Arantxa Gutiérrez

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.

Interleukin-7 in Plasma Correlates with CD4 T-Cell Depletion and May Be Associated with Emergence of Syncytium-Inducing Variants in Human Immunodeficiency Virus Type 1-Positive Individuals

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV-positive patients. There was a negative correlation between circulating IL-7 levels and CD4+ T-cell count in HIV-positive patients (r = −0.621;P < 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/μl than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.

Role of the Human Immunodeficiency Virus Type 1 Envelope Gene in Viral Fitness

ABSTRACT A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.

HIV-1 resistance to the gp41-dependent fusion inhibitor C-34.

The gp41 subunit of HIV-1 has been recently recognized as a target for antiviral therapy. C-34 is a peptide that mimics the heptad repeat 2 in the ectodomain of gp41. Here, we describe two HIV-1 strains selected after 5 and 17 passages in culture with increasing concentrations of C-34 (breakthrough concentration of 10 microg/ml). The HXB2-derived strain was more than 1000-fold resistant and contained a V38E mutation in the gp41 coding DNA sequence. The NL4-3-derived strain was more than 500-fold resistant and contained a L33S mutation in gp41. No cross-resistance to the RT inhibitor AZT, the HIV binding inhibitor dextran sulfate (DS), or the chemokine receptor antagonist ALX-40-4C was detected. These data indicate that HIV-1 can overcome C-34 inhibition through mutations at residues not involved in the formation of the hydrophobic cavity of gp41.

CXCR4 and SDF-1 expression in B-cell chronic lymphocytic leukemia and stage of the disease

The pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) has been linked to an overexpression of the chemokine receptor CXCR4 and increased in vitro functional response to its natural ligand CXCL12 (SDF-1). The CXCR4/SDF-1 system appears to be important for tissue localization and increased survival of B-CLL cells. The aim of our study was to examine if CXCR4 expression and SDF-1 blood levels were correlated to clinical and pathological stage of B-CLL. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) techniques were used to determine CXCR4 expression and SDF-1 plasma levels, respectively, in a cohort of 51 patients diagnosed with B-CLL to correlate these measurements with several parameters that define the clinical stage of the disease. We confirmed that CXCR4 was consistently expressed on circulating B-CLL cells with a fluorescence intensity that was five-fold greater than in cells from healthy volunteers. There was a correlation between CXCR4 expression and leukocyte count (r: 0.55, p<0.01), and CD19+/CD5+ cells (r: 0.63, p<0.01). Interestingly, the group of B-CLL patients showed lower SDF-1 plasma levels compared to the control group. However, there was no correlation between CXCR4 or SDF-1 expression and the clinical stage of disease or the pattern of bone marrow infiltration. The results obtained suggest that other factors, and not only alteration in the SDF-1/CXCR4 chemokine system, must account for marrow infiltration of neoplastic cells observed in B-CLL and that CXCR4 could be involved in other features that exhibit malignant B cells, such as increased survival, rather than in their homing or migration to the bone marrow.

Anti-Human Immunodeficiency Virus Activity of Novel Aminoglycoside-Arginine Conjugates at Early Stages of Infection

Conjugates of L-arginine with aminoglycosides have already been described as potent in vitro inhibitors of the HIV-1 Tat-trans-activation responsive element interaction. The polycationic nature of these agents leads us to suggest that they may be active against HIV-1 replication by inhibiting earlier stages of the virus life cycle. We have found that R4K and R3G, kanamycin A, and gentamicin C, conjugated with arginine, inhibited HIV-1 NL4-3 replication at EC50 values of 15 and 30 microM for R3G and R4K, respectively, without a detectable tonic effect on MT-4 cells at concentrations higher than 4000 and about 1000 microM, respectively. Both compounds inhibited the binding of a monoclonal antibody (12G5) directed to CXCR4 as well as the intracellular Ca2+ signal induced by the chemokine SDF-1alpha on CXCR4+ cells, suggesting that aminoglycoside-arginine conjugates interact with CXCR4, the coreceptor used by T-tropic, X4 strains of HIV-1. On the other hand, CB4K, a conjugate of kanamycin A with gamma-guanidinobutyric acid, structurally similar to R4K, failed to display any anti-HIV activity of CXCR4 antagonist activity. An HIV-1 strain that was made resistant to the known CXCR4 antagonist AMD3100 was cross-resistant to both R4K and R3G. However, unlike SDF-1alpha and R4K, R3G inhibited the binding of HIV-1 to MT-4 cells. Aminoglycoside-arginine conjugates inhibit HIV replication by interrupting the early phase of the virus life cycle, namely virus binding to CD4 cells and interaction with CXCR4. R3G and R4K may serve as prototypes of novel anti-HIV agents and should be further studied.

A bacteriophage Lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.

Interleukin-7-Dependent Production of RANTES That Correlates with Human Immunodeficiency Virus Disease Progression

ABSTRACT There is a relationship between CD4-T-cell number and circulating interleukin 7 (IL-7) levels in human immunodeficiency virus (HIV)-positive individuals. Here, we show that IL-7 induced a dose-dependent production of CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) in peripheral blood mononuclear cells (PBMC), ex vivo tonsil lymphoid tissue of HIV− individuals, and PBMC from HIV+ individuals, suggesting that IL-7 may regulate β-chemokine production in vivo. In a cross-sectional study of HIV+ individuals (n = 130), a weak but significant correlation between IL-7 and RANTES was noted (r = 0.379; P < 0.001). Remarkably, the correlation between IL-7 and RANTES increased to an r value of 0.798 (P < 0.001) if individuals with low CD4 cell counts (<200 cells/μl) were excluded from the analysis. Our results suggest that there is a relationship between IL-7 and the production of RANTES both in vitro and in vivo that is lost in immune-compromised patients (CD4 count of <200 cells/μl) but that could be restored by antiretroviral therapy. Unlike the case for IL-7, high levels of RANTES suggest an intermediate stage of HIV disease progression.

Resistance of the human immunodeficiency virus to the inhibitory action of negatively charged albumins on virus binding to CD4

Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120. Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4+ MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA. The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype. The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1alpha, which block HIV replication by interaction with the chemokine receptor CXCR4. Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4+ cells.

Stromal-cell-derived factor 1 prevents the emergence of the syncytium-inducing phenotype of HIV-1 in vivo.

In a correlative study, the mean plasma level of the chemokine stromal-cell-derived factor 1 (SDF-1) was lower in subjects with syncytium-inducing (SI) than in subjects with non-syncytium-inducing (NSI) HIV isolates, regardless of the CD4 cell count or when compared with HIV-negative individuals. Individuals with high SDF-1 had an 81% probability of having an NSI virus phenotype compared with individuals with lower SDF-1. Increased expression of SDF-1 may help explain why the more pathogenic SI HIV-1 variants do not appear in some individuals.