Enhance HIV-1 Replication Research Articles

Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes

BackgroundThe outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef’s complex pathogenic phenotype can be recapitulated.ResultsIntravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9) and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes.ConclusionWe conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following infection. However, CD4+CD8+ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

Mechanisms of interaction between protozoan parasites and HIV

This review summarizes the current knowledge on human immunodeficiency virus type 1 (hereinafter called HIV)/protozoan co-infections in the case of three important, although neglected, tropical diseases: malaria, trypanosomiasis (Chagas disease) and leishmaniasis. The HIV pandemic has modified the immunopathogenic, epidemiological and therapeutic aspects of these human diseases. In-vitro data suggests that HIV favors Leishmania infection, whereas different parasites have contrasting effects on HIV. However, many of the previous models are a limited representation of the complex interactions within the host; this situation is particularly the case when microbial products are used in place of live parasites. In the host, protozoan parasites generally enhance HIV replication and accelerate AIDS progression. HIV alters parasite pathogenesis, often worsening disease outcome. These aspects bring significant complications for the treatment of co-infected individuals.

Nef alleles from all major HIV-1 clades activate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner.

The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs) that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts). Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication.

Short Communication: Activating Transcription Factor 4 (ATF4) Promotes HIV Type 1 Activation

Activating transcription factor 4 (ATF4) is a central factor in the cellular response to multiple stresses, including altered metabolic conditions, anoxia and hypoxia, and redox stress. ATF4 is triggered by endoplasmic reticulum stress and consequent unfolded protein response. This report identifies for the first time ATF4 as a transcription factor upregulated by HIV-1 infection. Upregulation of ATF4 enhances HIV replication, by synergistic interactions with HIV Tat. Moreover, in specific cell lines ATF4 has a direct transactivating potential on the LTR, even in the absence of Tat. We also provide evidence that expression of ATF4 induces HIV reactivation in chronically infected cell lines. These results show for the first time that ATF4 induction might have an important role in HIV replication, and suggest that ATF4 might represent a convergent signaling molecule for different stressors important in regulating the HIV-1 cycle.

A novel role for Slit2/Robo1 axis in modulating HIV-1 replication in T cells

Slit2 is a secreted glycoprotein that has been shown to possess anti-inflammatory properties. In addition, Slit2 has been shown to modulate CXCR4-mediated functional effects in T cells. However, its role in HIV-1 pathogenesis is not known. The objective of the current study is to analyze the role of Slit2 in modulating HIV-1 replication in T cells. The effect of endogenous Slit2 expression of HIV-1 replication in T cells was studied by transient overexpression or downregulation of Slit2. The role of exogenous Slit2 was studied by analyzing the effect of soluble Slit2 protein on HIV-1 replication in T-cell lines and peripheral blood mononuclear cells (PBMCs). Studies on T-cell lines revealed a higher expression of Slit2 in Jurkat T cells compared with MT4 cells. We observed that downregulation of Slit2 in Jurkat T cells using Slit2-specific small inhibitor RNA enhanced HIV-1 replication. However, overexpression of Slit2 in MT4 cells and PBMCs reduced HIV-1 replication. As Slit2 is a secretory protein, we further analyzed the role of soluble Slit2 on HIV-1 virus replication using various cell lines and PBMCs. Our data indicated that exogenous Slit2 inhibited replication of both X4-tropic and R5-tropic HIV-1 viruses. Further studies revealed that Slit2 mediated its functional effects by binding to Robo1 receptor. Taken together, our results describe Slit2/Robo1 axis as a novel modulator of HIV-1 replication in T cells. These studies may contribute to the understanding of the immunopathogenesis of HIV-1 infection.

