Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.     

Function of human immunodeficiency virus type 1 nef gene product in virus replication

We comparatively analyzed the replication kinetics of wild-type (wt) and nef mutant human immunodeficiency virus type 1 (HIV-1) in several CD4-positive cell lines, in order to clarify the molecular function of Nef protein. The delayed growth of nef mutant virus was observed at the initial stage of replication in all cell lines examined. This phenomenon was greatly amplified in the absence of vpu gene. In order to determine the infection stage in viral replication cycle which is specifically affected on virus replication rate in the presence of the Nef protein, we first examined the difference between wt and nef mutant viruses in the virus production rate from transfected cells, and found that the both viruses were produced with equal efficiency. This result showed that Nef protein could be dispensable for virion production. Therefore, early infection stages were focused by single-round infection assay, and the nef mutant virus was found to be much less infectious than wt virus. This indicated that the effect of Nef protein was exhibited in the early phase of a virus replication cycle, during viral adsorption to integration. By entry assay using wt and nef mutant virions, it was revealed that the Nef protein was required for efficient viral entry. These data suggest that the Nef protein might play a role in efficient incorporation of the Env protein into the virions, leading to enhanced viral infectivity.     

Inhibition of human immunodeficiency virus type 1 replication in myelomonocytic cells derived from retroviral vector-transduced peripheral blood progenitor cells

PURPOSE: To investigate the potential antioxidative role of carnosine (beta-alanyl-L-histidine) through its interaction with radiation induced radicals. MATERIALS AND METHODS: Pulse radiolysis experiments were performed by a 12 MeV electron linear accelerator (LINAC) on carnosine aqueous solutions at different pHs. The Raman spectra of solid samples were obtained by a Bruker IFS 66 spectrometer. RESULTS: The protonation state of the imidazole ring was observed to affect the site(s) of .OH attack on carnosine as well as the decay rates of the resulting adducts. Reasonably, a base catalysed water elimination from the adducts leading to a resonance-stabilized radical, which absorbs at lambda = 270 nm, takes place. Raman spectra in slightly alkaline medium have indicated that carnosine is mainly present as tautomer I and, of consequence, positions C(2) and C(4) of the imidazole ring are the preferential sites for .OH attack. CONCLUSIONS: These studies have shown that carnosine is a good scavenger of .OH radicals giving rise to a quite stable intermediate which should be less reactive towards other biological components than .OH itself. Raman data have been helpful in predicting the preferential sites for the .OH attack.     

Induction of CD4 expression and human immunodeficiency virus type 1 replication by mutants of the interferon-inducible protein kinase PKR

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), and the IFN-inducible protein kinase PKR is thought to mediate this effect by regulating protein synthesis. Here we report that ectopic expression of dominant negative PKR mutants in Jurkat cells induces HIV-1 replication. Specifically, expression of CD4 is upregulated by the PKR mutants, and this correlates with an induction of HIV-1 binding and proviral DNA synthesis upon HIV-1 infection. Moreover, activation of NF-kappaB was induced by an RNA binding-defective mutant of PKR. Thus, it appears that PKR, in addition to translational control, is involved in HIV-1 replication by modulating virus binding through the regulation of CD4 expression and virus gene expression through the activation of NF-kappaB.     

Propentofylline inhibits production of TNFalpha and infection of LP-BM5 murine leukemia virus in glial cells

Much evidence points to the involvement of N-methyl-D-aspartate (NMDA) receptors in the development and maintainance of neuropathic pain. In neuropathic pain, there is generally involved a presumed opioid-insensitive component, which apparently can be blocked by NMDA receptor antagonists. However, in order to obtain complete analgesia, a combination of an NMDA receptor antagonist and an opioid receptor agonist is needed. Recent in vitro data have demonstrated that methadone, ketobemidone, and dextropropoxyphene, in addition to being opioid receptor agonists, also are weak noncompetitive NMDA receptor antagonists. Clinical anecdotes suggest that the NMDA receptor antagonism of these opioids may play a significant role in the pharmacological action of these compounds; however, no clinical studies have been conducted to support this issue. In the present commentary, we discuss evidence for the NMDA receptor antagonism of these compounds and its relevance for clinical pain treatment; an overview of structure-activity relationships for the relevant opioids as noncompetitive NMDA receptor antagonists also is given. It is concluded that although the finding that some opioids are weak noncompetitive NMDA receptor antagonists in vitro has created much attention among clinicians, no clinical studies have been conducted to evaluate the applicability of these compounds in the treatment of neuropathic pain conditions.     

Importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication

The recent development and use of protease inhibitors have demonstrated the essential role that combination therapy will play in the treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1). Past clinical experience suggests that due to the appearance of resistant HIV-1 variants, additional therapeutics will be required in the future. To identify new options for combination therapy, it is of paramount importance to pursue novel targets for drug development. Ribosomal frameshifting is one potential target that has not been fully explored. Data presented here demonstrate that small molecules can stimulate frameshifting, leading to an imbalance in the ratio of Gag to Gag-Pol and inhibiting HIV-1 replication at what appears to be the point of viral particle assembly. Thus, we propose that frameshifting represents a new target for the identification of novel anti-HIV-1 therapeutics.     

