Abasic Phosphorothioate Oligomers Inhibit HIV-1 Reverse Transcription and Block Virus Transmission across Polarized Ectocervical Organ Cultures

ABSTRACTIn the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2′ deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal applicationin vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

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HIV-1 accessory protein Vpr interacts with REAF/RPRD2 to mitigate its antiviral activity

10.1101/408161 ◽

2018 ◽

Cited By ~ 2

Author(s):

Joseph M Gibbons ◽

Kelly M Marno ◽

Rebecca Pike ◽

Wing-yiu Jason Lee ◽

Christopher E Jones ◽

...

Keyword(s):

T Cells ◽

Viral Replication ◽

Viral Entry ◽

Reverse Transcription ◽

Early Phase ◽

Early Stage ◽

Target Cells ◽

Accessory Protein ◽

Hiv 1

AbstractThe Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intactvpris more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.ImportanceFor at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

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High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs

Nature Communications ◽

10.1038/s41467-021-22628-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Betty Ha ◽

Kevin P. Larsen ◽

Jingji Zhang ◽

Ziao Fu ◽

Elizabeth Montabana ◽

...

Keyword(s):

Reverse Transcriptase ◽

Viral Entry ◽

Reverse Transcription ◽

Molecular Mechanisms ◽

Dissociation Kinetics ◽

Structural Mechanism ◽

Medium Resolution ◽

Kinetics Of ◽

Hiv 1

AbstractReverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

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In Vivo Analysis of Human T-Cell Leukemia Virus Type 1 Reverse Transcription Accuracy

Journal of Virology ◽

10.1128/jvi.74.20.9525-9531.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9525-9531 ◽

Cited By ~ 58

Author(s):

Louis M. Mansky

Keyword(s):

T Cell ◽

Mutation Rate ◽

Virus Type ◽

Reverse Transcription ◽

Leukemia Virus ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Hiv 1

ABSTRACT Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZα peptide gene region (the mutational target). The vector was replicated bytrans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 × 10−6 mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.

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Environmental Restrictions: A New Concept Governing HIV-1 Spread Emerging from Integrated Experimental-Computational Analysis of Tissue-Like 3D Cultures

Cells ◽

10.3390/cells9051112 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1112 ◽

Cited By ~ 1

Author(s):

Samy Sid Ahmed ◽

Nils Bundgaard ◽

Frederik Graw ◽

Oliver Fackler

Keyword(s):

In Silico ◽

Computational Analysis ◽

Virus Transmission ◽

Target Cells ◽

Virus Spread ◽

Transmission Modes ◽

3D Collagen ◽

Cell Densities ◽

Hiv 1

HIV-1 can use cell-free and cell-associated transmission modes to infect new target cells, but how the virus spreads in the infected host remains to be determined. We recently established 3D collagen cultures to study HIV-1 spread in tissue-like environments and applied iterative cycles of experimentation and computation to develop a first in silico model to describe the dynamics of HIV-1 spread in complex tissue. These analyses (i) revealed that 3D collagen environments restrict cell-free HIV-1 infection but promote cell-associated virus transmission and (ii) defined that cell densities in tissue dictate the efficacy of these transmission modes for virus spread. In this review, we discuss, in the context of the current literature, the implications of this study for our understanding of HIV-1 spread in vivo, which aspects of in vivo physiology this integrated experimental–computational analysis takes into account, and how it can be further improved experimentally and in silico.

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Low-level alternative tRNA priming of reverse transcription of HIV-1 and SIV in vivo

Retrovirology ◽

10.1186/s12977-019-0473-2 ◽

2019 ◽

Vol 16 (1) ◽

Author(s):

Christine M. Fennessey ◽

Celine Camus ◽

Taina T. Immonen ◽

Carolyn Reid ◽

Frank Maldarelli ◽

...

Keyword(s):

Reverse Transcription ◽

Low Level ◽

Hiv 1

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Extracellular vesicles from symbiotic vaginal lactobacilli inhibit HIV-1 infection of human tissues

Nature Communications ◽

10.1038/s41467-019-13468-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 13

Author(s):

Rogers A. Ñahui Palomino ◽

Christophe Vanpouille ◽

Luca Laghi ◽

Carola Parolin ◽

Kamran Melikov ◽

...

Keyword(s):

Extracellular Vesicles ◽

Viral Entry ◽

Ex Vivo ◽

Target Cells ◽

Isolated Cells ◽

Viral Attachment ◽

Vaginal Lactobacilli ◽

Male To Female ◽

Hiv 1

AbstractThe vaginal microbiota, dominated by Lactobacillus spp., plays a key role in preventing HIV-1 transmission. Here, we investigate whether the anti-HIV effect of lactobacilli is mediated by extracellular vesicles (EVs) released by these bacteria. Human cervico-vaginal and tonsillar tissues ex vivo, and cell lines were infected with HIV-1 and treated with EVs released by lactobacilli isolated from vaginas of healthy women. EVs released by L. crispatus BC3 and L. gasseri BC12 protect tissues ex vivo and isolated cells from HIV-1 infection. This protection is associated with a decrease of viral attachment to target cells and viral entry due to diminished exposure of Env that mediates virus-cell interactions. Inhibition of HIV-1 infection is associated with the presence in EVs of several proteins and metabolites. Our findings demonstrate that the protective effect of Lactobacillus against HIV-1 is, in part, mediated by EVs released by these symbiotic bacteria. If confirmed in vivo, this finding may lead to new strategies to prevent male-to-female sexual HIV-1 transmission.