Inhibition of HIV entry by extracellular glucuronoxylomannan of Cryptococcus neoformans

Cryptococcosis, caused by Cryptococcus neoformans, is the most common opportunistic fungal disease in HIV/AIDS patients. The prognosis of AIDS patients with Cryptococcus infection is very poor. One of the major characteristics in cryptococcosis patients is the presence of high concentrations of the cryptococcal capsule polysaccharide (CCP) in the serum and cerebrospinal fluid. CCP enhances HIV replication in H9 T-cells, but the mechanism is unknown. In this study, we tested whether extracellular glucuronoxylomannan (GXM), a major component of CCP, enhances HIV entry using replication-incompetent HIV and a cell line which expresses a stable amount of CD4 and both of the HIV co-receptors. Extracellular GXM had no effect on cell–cell fusion however; viral entry surprisingly was inhibited by GXM. Hence, any enhancement of replication must be due to an effect that occurs post-entry.

IFN-γ Mediates Enhancement of HIV Replication in Astrocytes by Inducing an Antagonist of the β-Catenin Pathway (DKK1) in a STAT 3-Dependent Manner

Typically, IFN-γ is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-γ induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-γ to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-γ relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous β-catenin signaling, a pathway that is a potent inhibitor of HIV replication, IFN-γ diminished β-catenin signaling in astrocytes by 40%, as evaluated by both active β-catenin protein expression and β-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-γ-mediated inhibition of β-catenin signaling was dependent on its ability to induce an antagonist of the β-catenin signaling pathway, Dickkopf-related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-γ to enhance HIV replication in astrocytes. These data demonstrated that IFN-γ induces HIV replication in astrocytes by antagonizing the β-catenin pathway. To our knowledge, this is the first report to point to an intricate cross-talk between IFN-γ signaling and β-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface.

Osteopontin enhances HIV replication and is increased in the brain and cerebrospinal fluid of HIV-infected individuals

Despite effective and widely available suppressive anti-HIV therapy, the prevalence of mild neurocognitive dysfunction continues to increase. HIV-associated neurocognitive disorder (HAND) is a multifactorial disease with sustained central nervous system inflammation and immune activation as prominent features. Inflammatory macrophages, HIV-infected and uninfected, play a central role in the development of HIV dementia. There is a critical need to identify biomarkers and to better understand the molecular mechanisms leading to cognitive dysfunction in HAND. In this regard, we identified through a subtractive hybridization strategy osteopontin (OPN, SPP1, gene) an inflammatory marker, as an upregulated gene in HIV-infected primary human monocyte-derived macrophages. Knockdown of OPN in primary macrophages resulted in a threefold decrease in HIV-1 replication. Ectopic expression of OPN in the TZM-bl cell line significantly enhanced HIV infectivity and replication. A significant increase in the degradation of the NF-κB inhibitor, IκBα and an increase in the nuclear-to-cytoplasmic ratio of NF-κB were found in HIV-infected cells expressing OPN compared to controls. Moreover, mutation of the NF-κB binding domain in the HIV-LTR abrogated enhanced promoter activity stimulated by OPN. Interestingly, compared to cerebrospinal fluid from normal and multiple sclerosis controls, OPN levels were significantly higher in HIV-infected individuals both with and without neurocognitive disorder. OPN levels were highest in HIV-infected individuals with moderate to severe cognitive impairment. Moreover, OPN was significantly elevated in brain tissue from HIV-infected individuals with cognitive disorder versus those without impairment. Collectively, these data suggest that OPN stimulates HIV-1 replication and that high levels of OPN are present in the CNS compartment of HIV-infected individuals, reflecting ongoing inflammatory processes at this site despite anti-HIV therapy.

Suppression and enhancement of HIV replication in macrophages by CD8+ lymphocytes (105.43)

