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Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi(+) RRE(+)), 3′ defective (Psi(+) RRE(−)), and 5′ defective (Psi(−) RRE(+)) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4(+) T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3′ defective HIV DNA were not significantly different from those of 5′ defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3′ defective HIV RNA (median 86 copies/10(6) cells; 94% of HIV RNA) was much more abundant than 5′ defective (2.1 copies/10(6) cells; 5.6%) or intact (0.6 copies/10(6) cells

Although there have been great advancements in the field of HIV treatment and prevention, there is no cure. There are two types of HIV: HIV-1 and HIV-2. In addition to genetic differences between the two types of HIV, HIV-2 infection causes a slower disease progression, and the rate of new HIV-2 infections has dramatically decreased since 2003. Like HIV-1, HIV-2 is capable of establishing latent infection in CD4+ T cells, thereby allowing the virus to evade viral cytopathic effects and detection by the immune system. The mechanisms underlying HIV latency are not fully understood, rendering this a significant barrier to development of a cure. Using RT-ddPCR, we previously demonstrated that latent infection with HIV-1 may be due to blocks to HIV transcriptional elongation, distal transcription/polyadenylation, and multiple splicing. In this study, we describe the development of seven highly-specific RT-ddPCR assays for HIV-2 that can be applied to the study of HIV-2 infections and latency. We designed and validated seven assays targeting different HIV-2 RNA regions along the genome that can be used to measure the degree of progression through different blocks to HIV-2 transcription and splicing. Given that HIV-2 is vastly understudied relative to HIV-1 and that it can be considered a model of a less virulent infection, application of these assays to studies of HIV-2 latency may inform new therapies for HIV-2, HIV-1, and other retroviruses.

The binding of nuclear proteins of hematopoietic cells to transcriptional enhancers of interferon-inducible genes has been studied before and after exposure to alpha-interferon. Mobility shift assays show that a complex formed with interferon-inducible transcriptional enhancers before interferon induction contains a 73- and 84-kDa protein. Amino acid sequencing of the oligoaffinity column purified 73- and 84-kDa proteins showed that they belonged to a family of DNA-binding proteins which have been previously identified to exhibit binding in a sequence nonspecific manner to the ends of fragmented DNA or the origin of replication of adenovirus Type 2 DNA and sequence-specific binding to the distal regions of the U1 small nuclear RNA promoter, the promoter of the transferrin receptor gene, and the transcriptional regulatory regions of HLA genes. Following exposure to alpha-interferon, more slowly migrating complexes appeared which contained a 48-kDa protein, a 95-kDa protein, and a 105-kDa protein which bound to the 9-27 transcriptional enhancer in a sequence-specific manner.

Great excitement surrounded the initial discovery of the Philadelphia chromosome translocation and the molecular rearrangement of the abl protooncogene which is generated by this translocation in chronic myelogenous leukemia (CML). In this review, we will discuss how molecular analysis of the biological abnormalities generated in the CML cells by this molecular rearrangement can be applied to new directions in therapy for this disease.

Cytoplasmic protein extracts from chronic myelogenous leukemia (CML) cells contained an activity that altered the electrophoretic mobility of complexes formed between nuclear proteins and the transcriptional enhancers of interferon (IFN)-inducible genes. Exposure of CML cells to IFN-alpha diminished the effect of the CML cytoplasmic proteins on these nuclear protein-DNA complexes. The presence of clinical responsiveness to IFN-alpha correlated with the sensitivity to the IFN- induced change in the electrophoretic mobility of nuclear protein-DNA complexes. These data suggest that the action of IFN-alpha in CML may be linked to a pathway that can result in posttranslational modification of nuclear proteins.

Anti-CD3 antibodies of some IgG subclasses are very potent T lymphocyte mitogens in vitro and, seemingly contradictorily, very effective immunosuppressive agents in vivo. Using hamster anti-murine CD3 monoclonal antibody, 2C11, as a model, we have found that 2C11.IgG, or its F(ab')2 fragment, coupled to microbeads can provide short-term and vigorous activation of T cells and expansion of the lymphoid system in vivo. In contrast to free 2C11.IgG, these conjugates do not kill mice and cause T cell depletion, and can enhance immune responses. This study suggests that properly modified anti-CD3 antibodies can serve as in vivo immune system enhancers potentially useful in the treatment of cancer and chronic infectious diseases.

