Endogenous HIV-1 Research Articles

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1.

HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-β conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-β variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (∼350 Å(2)) positively charged surface in the all-β XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors. The host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of noncytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8(+) T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-β fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-β XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells

Monocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

Multiplexing Seven miRNA-Based shRNAs to Suppress HIV Replication

Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

341. Activation of Latent HIV-1 by Targeted Suppression of an Endogenous HIV-1 Expressed Antisense Non-Coding RNA

341. Activation of Latent HIV-1 by Targeted Suppression of an Endogenous HIV-1 Expressed Antisense Non-Coding RNA

Adenovirus Vectors Block Human Immunodeficiency Virus–1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat

Heterologous viruses may transactivate or suppress human immunodeficiency virus (HIV)-1 replication. An adenovirus type 5 gene transfer vector (Ad5) HIV-1 vaccine was recently evaluated in a clinical trial, without efficacy. In this context, it is relevant to ask what effect Ad vectors have on HIV-1 replication, particularly in cells that are part of the innate immune system. Infection of HIV-1-infected human alveolar macrophages (AMs) obtained from HIV-1(+) individuals with an Ad vector containing no transgene (AdNull) resulted in dose-responsive inhibition of endogenous HIV-1 replication. HIV-1 replication in normal AMs infected with HIV-1 in vitro was inhibited by AdNull with a similar dose response. Ad reduced AM HIV-1 replication up to 14 days after HIV-1 infection. Fully HIV-1-infected AMs were treated with 3'-azido-3'-deoxythymidine, after which Ad infection still inhibited HIV-1 replication, suggesting a postentry step was affected. Substantial HIV-1 DNA was still produced after Ad infection, as quantified by TaqMan real-time PCR, suggesting that the replication block occurred after reverse transcription. AdNull blocked HIV-1 long terminal repeat (LTR) transcription, as assessed by an vesicular stomatitis virus G protein pseudotyped HIV-1 LTR luciferase construct. The formation of HIV-1 DNA integrated into the host chromosome was not inhibited by Ad, as quantified by a two-step TaqMan real-time PCR assay, implying a postintegration block to HIV-1 replication. These data indicate that Ad vectors are inhibitory to HIV-1 replication in human AMs based, in part, on their ability to inhibit LTR-driven transcription.

HIV-1-Specific CD4+ T-Cell Responses Are Not Associated With Significant Viral Epitope Variation in Persons With Persistent Plasma Viremia

To determine whether increased sequence variation occurs in regions of endogenous HIV-1 targeted by HIV-1-specific CD4 T cells. The presence of increased variation would be suggestive of immune evasion by HIV-1. We performed a cross-sectional study of untreated HIV-1-infected subjects measuring HIV-1-specific interferon (IFN)-gamma-secreting CD4 T-cell responses against epitopes in Gag p17 and p24 and concurrent endogenous plasma HIV-1 RNA epitope sequence variation. CD8- depleted IFNgamma enzyme-linked immunospot assays were used to identify regions of HIV-1 Gag recognized by CD4 T cells. Reverse transcriptase polymerase chain reaction and TA cloning were used to sequence endogenous plasma HIV-1 virus and identify variants. CD4 T-cell epitopes in Gag p17 and p24 were identified in 5 individuals, and concurrent sequence information on endogenous HIV-1 was obtained in 4 of these individuals. Endogenous plasma HIV-1 RNA sequencing revealed no intrapatient amino acid sequence variation through identified epitopes. In these chronically infected viremic subjects, circulating IFNgamma-secreting CD4 T-cell responses were directed against epitope sequences found in the predominant strain of endogenous circulating plasma HIV-1, suggesting that escape from CD4 T-cell responses is not a common process in vivo.

