Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-alpha/beta). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-alpha/beta pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-alpha/beta signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-alpha/beta receptor complex to block downstream signaling.
Human immunodeficiency virus type 1 plasma viruses from 29 entry inhibitor-naive patients were characterized for their susceptibilities to T-20, AMD3100, and RANTES. A strikingly wide range of susceptibilities to T-20 was observed that was influenced by coreceptor usage but not by the susceptibilities of the viruses to inhibitors that target the chemokine receptors or by polymorphisms in the gp41 N helix.The human immunodeficiency virus type 1 (HIV-1) entry process (2,19,27) can be inhibited by several drugs (3,14,22,23), which belong to three groups according to the step they inhibit: (i) inhibitors of the interaction between the viral surface glycoprotein (gp120) and CD4, which target the CD4-binding site on gp120; (ii) inhibitors of the interaction between gp120 and CCR5 or CXCR4 (e.g., chemokines and their derivatives or small organic molecules that antagonize chemokine receptor activity); and (iii) fusion inhibitors, which are peptides derived from the sequence of the viral transmembrane glycoprotein (gp41) that prevent the formation of a hairpin structure required for membrane fusion. One of these peptides, T-20 (enfuvirtide), is currently being evaluated in phase III clinical trials (15,25,26). The optimization of treatment strategies that include entry inhibitors will rely on the availability of methods capable of determining baseline viral susceptibility and acquired resistance to these drugs. In addition, the characterization of the determinants of baseline susceptibility and of acquired resistance to entry inhibitors may provide valuable information on the viral entry process and on the precise mechanism of action of these drugs.We have developed a recombinant virus assay that permits the assessment of viral susceptibility to entry inhibitors. We modified a pNL4-3 molecular clone by deleting the region of the envelope gene encoding gp120 and the ectodomain of gp41 (positions 6480 to 8263) and replacing it with a linker that contains a unique MluI restriction site (vector 43-⌬env). Recombinant virus was produced by cotransfection of 293-T cells with the MluI-linearized 43-⌬env vector and a reverse transcription-PCR product, amplified from patient plasma samples, which encompasses the deleted region and carries short overlaps that allow homologous recombination. Virus-containing supernatants were used to infect subconfluent U373MG-CD4 cells expressing either CCR5 or CXCR4 (17), in the absence or in the presence of increasing concentrations of entry inhibitors. These target cells carry an HIV-1 long terminal repeat-lacZ cassette, which allows the quantification of single cycle infectivity by a colorimetric assay based on HIV-1 Tat-induced expression of -galactosidase (24). The concentrations inhibiting 50% of virus infectivity (IC 50 s) were calculated by using the median-effect equation (6).The recombinant virus assay was first used to determine the baseline susceptibilities of subtype B primary viruses to the fusion inhibitor T-20 (American Peptide Company, Inc., Sunnyvale, Calif.). Plasma samples sel...
Acquired human immunodeficiency virus type 1(HIV-1) resistance to the fusion inhibitor enfuvirtide (ENF)is primarily associated with mutations within the highly conserved first heptad repeat (HR1) region of gp41. Viral env sequences, however, are remarkably variable, and the envelope genetic background could have an important impact on optimal expression of HR1 mutations. We have examined the genetic evolution of env sequences, ENF susceptibility, and Env replicative capacity in patients failing ENF treatment. Sequential plasma-derived virus populations, obtained from six patients initiating ENF treatment as part of a salvage therapy, were studied using a recombinant phenotypic assay evaluating the entire gp120 and the gp41 ectodomains. Regardless of major differences in the baseline ENF susceptibilities, viral populations with similar phenotypic ENF resistance (50% inhibitory concentration, >3,000 ng/ml) were selected under treatment in four of six patients. As expected, in all patients ENF-resistant viruses harbored one or more HR1 mutations (positions 36, 38, and 43). Interestingly, in five patients the emergence of resistance mutations was not associated with reduced Env replicative capacity. Phylogenetic analysis of env sequences in sequential samples from two patients showed that the HR1 mutations had emerged in the context of env quasi-species that were different from those prevalent at baseline. Thus, the envelope genetic context appears to play a critical role in the selection of HR1 mutations and the expression of ENF resistance, thereby conditioning the evolution of HIV-1 under fusion inhibitor selective pressure.Considerable effort is currently being devoted to the development of antiviral agents able to prevent human immunodeficiency virus type 1 (HIV-1) entry into target cells. The HIV-1 entry process is mediated by the trimeric viral envelope glycoproteins (Env) exposed at the surface of the virion (45). The HIV-1 entry is a multistep process (11,14,45). The sequential interaction of the surface subunit, gp120, with CD4 and a chemokine receptor (CCR5 or CXCR4) exposed on the cell membrane triggers conformational changes in the ectodomain of the transmembrane subunit, gp41, which ultimately lead to fusion between the viral and host cell membranes. Like most of the retroviral transmembrane proteins, the ectodomain of gp41 contains an N-terminal fusion peptide followed by two heptad repeat domains (HR1 and HR2), which are connected by a non-helical loop region of 25 to 30 amino acids. Membrane fusion is currently thought to result from the insertion of the fusion peptide into the cellular membrane, and the formation of a six-helix bundle in which the central trimeric HR1 coiled-coil forms three hydrophobic grooves onto which three HR2 domains pack in reverse orientation (4,5,40,43).Although several HIV-1 entry inhibitors have been evaluated in clinical trials and have shown promising prospects for therapy (1, 28), the only entry inhibitor licensed to date is the fusion inhibitor enfuvirtide (ENF; als...
Four heavily antiretroviral-experienced HIV-infected patients had significant plasma HIV-RNA reductions (>1 log) after beginning an Enfuvirtide (ENF)-based rescue regimen. However, all had viral rebound shortly thereafter, sustaining high levels of plasma viremia over 80 weeks. These patients developed rapidly genotypic and phenotypic resistance to ENF. Mutations within the HR1 env region were selected (N43D in three and G36V/D in one), resulting in high-level phenotypic resistance to ENF. Interestingly, two patients had a sustained CD4+ T-cell increase and two maintained stable CD4+ T-cell counts despite virologic failure under ENF. The possible mechanisms involved in this response were examined. Changes in virus tropism from R5 to R5/X4 were observed in two patients, in parallel with increases in ENF phenotypic resistance. Low levels of T-cell activation, T-cell turnover, and cytotoxic T lymphocyte (CTL) activity were found in all four patients. An overall increase in the proportion of viruses released from cells of the macrophage lineage was observed. In summary, single mutations at the HR1 env region result in significant loss of susceptibility to ENF. Despite virologic failure, these patients may maintain elevated CD4+ counts through a reduction in their overall immune activation.
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