Intravirion targeting of a functional anti-human immunodeficiency virus ribozyme directed to pol.
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Ribozymes are catalytic RNAs that offer several advantages as specific therapeutic genes against human immunodeficiency virus type 1 (HIV-1). Significant challenges in antiviral uses of ribozymes include (1) how best to express and to deliver this agent and (2) what is the best locale to target ribozymes against HIV-1 RNA. To explore the former, we have previously characterized several vector systems for efficient expression/delivery of anti-HIV-1 ribozymes (Dropulic et al., 1992; Dropulic and Jeang, 1994a; Smith et al., 1997). Here, to investigate an optimal locale for ribozyme-targeting, we asked whether it might be advantageous to direct ribozymes into HIV-1 virions as opposed to the more conventional approach of targeting ribozymes into infected cells. Two series of experiments were performed. First, we demonstrated that anti-HIV-1 ribozymes could indeed be packaged specifically and efficiently into virions. Second, we compared the virus suppressing activity of a packageable ribozyme with its counterpart, which cannot be packaged into HIV-1 virions. Our results showed that although both ribozymes cleaved HIV-1 genomic RNA in vitro with equivalent efficiencies, the former ribozyme demonstrated significantly higher virus-suppressing activity than the latter. These findings provide proof-of-principle that to combat productive HIV-1 replication, intravirion targeting is more effective than intracellular targeting of ribozymes. (+info)
KSHV-encoded CC chemokine vMIP-III is a CCR4 agonist, stimulates angiogenesis, and selectively chemoattracts TH2 cells.
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Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 3 genes that are homologous to cellular chemokines. vMIP-III, the product of open reading frame K4.1, is the most distantly related to human chemokines and has yet to be characterized. We have examined the interaction of vMIP-III with chemokine receptors, its expression in KS lesions, and its in ovo angiogenic properties. We show expression of vMIP-III in KS lesions and demonstrate the stimulation of angiogenesis by this chemokine, like vMIP-I and vMIP-II, in the chick chorioallantoic membrane assay. vMIP-III does not block human immunodeficiency virus entry through the coreceptors CCR3, CCR5, or CXCR4. However, vMIP-III is an agonist for the cellular chemokine receptor CCR4. CCR4 is expressed by TH2-type T cells. Consistent with this, vMIP-III preferentially chemoattracts this cell type. Because of these biologic properties and because it is expressed in KS lesions, vMIP-III may play an important role in the pathobiology of KS. (Blood. 2000;95:1151-1157) (+info)
Assembly and processing of human immunodeficiency virus Gag mutants containing a partial replacement of the matrix domain by the viral protease domain.
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We constructed human immunodeficiency virus (HIV) mutants by replacing the matrix domain with sequences encoding the viral protease or p6* and protease. The chimeras retaining matrix myristylation and processing signals underwent efficient autoprocessing with severely defective particle budding. The budding defects of the chimeras were rescued by suppressing the chimera protease activity either through addition of an HIV protease inhibitor or through inactivating the chimera protease via a substitution mutation of the catalytic aspartic acid residue. This resulted in the release of chimeric virus-like particles with the density of a wild-type retrovirus particle. In addition, the assembly-competent but processing-defective chimeras produced proteolytically processed particles with significant reverse transcriptase activity when a downstream native pol gene was present. These results suggest that HIV has the potential to adapt heterologous sequences in place of the matrix sequence without major effects on virus-like particle budding. In addition, the positions of the protease and substrate accessibility may contribute significantly toward avoiding a premature Gag or Gag-Pol process, which leads to severe defects in both particle budding and incorporation. (+info)
Intracellular metabolism of beta-L-2',3'-dideoxyadenosine: relevance to its limited antiviral activity.
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The intracellular metabolism of the beta-L- enantiomer of 2', 3'-dideoxyadenosine (beta-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 microM [2',3',8-(3)H]-beta-L-ddA resulted in an increased intracellular concentration of beta-L-ddA with time, demonstrating that these cells were able to transport beta-L-ddA. However, it did not result in the phosphorylation of beta-L-ddA to its pharmacologically active 5'-triphosphate (beta-L-ddATP). Five other intracellular metabolites were detected and identified as beta-L-2', 3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 +/- 0.95, 8.15 +/- 2.64, and 15.60 +/- 1.74 pmol/10(6) cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a beta-glucuronic derivative of beta-L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with beta-L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of beta-L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of beta-L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of beta-L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28). (+info)
Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1.
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Human immunodeficiency virus (HIV) enters cells in vitro via CD4 and a coreceptor. Which of 15 known coreceptors are important in vivo is poorly defined but may be inferred from disease-modifying mutations, as for CCR5. Here two single nucleotide polymorphisms are described in Caucasians in CX3CR1, an HIV coreceptor and leukocyte chemotactic/adhesion receptor for the chemokine fractalkine. HIV-infected patients homozygous for CX3CR1-I249 M280, a variant haplotype affecting two amino acids (isoleucine-249 and methionine-280), progressed to AIDS more rapidly than those with other haplotypes. Functional CX3CR1 analysis showed that fractalkine binding is reduced among patients homozygous for this particular haplotype. Thus, CX3CR1-I249 M280 is a recessive genetic risk factor in HIV/AIDS. (+info)
The role of HIV-related chemokine receptors and chemokines in human erythropoiesis in vitro.