The effect of commencing combination antiretroviral therapy soon after human immunodeficiency virus type 1 infection on viral replication and antiviral immune responses

Twelve subjects were treated with zidovudine, lamivudine, and ritonavir within 90 days of onset of symptoms of acute infection to determine whether human immunodeficiency virus type 1 (HIV-1) infection could be eradicated from an infected host. In adherent subjects, with or without modifications due to intolerance, viral replication was suppressed during the 24-month treatment period. Durable suppression reduced levels of HIV-1-specific antibodies and cytotoxic T lymphocyte responses in selected subjects. Proviral DNA in mononuclear cells uniformly persisted. The persistence of HIV-1 RNA expression in lymphoid tissues and peripheral blood mononuclear cells suggests that elimination of this residual pool of virus should be achieved before considering adjustments in antiretroviral therapeutic regimens. In addition, given the reduction in levels of virus-specific immune responses, it would seem prudent to consider enhancing these responses using vaccine strategies prior to the withdrawal of antiviral therapy.     

Inhibition of human immunodeficiency type 1 virus replication in monocytic U-937 cells by superinfection with Chlamydia trachomatis

Status epilepticus is a neurological emergency associated with substantial morbidity and mortality. Experimental and clinical investigations suggest that prolonged seizure activity is associated with injury to vulnerable neurons. Compounds with neuroprotective properties may minimize such injury. Existing methods of inducing experimental status epilepticus result in seizure activity of variable duration and neuronal injury of variable degree. To minimize such variability, status epilepticus may be stopped with anticonvulsants, but this limits the ability to screen for independent neuroprotective properties. We have developed a simple and reliable non-pharmacological model of limbic status epilepticus in which the duration of status epilepticus is under direct experimental control. Status epilepticus is induced by continuous, unilateral hippocampal stimulation. Using this model, the degree of hippocampal pyramidal cell injury varies in direct proportion to status epilepticus duration across a range of 15-140 min. A progressive sequence of EEG changes unfolds with increasing status epilepticus duration, resembling that seen in other models. This model may serve as a reference against which the effects of potential neuroprotective compounds can be studied.     

Contact with thymic epithelial cells as a prerequisite for cytokine-enhanced human immunodeficiency virus type 1 replication in thymocytes

We report here that human immunodeficiency virus type 1 (HIV-1)-infected human thymocytes, in the absence of any exogenous stimulus but cocultivated with autologous thymic epithelial cells (TEC), obtained shortly (3 days) after thymus excision produce a high and sustained level of HIV-1 particles. The levels and kinetics of HIV-1 replication were similar for seven distinct viral strains irrespective of their phenotypes and genotypes. Contact of thymocytes with TEC is a critical requirement for optimal viral replication. Rather than an inductive signal resulting from the contact itself, soluble factors produced in the mixed culture are responsible for this effect. Specifically, the synergistic effects of tumor necrosis factor, interleukin-1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor may account by themselves for the high level of HIV-1 replication in thymocytes observed in mixed cultures. In conclusion, the microenvironment generated by TEC-thymocyte interaction might greatly favor optimal HIV-1 replication in the thymus.     

Dynamics of viral replication in infants with vertically acquired human immunodeficiency virus type 1 infection

About one-third of vertically HIV-1 infected infants develop AIDS within the first months of life; the remainder show slower disease progression. We investigated the relationship between the pattern of HIV-1 replication early in life and disease outcome in eleven infected infants sequentially studied from birth. Viral load in cells and plasma was measured by highly sensitive competitive PCR-based methods. Although all infants showed an increase in the indices of viral replication within their first weeks of life, three distinct patterns emerged: (a) a rapid increase in plasma viral RNA and cell-associated proviral DNA during the first 4-6 wk, reaching high steady state levels (> 1,000 HIV-1 copies/10(5) PBMC and > 1,000,000 RNA copies/ml plasma) within 2-3 mo of age; (b) a similar initial rapid increase in viral load, followed by a 2.5-50-fold decline in viral levels; (c) a significantly lower (> 10-fold) viral increase during the first 4-6 wk of age. All infants displaying the first pattern developed early AIDS, while infants with slower clinical progression exhibited the second or third pattern. These findings demonstrate that the pattern of viral replication and clearance in the first 2-3 mo of life is strictly correlated with, and predictive of disease evolution in vertically infected infants.     

Common themes of antibody maturation to simian immunodeficiency virus, simian-human immunodeficiency virus, and human immunodeficiency virus type 1 infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Vasectomy and human immunodeficiency virus type 1 in semen

PURPOSE: Human immunodeficiency virus type 1 (HIV) is cultured more often from seminal cells than seminal plasma. Because vasectomy causes dramatic reductions in seminal cells and also eliminates secretions from proximal sites in the male reproductive tract, vasectomy may change the potential infectiousness of semen. MATERIALS AND METHODS: We used polymerase chain reaction (PCR) assays to measure HIV ribonucleic acid (RNA) in seminal plasma and HIV deoxyribonucleic acid (DNA) in seminal cells from 46 asymptomatic, seropositive men before and after vasectomy. RESULTS: HIV RNA levels in semen correlated only weakly with blood levels (r = 0.22, p = 0.03). Of 183 semen specimens assayed for cell-free HIV RNA and proviral DNA 37 (20%) were positive for HIV RNA only, 41 (22%) were positive for HIV DNA only, and 18 (10%) were positive for RNA and DNA. Thus, detection of HIV RNA in seminal plasma was not associated with detection of HIV DNA in seminal cells. HIV RNA was present in 23 of 82 specimens (28%) (mean 2.87 log copies/ml.) before vasectomy and in 38 of 121 specimens (31%) after vasectomy (mean 2.81 log copies/ml.). CONCLUSIONS: These findings suggest that direct measurement of HIV levels in semen is necessary to assess the potential for sexual transmission, most cell-free HIV in seminal plasma arises distal to the vas deferens, and vasectomy may have minimal impact on the infectiousness of HIV seropositive men on sexual partners.     

Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors

Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.