Protein-DNA interactions of the human myeloid cell nuclear differentiation antigen (MNDA) were examined in vivo in proliferating HL-60 promyelocytic leukemia cells and following induction of differentiation by 1,25-dehydroxyvitamin D3. Intact cells were treated with the reversible cross-linking agent cis-diamminedichloroplatinum(II) and the MNDA levels in the isolated protein-DNA complexes were determined. Less than 1% of the total intracellular level of MNDA was cross-linked to DNA in the noninduced proliferating HL-60 cells. Once the cells were induced to differentiate into monocytes, the amount of antigen cross-linked to the DNA increased to over 5% of the total intracellular level. The increased efficiency of cross-linking the MNDA to DNA was specific for monocyte-induced HL-60 differentiation, achieved with three inducers, and was not observed in association with granulocyte-induced differentiation. On a molar basis the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) was the most effective inducer of monocyte differentiation, followed by 1,25-dihydroxy-16-ene-23-ynecholecalciferol which was more effective than 1,25-dihydroxycholecalciferol. A cesium chloride gradient analysis of the nucleic acid-protein fraction isolation from cis-diamminedichloroplatinum(II)-treated, monocyte-induced HL-60 cells documented the authenticity of the association between the MNDA and DNA. The results indicate that a significant level of chromatin reorganization may accompany monocyte-induced differentiation that leads to much higher levels of MNDA-DNA cross-linking to DNA. The expression of the MNDA is restricted to human myeloid cells and the present results indicate that a fraction of this low abundance nuclear protein is specifically located near the DNA [within cis-diamminedichloroplatinum(II) cross-linking distance] and that this association may be modulated specifically during monocyte differentiation.

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to chromium salt for at least 4 h, and the amount of DNA-protein complexes increased with longer incubation times. Complex formation occurred only with chromium salt concentrations of 200 microM or greater, and maximal cross-linking was effected at 5 mM. Immunotransfer methodology employing antibodies to nuclear matrix fraction and lamins was used to identify some of the polypeptides comprising the cross-linked complexes. These studies indicated specificity of chromium-induced complex formation within the nuclear protein fractions assayed. Our results document the ability of chromate to produce specific DNA-protein cross-links in living cells.

Cross-linking of proteins to DNA in live, intact Novikoff ascites hepatoma cells exposed in vitro to different concentrations of CuSO4, Pb(NO3)2, HgCl2, and AlCl3 was studied. Protein-DNA complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by electrophoretic separation of proteins associated with the DNA-containing pellets. Concentration dependence experiments showed that the optimal cross-linking occurred at metal concentration of 0.5 mM for CuSO4, HgCl2, and AlCl3 while the optimal cross-linking for Pb(NO3)2 was at 5 mM. For some metals at concentrations higher than optimal, the amounts of cross-linked proteins decreased significantly. Immunochemical analysis of the cross-linked proteins using antibodies to matrix, chromatin, lamins, and cytokeratin fractions demonstrated that some, but not all, members of these protein families became cross-linked to the DNA. Each metal exhibited a cross-linking pattern of its own, different from those of the other metals. Radioactive labeling experiments showed that all the metals tested became associated with the DNA-protein pellets within 1 h after their addition to the incubation medium. However, hexavalent chromium required more than 2 h before appearing in the DNA-protein pellets in significant amounts.

When cells are briefly exposed to cis-diamminedichloroplatinum (II) before lysis in high sodium dodecyl sulfate-urea solutions, the high molecular-weight nucleic acids pelleted by ultracentrifugation contain an increased level of bound proteins when compared to a similar fraction from untreated cells. Subsequent shearing of the pelleted DNA followed by treatment with DNase permits electrophoretic and immunoblot analysis of the crosslinked proteins. In the present study such experiments were carried out with reference to nuclear envelope pore complex proteins in the differentiating L8 rat skeletal muscle cells. The results show that (i) whereas the major lamin proteins crosslinked to DNA in both myoblast and myotubes, lamin B is crosslinked to a greater extent to DNA in myotubes; (ii) a 62-kDa lectin-binding glycoprotein is apparently situated differently with respect to DNA in myotube nuclei; and (iii) the crosslinking pattern of the nuclear matrix proteins to DNA is qualitatively similar in myoblast and myotubes. In addition, lamin C′, a modified form of lamin C, not observed in intact nonmuscle cells previously [ Glass et al. (1985) J. Biol. Chem. 260, 1895–1900], exists as a native component of the nuclear lamina in rat skeletal myotubes but not in myoblasts. These results point to significant structural alterations in the proteins of the nuclear lamina-pore complex during myogenesis.

The in vivo cross-linking of cytokeratins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 and cis-diamminedichloroplatinum(II) (cis-DDP) was studied. Cytokeratin-DNA complexes were obtained by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate. The cytokeratins were identified electrophoretically and immunologically by use of polyclonal and monoclonal antibodies. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to K2CrO4 for at least 4 h, and the amount of keratin-DNA complexes increased with the incubation time. Each of the three Novikoff ascites hepatoma cytokeratins (p39, p49, and p56) showed a different apparent rate of cross-link formation with DNA. Cytokeratin-DNA complexes were detectable in our system only with K2CrO4 concentrations of 200 microM or greater, and saturation in cross-linking was effected at approximately 2 mM. Higher K2CrO4 concentrations (up to 5 mM) did not produce further significant increases in the amount of cross-linked cytokeratins. Chromium and cis-DDP cross-linked the same cytokeratins at approximately the same ratios; however, both agents cross-linked the major cytokeratins selectively, since not all cytokeratins present in Novikoff hepatoma cell lysates could be cross-linked to DNA. Further evidence of DNA-cytokeratin complexes was obtained by CsCl gradient centrifugation. Our results document the ability of chromate and cis-DDP to produce DNA-cytokeratin cross-links in vivo and show that in live Novikoff hepatoma cells some, but not all, of the components of intermediate filaments are within cross-linking distance of DNA.

