Earl E. Henderson

μ-opioid modulation of HIV-1 coreceptor expressionand HIV-1 replication

A substantial proportion of HIV-1-infected individuals are intravenous drug users (IVDUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the μ-opioid receptor. Our results show that DAMGO, a selective μ-opioid agonist, increases CXCR4 and CCR5 expression in both CD3+ lymphoblasts and CD14+ monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the μ-opioid receptor based on the ability of a μ-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of μ-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.

Selective inactivation of CCR5 and decreased infectivity of R5 HIV-1 strains mediated by opioid-induced heterologous desensitization.

The opiates are well‐established immunomodulatory factors, and recent evidence suggests that μ‐ and δ‐opioid receptor ligands alter chemokine‐driven chemotactic responses through the process of heterologous desensitization. In the present report, we sought to examine the capacity of μ‐ and δ‐opioids to modulate the function of chemokine receptors CCR5 and CXCR4, the two major human immunodeficiency virus (HIV) coreceptors. We found that the chemotactic responses to the CCR1/5 ligand CCL5/regulated on activation, normal T expressed and secreted, but not the CXCR4 ligand stromal cell‐derived factor‐1α/CXCL12 were inhibited following opioid pretreatment. Studies were performed with primary monocytes and Chinese hamster ovary cells transfected with CCR5 and the μ‐opioid receptor to determine whether cross‐desensitization of CCR5 was a result of receptor internalization. Using radiolabeled‐binding analysis, flow cytometry, and confocal microscopy, we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization. Despite this, we found that the cross‐desensitization of CCR5 by opioids was associated with a decrease in susceptibility to R5 but not X4 strains of HIV‐1. Our findings are consistent with the notion that impairment of the normal signaling activity of CCR5 inhibits HIV‐1 coreceptor function. These results have significant implications for our understanding of the effect of opioids on the regulation of leukocyte trafficking in inflammatory disease states and the process of coreceptor‐dependent HIV‐1 infection. The interference with HIV‐1 uptake by heterologous desensitization of CCR5 suggests that HIV‐1 interaction with this receptor is not passive but involves a signal transduction process.

μ-Opioid Induction of Monocyte Chemoattractant Protein-1, RANTES, and IFN-γ-Inducible Protein-10 Expression in Human Peripheral Blood Mononuclear Cells

Strong evidence for the direct modulation of the immune system by opioids is well documented. μ-Opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-γ-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [d-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a μ-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Opioids, Receptors, and Immunity

The results achieved by those seeking to determine whether opioids and other drugs of abuse can affect immunity are quite astonishing given the short period of time that research has focused on this area. Despite the fact that there is no longer any question that opioids produce a variety of effects on the immune system, the extent and significance of these changes in the drug-abusing population remains to be determined. Whether or not the findings in mice and in in vitro preparations can be extrapolated to man is not yet known. Of major significance is the question of whether the endogenous opioid system is involved in immunoregulation. Given the multitude of drugs taken by drug abusers and the varying patterns of drug administration, the significance of the findings in the literature is still an open question. However, it is only by continuing studies such as those discussed at this meeting that we will find the answers.

Inhibition of Epstein-Barr virus-associated nuclear antigen (EBNA) induction by (2′,5′)oligoadenylate and the cordycepin analog: Mechanism of action for inhibition of EBV-induced transformation

Abstract We have previously shown that interferon inhibits Epstein-Barr virus-induced host cell DNA synthesis and morphologic transformation of human adult peripheral blood leukocytes and that core (2′,5′)oligoadenylate and the cordycepin analog can replace interferon with respect to the blockage of EBV-induced functions. The present communication describes the inhibition of Epstein-Barr virus-associated nuclear antigen (EBNA) in EBV-infected human adult peripheral blood leukocytes and the inhibition of EBV-induced morphologic transformation of human umbilical cord leukocytes by interferon, core (2′,5′)oligoadenylate and the core cordycepin analog. These data suggest that the antiviral mechanism of interferon, core (2′,5′)oligoadenylate and the core cordycepin analog is through the inhibition of EBV early functions, possibly transcription or translation of EBNA.