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In order to better define the role of HIV-related chemokines in human erythropoiesis we studied: A) the expression of chemokine receptors, both on human CD34(+) cells which include erythroid progenitors and on more mature erythroid cells; B) the functionality of these receptors by calcium flux, chemotaxis assay and phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 (ERK1/ERK2) and AKT, and finally C) the influence of chemokines on BFU-E formation. We found that HIV-related chemokine receptor CXCR4, but not CCR5, is detectable on human CD34(+) BFU-E cells. CXCR4 surface expression decreased during erythroid maturation, although CXCR4 mRNA was still present in cells isolated from differentiated erythroid colonies. SDF-1, a CXCR4 ligand, induced calcium flux and phosphorylation of MAPK (p42/44) and AKT in CD34(+)KIT(+) bone marrow mononuclear cells which contain BFU-E, as well as chemotactic activity of both human CD34(+) BFU-E progenitors and erythroid cells isolated from day 2-6 BFU-E colonies. Responsiveness to SDF-1 decreased when the cells differentiated to the point of surface expression of the erythroid-specific marker Glycophorin-A. In contrast, the CCR5 ligands (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and RANTES) did not activate calcium flux, MAPK and AKT phosphorylation or chemotaxis of CD34(+)KIT(+) cells or cells isolated from the BFU-E colonies. Interestingly, none of the chemokines tested in this study had any effect on BFU-E colony formation. In conclusion, only CXCR4 is functional, and its specific ligand SDF-1 may therefore play an important role in the homing and/or retention of early erythroid precursors in the bone marrow environment. (+info)
Constitutive expression of stromal derived factor-1 by mucosal epithelia and its role in HIV transmission and propagation.
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HIV particles that use the chemokine receptor CXCR4 as a coreceptor for entry into cells (X4-HIV) inefficiently transmit infection across mucosal surfaces [1], despite their presence in seminal fluid and mucosal secretions from infected individuals [2] [3] [4]. In addition, although intestinal lymphocytes are susceptible to infection with either X4-HIV particles or particles that use the chemokine receptor CCR5 for viral entry (R5-HIV) during ex vivo culture [5], only systemic inoculation of R5-chimeric simian-HIV (S-HIV) results in a rapid loss of CD4(+) intestinal lymphocytes in macaques [6]. The mechanisms underlying the inefficient capacity of X4-HIV to transmit infection across mucosal surfaces and to infect intestinal lymphocytes in vivo have remained elusive. The CCR5 ligands RANTES, MIP-1alpha and MIP-1beta suppress infection by R5-HIV-1 particles via induction of CCR5 internalization, and individuals whose peripheral blood lymphocytes produce high levels of these chemokines are relatively resistant to infection [7] [8] [9]. Here, we show that the CXCR4 ligand stromal derived factor-1 (SDF-1) is constitutively expressed by mucosal epithelial cells at sites of HIV transmission and propagation. Furthermore, CXCR4 is selectively downmodulated on intestinal lymphocytes within the setting of prominent SDF-1 expression. We postulate that mucosally derived SDF-1 continuously downmodulates CXCR4 on resident HIV target cells, thereby reducing the transmission and propagation of X4-HIV at mucosal sites. Moreover, such a mechanism could contribute to the delayed emergence of X4 isolates, which predominantly occurs during the later stages of the HIV infection. (+info)
Strong human immunodeficiency virus (HIV)-specific CD4+ T cell responses in a cohort of chronically infected patients are associated with interruptions in anti-HIV chemotherapy.
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Virus-specific CD4+ T-helper cell function is important in controlling human immunodeficiency virus (HIV) infection but is impaired in patients with progressive HIV disease. It has been reported that after highly active antiretroviral therapy (HAART), HIV-specific lymphoproliferative responses remain absent, whereas responses to non-HIV microbial antigens are restored. However, in analyzing immune responses in a cohort of chronically infected adults on HAART, we observed strong HIV-specific CD4+ T cell responses of Th-1 phenotype in 11 of 22 patients. The magnitude and frequency of HIV-specific lymphoproliferative responses was strongly associated with previous interruptions in HAART (P=.001). In contrast, the magnitude of CD8+ T cell responses to HIV Gag, Pol, Env, and Nef was similar in patients who had and those who had not interrupted HAART. We conclude that (1) a significant proportion of chronically HIV-infected patients on HAART can generate strong HIV-specific CD4+ and CD8+ T cell immunity and (2) transient interruptions in antiviral treatment may prime or boost HIV-specific CD4+ T-helper responses. (+info)