p130 species. It has now been shown that the EIA protein of adenovirus is able to bind to all three pRb-related proteins through sequences in conserved region 1 and 2. We have shown previously that polyomavirus large T-antigen also interacts with pRb and p107 in vitro. The pRb and p107 binding domains reside between residues 141, 158 which include conserved region 2. In the present study, we demonstrate that polyomavirus large T-antigen also interacted with p130 in vitro through the same sequences in conserved region 2.

Polyomavirus large T antigen zinc finger is not required for efficient cellular immortalization of primary rat embryo fibroblasts.

The polyomavirus large T antigen contains a zinc finger domain required for the formation of hexamers involved in viral DNA replication. Since mutations within the zinc finger domains of transforming proteins like SV40 large T antigen and human papilloma virus E7 protein generally decrease their overall transforming activity, we have examined the ability of a mutant polyomavirus large T antigen that harbors a deletion in sequences within the zinc finger motif to immortalize primary rat embryo fibroblasts. In contrast to result obtained with SV40 large T antigen and the human papilloma virus E7 protein we show that deletion of the entire zinc finger motif enhances the immortalization efficiency of this mutant T antigen.

Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site. Implications for the role of lysine 136 in DNA binding.

We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine epsilon-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.

Functional implications of mutations within polyomavirus large T antigen Rb-binding domain: effects on pRb and p107 binding in vitro and immortalization activity in vivo.

In this study, we have extensively modified the Rb-binding domain of polyomavirus large T antigen. Mutant polyomavirus large T antigens were tested for their ability to bind pRb and p107 in vitro and assayed for their capacity to immortalize primary rat embryo fibroblasts in vivo. Polyomavirus large T antigen bound pRb and p107 through a common region located between amino acids 141 to 158, containing the consensus Rb-binding sequence D/N-L-X-C-X-E. Substitution of any amino acid within the core Rb-binding sequence abolished pRb and p107 binding in vitro and immortalization activity in vivo. Substitution of amino acids outside the core Rb-binding sequence reduced pRb and p107 binding in vitro and decreased or abolished immortalization of rat embryo fibroblasts in vivo. Although duplication of the Rb-binding domain within the polyomavirus large T antigen results in a molecule that can bind at least twice as much pRb and p107 in vitro, this mutant displayed an essentially wild-type level of immortalization activity. More importantly, we found that the addition of acidic residues within the casein kinase II consensus phosphorylation region flanking the Rb-binding domain, or the deletion of amino acids 256 to 272, increased the immortalizing activity of the mutant polyomavirus large T antigen. These two mutants displayed a greater than wild-type level of pRb binding in vitro, while in contrast, a decreased affinity for p107 binding in vitro was observed. Together, these results indicate that while pRb binding appears to be an essential event for immortalization, there is no tight correlation between the frequency of immortalization and the absolute level of pRb binding in vitro, indicating that other large T antigen functions are important for cellular immortalization.