The very sensitive and reliable silver staining method to visualize proteins in polyacrylamide gels described by Wray et al. (Anal. Biochem. (1981) 118, 197-203) fails when the protein sample contains nucleic acids and/or metals. By washing the polyacrylamide gels in acetic acid and repeatedly in methanol immediately following electrophoresis and then using the procedure of Wray et al., many gels otherwise unstainable may be stained with a high degree of reliability. This method allows visualization of a minute amount of proteins in samples containing high amounts of DNA and metals.

Crosslinking of proteins to DNA was studied in live intact Novikoff ascites hepatoma cells exposed in vitro to salts of chromium VI, III, and II, nickel II, cadmium II, and to CoCl2, As2O3, and AlK(SO4)2. DNA-protein complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by polyacrylamide gel electrophoresis. Hexavalent chromium compounds formed DNA-protein complexes very efficiently. The trivalent, poorly soluble, cupric chromite was nearly as efficient crosslinker as hexavalent Cr, perhaps because phagocytosis facilitated its entry into the cells. The more basic divalent form produced hardly any crosslinks. Most of the crosslinked proteins were common to all of the chromium salts employed. Nickel salts formed DNA-protein crosslinks less efficiently. Most proteins crosslinked by this metal had a high molecular weight ranging from 94,000 to 200,000. There was little qualitative difference between the crosslinked protein patterns for several various nickel (II) salts. Similar results were obtained for cells incubated with cadmium salts. Most of the proteins crosslinked by cadmium had high molecular weights and were similar to those crosslinked by nickel (II). Relatively weak, but significant, crosslinking was also observed when the Novikoff hepatoma cells were exposed to CoCl2, As2O3, or AlK(SO4)2.

Cross-linking of proteins to DNA in live, intact Novikoff ascites hepatoma cells exposed in vitro to different concentrations of CuSO4, Pb(NO3)2, HgCl2, and AlCl3 was studied. Protein-DNA complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by electrophoretic separation of proteins associated with the DNA-containing pellets. Concentration dependence experiments showed that the optimal cross-linking occurred at metal concentration of 0.5 mM for CuSO4, HgCl2, and AlCl3 while the optimal cross-linking for Pb(NO3)2 was at 5 mM. For some metals at concentrations higher than optimal, the amounts of cross-linked proteins decreased significantly. Immunochemical analysis of the cross-linked proteins using antibodies to matrix, chromatin, lamins, and cytokeratin fractions demonstrated that some, but not all, members of these protein families became cross-linked to the DNA. Each metal exhibited a cross-linking pattern of its own, different from those of the other metals. Radioactive labeling experiments showed that all the metals tested became associated with the DNA-protein pellets within 1 h after their addition to the incubation medium. However, hexavalent chromium required more than 2 h before appearing in the DNA-protein pellets in significant amounts.

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.

When Novikoff hepatoma-bearing rats were given injections of a therapeutic dose of cis-diamminedichloroplatinum(II) (cis-DDP) (7 mg/kg), DNA-protein cross-links could be detected by using antisera to dehistonized chromatin, nuclear matrix, or Novikoff hepatoma cytoskeletal preparation. The extent of cross-linking increased in time up to 24 h after the injection, after which time the DNA-protein cross-links were gradually repaired, with no cross-links detectable at 72 h. trans-DDP in equitoxic (40 mg/kg) dose was very efficient in forming DNA-protein cross-links. Although formed more rapidly, these trans-DDP-mediated cross-links were repaired faster, within 48 h after the injection. The repair of cross-links at equimolar trans-DDP dose (7 mg/kg) was even more rapid. The principal proteins cross-linked to the DNA by both cis- or trans-DDP were Novikoff hepatoma cytokeratins (Mr 39,000, 49,000, 56,000, and an additional protein band reacting with the antiserum to Novikoff hepatoma cytoskeletal preparation at Mr approximately 68,000).

The binding of nuclear proteins of hematopoietic cells to transcriptional enhancers of interferon-inducible genes has been studied before and after exposure to alpha-interferon. Mobility shift assays show that a complex formed with interferon-inducible transcriptional enhancers before interferon induction contains a 73- and 84-kDa protein. Amino acid sequencing of the oligoaffinity column purified 73- and 84-kDa proteins showed that they belonged to a family of DNA-binding proteins which have been previously identified to exhibit binding in a sequence nonspecific manner to the ends of fragmented DNA or the origin of replication of adenovirus Type 2 DNA and sequence-specific binding to the distal regions of the U1 small nuclear RNA promoter, the promoter of the transferrin receptor gene, and the transcriptional regulatory regions of HLA genes. Following exposure to alpha-interferon, more slowly migrating complexes appeared which contained a 48-kDa protein, a 95-kDa protein, and a 105-kDa protein which bound to the 9-27 transcriptional enhancer in a sequence-specific manner.