Abstract CD8+ cytolytic T cells can suppress HIV replication by a variety of lytic and nonlytic mechanisms, and the relative clinical importance of particular CTL functions is not fully understood. Therefore studies have utilized various in vitro viral suppression assays as an indication of the antiviral efficacy. We measured antiviral activities of CD8+ lymphocytes from elite controllers who spontaneously suppress HIV without medication, from chronically infected HIV+ donors, and from HIVneg donors. Bulk CD8+ lymphocytes containing a CD56+ fraction suppressed HIV replication in autologous macrophages, even for HIVneg donors. CD56-depleted CD8 T cells from HIV+ donors also suppressed HIV replication in macrophage targets, with elite controller T cells exerting significantly greater suppression than progressor T cells. CD56-depleted CD8 T cells from HIVneg donors did not suppress but rather enhanced HIV-replication specifically in macrophages. Finally, when CD8 T cells and HIV-infected macrophages from elite controllers were separated by a transwell membrane, CD8 T cells did not suppress but rather enhanced HIV replication, indicating that HIV suppression was cell-contact dependent while soluble factors may be responsible for enhanced HIV replication. In summary CD8+CD56+ cells have innate antiviral activity in both elite controllers and uninfected donors, while CD8+ T cells have specific antiviral activity in HIV+ donors. In some cases inflammatory CTL may enhance viral replication.

Identification of Personal Lubricants That Can Cause Rectal Epithelial Cell Damage and Enhance HIV Type 1 Replicationin Vitro

Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (p<0.0001). A common ingredient in three of these preparations is polyquaternium-15. In vitro testing of a chemically related compound (MADQUAT) confirmed that this similarly augmented HIV-1 replication. Most of the lubricants were found to be hyperosmolar and the TEER value dropped approximately 60% 2 h after exposure to all lubricants tested. Cells treated with Carraguard, saline, and cell controls maintained about 100% initial TEER value after 2-6 h. We have identified four lubricants that significantly increase HIV-1 replication in vitro. In addition, the epithelial damage caused by these and many other lubricants may have implications for enhancing HIV transmission in vivo. These data emphasize the importance of performing more rigorous safety testing on these products.

Inhibition of Anti-HIV MicroRNA Expression: A Mechanism for Opioid-Mediated Enhancement of HIV Infection of Monocytes

Inhibition of Anti-HIV MicroRNA Expression: A Mechanism for Opioid-Mediated Enhancement of HIV Infection of Monocytes

Evaluation of the effect of pyrimethamine, an anti-malarial drug, on HIV-1 replication

Co-infection of human immunodeficiency virus (HIV) with malaria is one of the pandemic problems in Africa and parts of Asia. Here we investigated the impact of pyrimethamine (PYR) and two other clinical anti-malarial drugs (chloroquine [CQ] or artemisinin [ART]) on HIV-1 replication. Peripheral blood mononuclear cells (PBMCs) or MT-2 cells were infected with HIV NL4.3 strain and treated with different concentrations of the anti-malarial drugs. HIV-1 replication was measured using p24 ELISA. We show that 10 μM CQ and ART inhibited HIV-1 replication by 76% and 60% in PBMCs, respectively, but not in MT-2 cells. In contrast, 10 μM PYR enhanced HIV-1 replication in MT-2 cells by >10-fold. A series of molecular mechanism studies revealed that PYR increased intracellular HIV gag proteins without affecting the promoter or the reverse transcriptase activity. The effect of PYR was independent of HTLV-1 produced by MT-2 cells. Of interest, PYR treatment led to S-phase accumulation and increased AZT and d4T antiviral activity by ∼4-fold. Taken together, we show that PYR significantly enhances HIV-1 replication by affecting the cellular machinery. Our results could be relevant for the management of malaria and HIV particularly in regions where HIV-1 and malaria epidemics overlap.

Suppressor of Cytokine Signaling 3 Inhibits Antiviral IFN-β Signaling To Enhance HIV-1 Replication in Macrophages