994. E1 Adenovirus Gene Transfer Vectors Inhibit HIV-1 Replication in Human Alveolar Macrophages

E1 adenovirus (Ad) vector infection of human alveolar macrophages (AM) inhibits HIV-1 replication independently of any transgene (Rice et al, Am J Respir Cell Mol Biol 2002; 27: 214-219) when the AM are infected with HIV-1 in vitro. We have previously shown that deletions of E4, Ad DNA polymerase or preterminal protein had no effect on the ability of Ad to block HIV-1 replication, empty Ad capsids do not block HIV-1 replication, and Ad acts, in part, by suppressing transcription from the HIV-1 long terminal repeats (LTR). The inhibition of HIV-1 replication by Ad is dose-dependent. We hypothesized that Ad would cause a similar dose-dependent inhibition of endogenous HIV-1 replication in AM obtained from HIV-1+ individuals. To address this question, we examined the shape of the dose-response curve for submaximal Ad inhibition of HIV-1 replication in AM. AM (6|[times]|105 cells/well) from a normal individual were infected with AdNull (0 to 2.5 |[times]| 104 pu/cell) or treated with azidothymidine (AZT, 10 |[mu]|g/ml), prior to infection with HIV-1 (103 TCID50/well). Conditioned media was collected twice weekly, and HIV-1 replication was quantified by measuring p24 in the conditioned media by ELISA. On day 16 post-infection, at a time of linear increases in p24, indicating active HIV-1 replication, p24 levels were (ng/ml) No Ad: 19.4; Ad doses in pu/cell: 102: 17.2 ; 5|[times]|102: 12.5; 103: 5.2; 5|[times]|103: 0.72; 104 0.11; 2.5|[times]|104: 0.04; AZT: 0.04. We compared these results with Ad effects on HIV-1 spontaneously elaborated from AM recovered from an HIV-1 positive individual. AM (8 |[times]| 105 cells/well) were infected with AdNull (0 to 2.5 |[times]| 104 pu/cell) or treated with AZT. p24 levels in the culture increased linearly over 4 days. On day 4 post-infection with Ad, p24 levels were No Ad: 44.2; 102: 32.4 ; 5|[times]|102: 24.7; 103: 10.7; 5|[times]|103: 1.70; 104: 0.27; 2.5|[times]|104: 0.08; AZT: 0.08. The dose-response for inhibitory effects of Ad on HIV-1 replication was similar under both conditions over several logs of Ad doses. In a separate experiment, Ad inhibition of HIV-1 replication in AM obtained from another HIV+ individual was evaluated as a function of Ad dose in half-log increments spanning the range from 102 |[minus]|3|[times]|104 pu/cell and yielding a range of p24 levels on day 4 ranging from 1.1-26.2. When the percent inhibition of HIV-1 replication was plotted as a function of log [Ad dose], the relationship was nearly linear, R2=0.96 over a 2 log range of doses. These data indicate that Ad inhibits endogenous HIV-1 replication in AM with a dose-response relationship similar to that observed when normal AM are infected with HIV-1 in vitro.

Expression of endogenous HIV-1 crossreactive antigens within normal human extravillous trophoblast cells

Expression of intact endogenous retroviruses by normal placental villous trophoblast and immuno-crossreactivity of villous trophoblast with anti-retroviral antisera have been documented. The nature and/or potential function of these particles/proteins has not yet been fully defined. We previously reported that monoclonal antibodies directed against HIV-1 envelope and gag proteins react with normal human villous trophoblast. In this study, we report that extravillous trophoblast (EVT) from second- and third-trimester tissue are also cross-reactive with anti-HIV-1 gp120 160 and p17 18 antibodies. We document a differential expression of such cross-reactive epitopes between mononuclear EVT and placental bed giant cells. Mononuclear EVT principally displayed reactivity throughout the cytoplasm with little or no difference between cells, whereas placental bed giant cells displayed distinct localization of labeling to limited areas of cytoplasm. This pattern of reactivity apparently correlates with trophoblast morphological differentiation and with our earlier observations concerning villous trophoblast. These data illustrate that retrovirus-associated epitopes are expressed by trophoblast throughout the normal human placenta and that this distribution is related to morphologic differentiation of these cells.