HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.     

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.     

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.     

Inhibition of early steps of HIV-1 replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.     

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by a two-amino-acid insertion in HIV-1 Vif from a nonprogressing mother and child

We studied a 15-year-old girl, patient X, who has maintained consistently low plasma loads of human immunodeficiency virus type 1 (HIV-1) RNA, as well as normal and stable CD4(+) T-cell concentrations. She has presented no clinical manifestations of AIDS, despite having only received zidovudine monotherapy for a part of her life. Patient X's HIV-positive mother (patient Y) has also not progressed to AIDS and has never been treated with antiretroviral agents. HIV-1 isolated from patient X replicated poorly in human peripheral blood mononuclear cells (PBMC). In order to map the determinant of the poor growth of patient X's isolate, viral sequences from patient X were determined and examined for insertion or deletion mutations. These sequences contained a two-amino-acid insertion mutation in the Vif gene, which was also observed in uncultured PBMC acquired at different times. Furthermore, Vif sequences harbored by patient Y contained the identical mutation. These observations suggest that polymorphic HIV-1 was transmitted to patient X perinatally 15 years previously and has been maintained since that time. Recombinant HIV-1, engineered with Vif sequences from patient X, replicated in PBMC to levels approximately 20-fold lower than that of wild type. Removal of the insertion mutation from this recombinant restored replication efficiency to wild-type levels, while introduction of the insertion mutation into wild-type Vif sequences resulted in greatly decreased replication. Furthermore, Vif protein from patient X's HIV-1 was aberrantly cleaved, suggesting a mechanism for loss of Vif function. Since HIV-1 containing these sequences replicates poorly, the implication is that the two-amino-acid insertion mutation in Vif contributes significantly to the nonprogressor status of this mother and child. Further studies of these sequences might provide information regarding contributions of Vif structure and/or function to HIV-1 virulence.     

HIV-1 infection and HIV-1 Tat protein permit the survival and replication of a non-pathogenic trypanosomatid in macrophages through TGF-beta1 production

Monoxenic trypanosomatids, which usually are non-pathogenic in humans, have been detected in AIDS patients, but the mechanisms underlying the establishment of these protozoa in HIV-1-infected individuals are poorly understood. Here we addressed the role of HIV-1 and the HIV-1 Tat protein in the replication of the monoxenic trypanosomatid Blastocrithidia culicis in HIV-1-infected primary human macrophages. We found that HIV-1 and B. culicis replication augmented almost three times in co-infected macrophages, and that Tat antiserum significantly reduced the exacerbated protozoan growth. Exposure of B. culicis only infected macrophages to Tat protein also resulted in enhanced protozoan proliferation, reaching a twofold increase at 100 ng/mL. Electron microscopy analysis revealed that B. culicis and HIV-1 co-habit the same cells, and showed protozoan dividing forms inside macrophages. Protozoan replication diminished when B. culicis only infected macrophages were treated with Tat protein in the presence of anti-TGF-beta1 antibodies, suggesting a participation of this cytokine in the augmentation of protozoan multiplication. In fact, exogenous TGF-beta1 promoted the trypanosomatid replication in macrophages. Overall, our results suggest that HIV-1 infection deactivates the macrophage microbicidal activity, permitting the survival and multiplication of an otherwise non-pathogenic protozoan in these cells, a process partially mediated by Tat protein, via TGF-beta1 secretion.     

Inhibition of HIV-1 replication by ribozymes that show poor activity in vitro.

Self-cleaving RNAs (ribozymes) can be engineered to cleave target RNAs of choice in a sequence-specific manner (1). Consequently, they could be used to inhibit virus replication or to analyse host gene function in vivo. However, ribozymes that are catalytic in vitro are generally disappointing when analysed in cells unless expressed at high levels relative to their target RNAs (2, 3). Here we provide evidence that this can be overcome by optimizing ribozyme structure using cellular rather than cell-free assays. We show that ribozymes of relatively long flanking complementary regions (FCRs), while poor catalysts in vitro, can produce profound inhibition of HIV replication in cells. By examining a series of ribozymes in which the FCRs vary from 9 to 564 nucleotides, we establish that the optimum length for activity in the cell is > or = 33 nucleotides.     

Macrophage colony-stimulating factor antagonists inhibit replication of HIV-1 in human macrophages

Macrophages infected with HIV-1 produce high levels of M-CSF and macrophage-inflammatory protein-1alpha (MIP-1alpha). M-CSF facilitates the growth and differentiation of macrophages, while the chemotactic properties of MIP-1alpha attract both T lymphocytes and macrophages to the site of HIV infection. Studies described in this work indicate M-CSF may function in an autocrine/paracrine manner to sustain HIV replication, and data suggest possible therapeutic strategies for decreasing viral load following HIV infection. We show that macrophage infection with measles virus or respiratory syncytial virus, in contrast to HIV-1, results in production of MIP-1alpha, but not M-CSF. Thus, M-CSF appears to be specifically produced upon infection of macrophages with HIV-1. Furthermore, addition of M-CSF antagonists to HIV-1-infected macrophages, including anti-M-CSF monoclonal or polyclonal Abs or soluble M-CSF receptors, dramatically inhibited HIV-1 replication and reduced production of MIP-1alpha. Our results suggest that biologic antagonists for M-CSF may represent novel strategies for inhibiting the spread of HIV-1 by 1) blocking virus replication in macrophages, 2) reducing recruitment of HIV-susceptible T cells and macrophages by MIP-1alpha, and 3) preventing the establishment and maintenance of infected macrophages as a reservoir for HIV.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.     

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.     

Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives.

Several synthetic 2'-O-methyl-RNA oligomers and their derivatives have been evaluated for inhibitory effect against HIV-induced cytopathic effect and expression of the virus specific antigen in cultured MT-4 cells. In this study, oligo(2'-O-methyl)ribonucleoside phosphorothioates showed a potent inhibitory activity with size dependency (25-mer showed it at 1 microM), but by contrast both 2'-O-methylribo- and deoxy-oligomers with normal phosphate linkages failed to inhibit. However, it should be noted that the patched oligo(2'-O-methyl)ribonucleotide (20-mer), in which five linkages at 5'- and three linkages at 3'-ends of normal phosphates were replaced with thiophosphates, has recovered the substantial inhibitory effect. These results show that the size of oligomer and phosphorothioate linkages, probably resistant to exolytic nucleases, are essential for exhibiting antiviral activity.