HIV-1 replication within macrophages of the CNS often results in cognitive and motor impairment, which is known as HIV-associated dementia (HAD) in its most severe form. IFN-beta suppresses viral replication within these cells during early CNS infection, but the effect is transient. HIV-1 eventually overcomes this protective innate immune response to resume replication through an unknown mechanism, initiating the progression toward HAD. In this article, we show that Suppressor of Cytokine Signaling (SOCS)3, a molecular inhibitor of IFN signaling, may allow HIV-1 to evade innate immunity within the CNS. We found that SOCS3 is elevated in an in vivo SIV/macaque model of HAD and that the pattern of expression correlates with recurrence of viral replication and onset of CNS disease. In vitro, the HIV-1 regulatory protein transactivator of transcription induces SOCS3 in human and murine macrophages in a NF-kappaB-dependent manner. SOCS3 expression attenuates the response of macrophages to IFN-beta at proximal levels of pathway activation and downstream antiviral gene expression and consequently overcomes the inhibitory effect of IFN-beta on HIV-1 replication. These studies indicate that SOCS3 expression, induced by stimuli present in the HIV-1-infected brain, such as transactivator of transcription, inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages. This consequence of SOCS3 expression in vitro, supported by a correlation with increased viral load and onset of CNS disease in vivo, suggests that SOCS3 may allow HIV-1 to evade the protective innate immune response within the CNS, allowing the recurrence of viral replication and, ultimately, promoting progression toward HAD.

Total Antioxidant Capacity of Saliva in Children with HIV

Several recent reports have indicated high levels of reactive oxygen species, causing oxidative stress, in the pathogenesis of HIV infection. Oxidative stress may lead to enhanced HIV replication in infected cells and may also aggravate the immunodeficiency by reduction of cellular immunity and possibly by increased programmed cell death of lymphocytes. Saliva can constitute a first line of defense against free radical mediated oxidative stress. The use of saliva as a diagnostic fluid has become somewhat of a translational research success story. Technologies are now available enabling saliva to be used to diagnose disease and predict disease progression. The antioxidant capacity of saliva was investigated in 68 children who were divided into two groups. 34 children who were investigated were diagnosed as having HIV infection and the other group consisted of children who reported to the department and served as healthy controls. Total antioxidant capacity of saliva was evaluated by spectrophotometric assay. The results indicated that the total antioxidant capacity (TAC) of saliva decreased in children with HIV infection. TAC was seen to increase with the age of the children.

Stimulation of HIV-1 Replication in Immature Dendritic Cells in Contact with Primary CD4 T or B Lymphocytes

Sexual transmission is the major route of HIV-1 infection worldwide. Dendritic cells (DCs) from the mucosal layers are considered to be the initial targets of HIV-1 and probably play a crucial role in HIV-1 transmission. We investigated the role of cell-to-cell contact between HIV-1-exposed immature DCs and various lymphocyte subsets in the stimulation of HIV-1 replication. We found that HIV-1 replication and production in DCs were substantially enhanced by the coculture of DCs with primary CD4 T or nonpermissive B lymphocytes but not with primary activated CD8 T lymphocytes or human transformed CD4 T lymphocytes. Most of the new virions released by cocultures of HIV-1-exposed immature DCs and primary B lymphocytes expressed the DC-specific marker CD1a and were infectious for both immature DCs and peripheral blood mononuclear cells (PBMCs). Cocultured DCs thus produced large numbers of infectious viral particles under these experimental conditions. The soluble factors present in the supernatants of the cocultures were not sufficient to enhance HIV-1 replication in DCs, for which cell-to-cell contact was required. The neutralizing monoclonal antibody IgG1b12 and polyclonal anti-HIV-1 sera efficiently blocked HIV-1 transfer to CD4 T lymphocytes but did not prevent the increase in viral replication in DCs. Neutralizing antibodies thus proved to be more efficient at blocking HIV-1 transfer than previously thought. Our findings show that HIV-1 exploits DC-lymphocyte cross talk to upregulate replication within the DC reservoir. We provide evidence for a novel mechanism that may facilitate HIV-1 replication and transmission. This mechanism may favor HIV-1 pathogenesis, immune evasion, and persistence.

Tuberculosis in HIV-infected patients in the HAART era

Summary Tuberculosis in one of the most common opportunistic infections in HIV infected patients. HIV promotes progression of Mycobacterium tuberculosis latent infection to active disease and in turn M tuberculosis enhances HIV replication. During eight year period 51 cases of tuberculosis was recognized in Department of Infectious Diseases and Hepatology in Bydgoszcz. Only 4 patients were on antiretroviral therapy and of those who never received antiretroviral treatment; 70% had a CD4 count >100 cells/ml at the time of tuberculosis, and the high proportion (25.4%) of patients were diagnosed with HIV infection concurrently with tuberculosis. Diagnosis was confirmed by isolation of Mycobacterium tuberculosis and by the detection of nucleic acids of pathogen in 96.1% patients. The sputum smear was positive only in 5 cases of pulmonary tuberculosis and in three cases of disseminated tuberculosis was the most common clinical manifestation of disease in study population. A total of 8 patients with pulmonary involvement the chest radiograph examination was normal. Tuberculosis in HIV infected patients is in spite of availability of antiretroviral therapy a serious medical and epidemiological problem.

Nef, macrophages and B cells: a highway for evasion

Nef, macrophages and B cells: a highway for evasion

CXCL16 in HIV infection – a link between inflammation and viral replication

While some chemokines are thought to be protective in HIV-infected individuals by their ability to block HIV entry into T cells and macrophages, chemokines could also have harmful effects in HIV infection through their ability to promote inflammation. Here, we examined the regulation and the effects of CXCL16, a newly discovered chemokine of the CXC family, in HIV-infected patients. We examined serum levels of CXCL16 in clinically well-defined subgroups of HIV-infected individuals both before (n = 62) and during HAART (n = 40) as well as in age- and sex-matched healthy controls (n = 30). We also examined the effects of CXCL16 on inflammatory and anti-inflammatory cytokines and HIV replication in peripheral blood mononuclear cells (PBMC). Our main and novel findings were: (i) HIV-infected patients had significant raised CXCL16 levels according to disease severity and progression. (ii) During HAART, the immunological improvement was accompanied by a modest increase in CXCL16 level. (iii) While soluble CXCL16 promoted an anti-inflammatory response in PBMC from those on successful HAART, it induced an inflammatory response and enhanced HIV replication in PBMC from those with high viral load irrespectively of ongoing HAART. (iv) Recombinant HIV-tat protein significantly increased CXCL16 release in THP-1 macrophages. Our findings suggest a complex interaction between CXCL16 and HIV, promoting both inflammatory and anti-inflammatory effects as well as HIV replication, partly dependent on accompanying HIV replication.

P19-19. MVA vaccines are efficiently cross-presented by DCs and do not enhance HIV replication in DC/T cell cultures

P19-19. MVA vaccines are efficiently cross-presented by DCs and do not enhance HIV replication in DC/T cell cultures

Platelet factor 4 (CXCL4) facilitates human macrophage infection with HIV-1 and potentiates virus replication

Platelet factor 4 (CXCL4), a member of the CXC chemokine subfamily released in high amounts by activated platelets, has been identified as a monocyte survival factor that induces monocyte differentiation into macrophages. Although CXCL4 has been shown to have biological effects unique to chemokines, nothing is known about the role of CXCL4-derived human macrophages or CXCL4 in human immunodeficiency virus (HIV) disease. In this study, CXCL4-derived macrophages are compared with macrophage-colony stimulating factor (M-CSF)-derived macrophages for their ability to support HIV-1 replication. We show that CXCL4-derived macrophages can be infected with macrophage-tropic HIV-1 that uses either CC-chemokine receptor 5 (CCR5) or CXC-chemokine receptor 4 (CXCR4) as a co-receptor for viral entry. We also find that M-CSF and the chemokines, monocyte chemoattractant protein 1 (MCP-1; CCL2) and macrophage-inflammatory-protein-1-alpha (MIP-1alpha; CCL3) are produced upon R5- and X4-tropic HIV-1 replication in both M-CSF- and CXCL4-derived human macrophages. In addition, CXCL4 added to M-CSF-derived macrophages after virus adsorption and maintained throughout the infection enhances HIV-1 replication. We thus propose a novel role for CXCL4 in HIV disease.