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The leukotriene B4receptor functions as a novel type of coreceptor mediating entry of primary HIV-1 isolates into CD4-positive cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.16.9530 ◽

1998 ◽

Vol 95 (16) ◽

pp. 9530-9534 ◽

Cited By ~ 40

Author(s):

Christer Owman ◽

Alfredo Garzino-Demo ◽

Fiorenza Cocchi ◽

Mikulas Popovic ◽

Alan Sabirsh ◽

...

Keyword(s):

Chemokine Receptor ◽

Viral Entry ◽

Chemokine Receptors ◽

Structural Similarity ◽

Target Cells ◽

Natural Ligand ◽

Hiv 1 ◽

Entry Efficiency ◽

Receptor Family

The recently cloned human chemoattractant receptor-like (CMKRL)1, which is expressedin vivoin CD4-positive immune cells, has structural homology with the two chemokine receptors C-C chemokine receptor (CCR)5 and C-X-C chemokine receptor (CXCR)4, which serve as the major coreceptors necessary for fusion of the HIV-1 envelope with target cells. In view of the structural similarity, CMKRL1 was tested for its possible function as another HIV-1 coreceptor after stable expression in murine fibroblasts bearing the human CD4 receptor. The cells were infected with 10 primary clinical isolates of HIV-1, and entry was monitored by semiquantitative PCR of viral DNA. The efficiency of the entry was compared with the entry taking place in CD4-positive cells expressing either CCR5 or CXCR4. Seven of the isolates used CMKRL1 for viral entry; they were mainly of the syncytium-inducing phenotype and also used CXCR4. Entry efficiency was higher with CMKRL1 than with CXCR4 for more than half of these isolates. Three of the ten isolates did not use CMKRL1; instead, entry was mediated by both CCR5 and CXCR4. The experiments thus indicate that CMKRL1 functions as a coreceptor for the entry of HIV-1 into CD4-positive cells. In the course of this study, leukotriene B4was shown to be the natural ligand for this receptor (now designated BLTR), which therefore represents a novel type of HIV-1 coreceptor along with the previously identified chemokine receptors. BLTR belongs to the same general chemoattractant receptor family as the chemokine receptors but is structurally more distant from them than are any of the previously described HIV-1 coreceptors.

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Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors

Journal of Virology ◽

10.1128/jvi.01548-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9639-9652 ◽

Cited By ~ 18

Author(s):

Mako Toyoda ◽

Yoko Ogata ◽

Macdonald Mahiti ◽

Yosuke Maeda ◽

Xiaomei T. Kuang ◽

...

Keyword(s):

Viral Replication ◽

Viral Entry ◽

Immune Escape ◽

Elite Controllers ◽

Functional Impairments ◽

Disease Phenotypes ◽

Infected Cells ◽

Entry Receptor ◽

Hiv 1

ABSTRACTHIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified viain vitromutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here,nefclones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nef's ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linkedin vivo. Moreover, impairments in Nef's receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks.IMPORTANCEHIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressurein vivoremains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes, including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicated that downregulation of HIV-1 entry receptors, particularly CCR5, is impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein functionin vivo.

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Migration ofAntigen-Specific T Cells Away from CXCR4-Binding Human ImmunodeficiencyVirus Type 1gp120

Journal of Virology ◽

10.1128/jvi.78.10.5184-5193.2004 ◽

2004 ◽

Vol 78 (10) ◽

pp. 5184-5193 ◽

Cited By ~ 18

Author(s):

Diana M. Brainard ◽

William G. Tharp ◽

Elva Granado ◽

Nicholas Miller ◽

Alicja K. Trocha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Cell ◽

Active Movement ◽

Target Cells ◽

Cell Mediated Immunity ◽

Cell Trafficking ◽

Hiv 1

ABSTRACT Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.

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Factors Important to the Prioritization and Development of Successful Topical Microbicides for HIV-1

Molecular Biology International ◽

10.1155/2012/781305 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

Karen W. Buckheit ◽

Robert W. Buckheit

Keyword(s):

Clinical Trials ◽

Ex Vivo ◽

Sexual Transmission ◽

Virus Transmission ◽

Preclinical Development ◽

Sexual Transmission Of Hiv ◽

The Right ◽

Hiv 1

Significant advancements in topical microbicide development have occurred since the prevention strategy was first described as a means to inhibit the sexual transmission of HIV-1. The lack of clinical efficacy of the first generation microbicide products has focused development attention on specific antiretroviral agents, and these agents have proven partially successful in human clinical trials. With greater understanding of vaginal and rectal virus infection, replication, and dissemination, better microbicide products and delivery strategies should result in products with enhanced potency. However, a variety of development gaps exist which relate to product dosing, formulation and delivery, and pharmacokinetics and pharmacodynamics which must be better understood in order to prioritize microbicide products for clinical development. In vitro, ex vivo, and in vivo models must be optimized with regard to these development gaps in order to put the right product at the right place, at the right time, and at the right concentration for effective inhibition of virus transmission. As the microbicide field continues to evolve, we must harness the knowledge gained from unsuccessful and successful clinical trials and development programs to continuously enhance our preclinical development algorithms.

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