Altered HIV expression and EBV-induced transformation in coinfected PBLs and PBL subpopulations

Human immunodeficiency virus (HIV) IIIB expression and Epstein-Barr virus (EBV) B95.8-induced transformation were studied during coinfection. Coinfection of peripheral blood lymphocyte (PBL) cultures with HIV and EBV resulted in down-regulation of HIV expression. EBV-induced and spontaneous transformation were markedly reduced in PBL cultures exposed to HIV before EBV. On the other hand, transformation was enhanced when PBL cultures were infected with HIV either simultaneous to or after EBV. Reconstitution of EBV-infected B cell cultures with autochthonous T cells demonstrated that HIV-infected T cells had a reduced ability to inhibit EBV-induced transformation. PHA stimulation of HIV-infected T cells eliminated their ability to inhibit EBV-induced transformation. Lymphoblastoid cell lines (LCLs) established from coinfected PBLs expressed B cell markers and were EBV positive, while a large proportion of the LCLs expressed HIV antigens, released reverse transcriptase activity into the supernatant, and produced syncytia when cocultivated with indicator cell line SupT1. HIV provirus could be detected in LCLs established from coinfected cultures by PCR amplification using specific sets of amplimers for gag and env genes of HIV. To more closely examine the role of various cell types in lymphocyte transformation and HIV replication during coinfection, experiments were carried out using subpopulations enriched for either B or T cells. Simultaneous coinfection of purified B cells with EBV and HIV resulted in a marked reduction of HIV expression, whereas EBV-induced transformation was enhanced. In contrast, spontaneous B cell transformation was inhibited by HIV. A proportion of LCLs established from purified B cells coinfected with EBV and HIV expressed HIV antigens, released reverse transcriptase activity, and produced syncytia on SupT1 cells. These results demonstrate that the IIIB strain of HIV and B95.8 strain of EBV can interact during coinfection of B cells to alter the course of virus expression.

Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transforme with the denV gene of bacteriophage T4

The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities. Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers. These nicks may serve as substrates to initiate excision repair of pyrimidine dimers by endogenous enzymes. In the present study, murine fibroblasts stably transfected with denV were able to remove 50-80% of UVR-induced pyrimidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair. For both control and denV-transfected cells, repair continued for at least 24 h after exposure. When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired. The enhanced pyrimidine dimer repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival.

2′,5′-Adenylate and Cordycepin Trimer Cores: Metabolic Stability and Evidence for Antimitogenesis without 5′-Rephosphorylation

Abstract The effect of the 2′,5′-adenylate and cordycepin trimer cores on DNA and protein synthesis in human umbilical cord lymphocytes, lymphoblasts, peripheral blood lymphocytes and Epstein-Barr virus infected lymphocytes and their metabolism in tissue culture medium have been studied. [32P]Adenylate and [32P]- and [3H]cordycepin trimer cores were synthesized either enzymatically or chemically and added to cells in culture. The half-lives of the 2′,5′-A3 core and 2′,5′-3′dA3 core in tissue culture were 3 and 17 hr, respectively. Chromatographic analysis of the TCA-soluble extracts of the lymphocytes and lymphoblasts treated with 2′,5′-[3H]A3 showed that 0.25% of the 32P was identified as AMP, ADP, ATP and inorganic phosphate. By the more sensitive 2′,5′-p3A4[32P]pCp radiobinding assay, 2′,5′-A3 was detected in the TCA supernatants; however, there was no 5′-rephosphorylation. With the [3H]- and [32P]cordycepin trimer core, 0.55% and 1.3% of the radioactivity was in the TCA soluble extracts. Although ther...

Host cell reactivation of CAT-expression vectors as a method to assay for cloned DNA-repair genes.

We demonstrate the feasibility of using passive host-cell reactivation of a shuttle-vector pRSVcat to detect cloned DNA-repair genes. As models, a transient expression vector, pRSVdenV, and a positive-selection vector, pRSVdenV/SVgpt, were constructed containing the T4 coliphage denV gene, coding for an ultraviolet-specific endonuclease, under promotion of the Rous sarcoma virus (RSV) long-terminal repeat. Cotransfection of one or three copies of pRSVdenV per UV-irradiated pRSVcat molecule into xeroderma pigmentosum (XP) cells (XP12Ro[M1]) resulted in a dramatic increase in transient expression of chloramphenicol acetyl transferase (CAT) activity. XP clones stable transformed by pRSVdenV/SVgpt but not the parent cell line rescued CAT activity from this UV-irradiated reporter gene. The ability to express CAT activity from a UV-irradiated pRSVcat correlated with the presence of the structural denV gene as detected by Southern blot analysis. Post-UV irradiation colony-forming ability and DNA nucleotide excision-repair synthesis were partially restored in XP clones which rescued CAT activity. These results demonstrate the feasibility of using the cloned denV gene with its well characterized pyrimidine cyclobutane dimer-specific endonuclease activity to reconstitute UV-induced DNA repair in human cells deficient in DNA repair. Measuring CAT expression from pRSVcat affords a rapid, sensitive procedure to screen for functional cloned DNA-repair genes and to test mutant cells for defects in DNA repair.

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.