Due to their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of lethal mutagenesis that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base-pairs in DNA duplexes containing or not the nucleoside analogues: a promising candidate should display a small destabilisation of the matched base-pair compared to the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base-pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesised base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture since only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency, by 3.1 and 3.4 fold, respectively.
Plasmacytoid dendritic cells (pDC) expressing Fc?RIIa are antigen-presenting cells able to link innate and adaptive immunity and producing various cytokines and chemokines. Although highly restricted, they are able to replicate HIV-1. We determined the activity of anti-HIV-1 neutralizing antibodies (NAb) and non-neutralizing inhibitory antibodies (NNIAb) on the infection of primary pDC by HIV-1 primary isolates and analyzed cytokines and chemokines production. Neutralization assay was performed with primary pDC in the presence of serial antibodies (Ab) concentrations. In parallel, we measured the release of cytokines and chemokines by ELISA and CBA Flex assay. We found that NAb, but not NNIAb, inhibit HIV-1 replication in pDC. This inhibitory activity was lower than that detected for myeloid dendritic cells (mDC) infection and independent of Fc?RIIa expressed on pDC. Despite the complete protection, IFN-? production was detected in the supernatant of pDC treated with NAb VRC01, 4E10, PGT121, 10-1074, 10E8, or polyclonal IgG44 but not with NAb b12. Production of MIP-1?, MIP-1?, IL-6, and TNF-? by pDC was also maintained in the presence of 4E10, b12 and VRC01. These findings suggest that pDC can be protected from HIV-1 infection by both NAb and IFN-? release triggered by the innate immune response during infection.
Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-?) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-? secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.
Human immunodeficiency virus type 1 (HIV-1) replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and simian immunodeficiency virus (SIV) from sooty mangabeys (SIVsm) or from macaques (SIVmac) but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T and B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome under coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC-lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with increased HIV-1 replication in DCs. Moreover, in infected DC-T lymphocyte cocultures, DCs acquired maturation status and secreted type 1 interferon (alpha interferon [IFN-?]). The blockade of DC-lymphocyte cross talk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, cross talk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture.
The chronology of HIV infection in mucosal tissue after sexual transmission is unknown. Several potential HIV target cells are present at these sites, including dendritic cells, macrophages, and CD4(+) T lymphocytes. Dendritic cells and macrophages are antigen-presenting cells (APCs) and are thus involved in cross-talk with T cells. This close contact may favor efficient HIV-1 transfer to T lymphocytes, resulting in rapid HIV-1 dissemination.
The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix ?H in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.
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.
With the goal of limiting HIV-1 proliferation by increasing the mutation rate of the viral genome, we synthesized a series of pyrimidine nucleoside analogues modified in position 5 of the aglycone moiety but unmodified on the sugar part. The synthetic strategies allow us to prepare the targeted compounds directly from commercially available nucleosides. All compounds were tested for their ability to reduce HIV-1 proliferation in cell culture. Two of them (5-hydroxymethyl-2-dU (1c) and 5-hydroxymethyl-2-dC (2c)) displayed a moderate antiviral activity in single passage experiments. The same two compounds plus two additional ones (5-carbamoyl-2-dU (1a) and 5-carbamoylmethyl-2-dU (1b)) were potent inhibitors of HIV-1 RT activity in serial passage assays, in which they induced a progressive loss of HIV-1 replication. In addition, viruses collected after seven passages in the presence of 1c and 2c replicated very poorly after withdrawal of these compounds, consistent with the accumulation of deleterious mutations in the HIV-1 genome.
Nine anionic water-soluble calixarene species, incorporating sulfonate, carboxylate or phosphonate groups, six of them incorporating two 2,2-bithiazole subunits in alternate position at the lower rim, have been synthesised and evaluated as anti-HIV agents on various HIV strains and cells of the lymphocytic lineage (HIV-1 III B/MT4, HIV-1 LAI/CEM-SS, HIV-1 Bal/PBMC), using AZT as reference compound. A toxicity was detected for a minority of compounds on PBMC whereas for the others no cellular toxicity was measured at concentrations up to 100 microM. Most of the compounds have an antiviral activity in a 10-50 microM range, and one of them, sulfonylated, displays its activity, whatever the tropism of the virus, at a micromolar concentration.
Dendritic cells (DCs) support only low levels of HIV-1 replication, but have been shown to transfer infectious viral particles highly efficiently to neighboring permissive CD4 T lymphocytes. This mode of cell-to-cell HIV-1 spread may be a predominant mode of infection and dissemination. In the present study, we analyzed the kinetics of fusion, replication, and the ability of HIV-1-specific Abs to inhibit HIV-1 transfer from immature DCs to autologous CD4 T lymphocytes. We found that neutralizing mAbs prevented HIV-1 transfer to CD4 T lymphocytes in trans and in cis, whereas nonneutralizing Abs did not. Neutralizing Abs also significantly decreased HIV-1 replication in DCs, even when added 2 hours after HIV-1 infection. Interestingly, a similar inhibition of HIV-1 replication in DCs was detected with some nonneutralizing Abs and was correlated with DC maturation. We suggest that the binding of HIV-1-specific Abs to Fc?Rs leads to HIV-1 inhibition in DCs by triggering DC maturation. This efficient inhibition of HIV-1 transfer by Abs highlights the importance of inducing HIV-specific Abs by vaccination directly at the mucosal portal of HIV-1 entry to prevent early dissemination after sexual transmission.
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