Hélène Dutartre

General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2′-Modified Nucleotide Analogues

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2′-C-methyl CTP (2′-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2′-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2′-modified nucleotide analogues. 2′-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2′-O-methyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that “2′-conformer” analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.

A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

ABSTRACT We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.

Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl β‐glucoside and sucrose utilization respectively. Their RNA‐binding activity is carried by the N‐terminal domain (CAT), and is regulated by two similar C‐terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl β‐glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT‐PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP‐mediated inhibition process was found to essentially involve His100 of LicT‐PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by P∼BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII‐mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.

The Imidazopyrrolopyridine Analogue AG110 Is a Novel, Highly Selective Inhibitor of Pestiviruses That Targets the Viral RNA-Dependent RNA Polymerase at a Hot Spot for Inhibition of Viral Replication

ABSTRACT Ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-carboxylate (AG110) was identified as a potent inhibitor of pestivirus replication. The 50% effective concentration values for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect, viral RNA synthesis, and production of infectious virus were 1.2 ± 0.5 μM, 5 ± 1 μM, and 2.3 ± 0.3 μM, respectively. AG110 proved inactive against the hepatitis C virus and a flavivirus. AG110 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carry the E291G mutation in the viral RNA-dependent RNA polymerase (RdRp). AG110-resistant virus is cross-resistant to the cyclic urea compound 1453 which also selects for the E291G drug resistance mutation. Moreover, BVDV that carries the F224S mutation (because of resistance to the imidazopyridine 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine [BPIP]and VP32947) is also resistant to AG110. AG110 did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). Molecular modeling revealed that E291 is located in a small cavity near the tip of the finger domain of the RdRp about 7 Å away from F224. Docking of AG110 in the crystal structure of the BVDV RdRp revealed several potential contacts including with Y257. The E291G mutation might enable the free rotation of Y257, which might in turn destabilize the backbone of the loop formed by residues 223 to 226, rendering more mobility to F224 and, hence, reducing the affinity for BPIP and VP32947. It is concluded that a single drug-binding pocket exists within the finger domain region of the BVDV RdRp that consists of two separate but potentially overlapping binding sites rather than two distinct drug-binding pockets.

Viral Source-Independent High Susceptibility of Dendritic Cells to Human T-Cell Leukemia Virus Type 1 Infection Compared to That of T Lymphocytes

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1)-infected CD4+ T cells and dendritic cells (DCs) are present in peripheral blood from HTLV-1 carriers. While T-cell infection requires cell-cell contact, DCs might be infected with cell-free virus, at least in vitro. However, a thorough comparison of the susceptibilities of the two cell types to HTLV-1 infection using cell-associated and cell-free viral sources has not been performed. We first determined that human primary monocyte-derived dendritic cells (MDDCs) were more susceptible to HTLV-1 infection than their autologous lymphocyte counterparts after contact with chronically infected cells. Next, a comparison of infection efficiency using nonconcentrated or concentrated supernatants from infected cells as well as purified viral biofilm was performed. Integrated provirus was found after exposure of MDDCs or primary lymphocytes to viral biofilm but not to a viral supernatant. Using a large series of primary cell samples (n = 21), we demonstrated a higher proviral load in MDDCs exposed to viral biofilm than in lymphocytes. This higher susceptibility is correlated to a higher expression of neuropilin-1 on MDDCs than on autologous activated T lymphocytes. Moreover, we show that MDDCs infected with viral biofilm can transmit the virus to lymphocytes. In conclusion, MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro, supporting a model in which DC infection might represent an important step during primo-infection in vivo. IMPORTANCE HTLV-1 is able to infect several cell types, but viral DNA is mainly found in T lymphocytes in vivo. This supports a model in which T lymphocytes are the main target of infection. However, during the primo-infection of new individuals, incoming viruses might first encounter dendritic cells (DCs), the specialized immune cells responsible for the antiviral response of the host. HTLV-1 cell-free purified viruses can infect dendritic cells in vitro, while T-cell infection is restricted to cell-to-cell transmission. In order to understand the sequence of HTLV-1 dissemination, we undertook a direct comparison of the susceptibilities of the two cell types using cell-associated and cell-free viral sources. We report here that MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro and are able to efficiently transmit the virus to lymphocytes. Our results suggest that DCs may represent a true viral reservoir, as the first cell type to be infected in vivo.

Alpha Interferon Restricts Human T-Lymphotropic Virus Type 1 and 2 De Novo Infection through PKR Activation

ABSTRACT Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses.

ADAR1 enhances HTLV-1 and HTLV-2 replication through inhibition of PKR activity

BackgroundThe role of innate immunity in general and of type I interferon (IFN-I) in particular in HTLV-1 pathogenesis is still a matter of debate. ADAR1-p150 is an Interferon Stimulated Gene (ISG) induced by IFN-I that can edit viral RNAs. We therefore investigated whether it could play the role of an anti-HTLV factor.ResultsWe demonstrate here that ADAR1 is also expressed in the absence of IFN stimulation in activated primary T-lymphocytes that are the natural target of this virus and in HTLV-1 or HTLV-2 chronically infected T-cells. ADAR1 expression is also increased in primary lymphocytes obtained from HTLV-1 infected individuals. We show that ADAR1 enhances HTLV-1 and HTLV-2 infection in T-lymphocytes and that this proviral effect is independent from its editing activity. ADAR1 expression suppresses IFN-α inhibitory effect on HTLV-1 and HTLV-2 and acts through the repression of PKR phosphorylation.DiscussionThis study demonstrates that two interferon stimulated genes, i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. The balanced expression of those proteins could determine the fate of the viral cycle in the course of infection.

HTLV-1, the Other Pathogenic Yet Neglected Human Retrovirus: From Transmission to Therapeutic Treatment

Going back to their discovery in the early 1980s, both the Human T-cell Leukemia virus type-1 (HTLV-1) and the Human Immunodeficiency Virus type-1 (HIV-1) greatly fascinated the virology scene, not only because they were the first human retroviruses discovered, but also because they were associated with fatal diseases in the human population. In almost four decades of scientific research, both viruses have had different fates, HTLV-1 being often upstaged by HIV-1. However, although being very close in terms of genome organization, cellular tropism, and viral replication, HIV-1 and HTLV-1 are not completely commutable in terms of treatment, especially because of the opposite fate of the cells they infect: death versus immortalization, respectively. Nowadays, the antiretroviral therapies developed to treat HIV-1 infected individuals and to limit HIV-1 spread among the human population have a poor or no effect on HTLV-1 infected individuals, and thus, do not prevent the development of HTLV-1-associated diseases, which still lack highly efficient treatments. The present review mainly focuses on the course of HTLV-1 infection, from the initial infection of the host to diseases development and associated treatments, but also investigates HIV-1/HTLV-1 co-infection events and their impact on diseases development.

Quantitative Analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) Infection Using Co-Culture with Jurkat LTR-Luciferase or Jurkat LTR-GFP Reporter Cells

Unlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression.