Nancy Vander Heyden

Inhibition of HIV and SIV infectivity by blockade of α-glucosidase activity

Abstract Processing of HIV and SIV envelope oligosaccharides is critical for proper intracellular trafficking and function. An inhibitor of α-glucosidases I and 11, N -butyl deoxynojirimycin ( N -BuDNJ), retards HIV-1 and SIVmac spread in lymphocytes and monocytes by diminishing virus infectivity, and also causes a reduction in syncytia formation between infected cells and uninfected lymphocytes. N-BuDNJ retards envelope processing from the precursor form to the mature surface (SU) and transmembrane proteins in HIV-1- and SIVmac-infected cells, as well as in cells infected with vaccinia-HIV-1 envelope recombinant virus. However, no significant reduction is seen in the amount of SU in released virus particles, though the virus particle-associated SU from N-BuDNJ-treated cells has an altered electrophoretic mobility. In contrast, N-BuDNJ had no effect on GAG protein synthesis and processing. These findings demonstrate a critical requirement for oligosaccharide processing by α-glucosidases I and It for HIV-1 and SIVmac envelope processing and fusogenicity.

The C-Terminal Proline-Rich Tail of Human Immunodeficiency Virus Type 2 Vpx Is Necessary for Nuclear Localization of the Viral Preintegration Complex in Nondividing Cells

ABSTRACT Human immunodeficiency virus type 2 (HIV-2), like other lentiviruses, is capable of infecting nondividing T cells and macrophages. The present work shows that in HIV-2-infected cells, Vpx is necessary for efficient nuclear import of the preintegration complex. In agreement with this finding, the subcellular localization of a GFP-Vpx fusion protein was found to be predominantly nuclear. However, deletion of the proline-rich C-terminal 11 residues of Vpx resulted in a shift of the fusion protein to the cytoplasm. Furthermore, the same deletion in the context of the provirus resulted in a decrease in nuclear import of the preintegration complex and attenuated replication in macrophages.

Alpha Interferon Inhibits Human T-Cell Leukemia Virus Type 1 Assembly by Preventing Gag Interaction with Rafts

ABSTRACT Alpha-2a interferon (IFN-α2a) has beneficial clinical effects on human T-cell leukemia virus type 1 (HTLV-1) infection, but its antiviral mechanism of action is unknown. Antiviral effects of IFN-α2a were studied in 293T cells expressing HTLV-1 proviral DNA and in HTLV-1-infected cells (HOS/PL, MT2, and HUT102). In 293T cells, an 50% inhibitory concentration of 10 U of IFN-α2a/ml was determined by p19 antigen ELISA. Analysis of IFN-treated cells demonstrated no defect in viral protein synthesis but did show a decrease in the level of released virus, as determined by immunoblot assays. Electron microscopy studies of IFN-treated cells revealed neither a defect in the site of virus budding nor tethering of virus particles at the plasma membrane, thus arguing against an effect on virus release. Cell fractionation studies and confocal microscopy showed no effect of IFN on Gag association with membranes. However, the level of Gag association with lipid rafts was decreased, suggesting a role of IFN in inhibiting HTLV-1 assembly.

Analysis of the function of viral protein X (VPX) of HIV-2

To investigate the function of vpx, a gene in HIV-2 and SIV, but not in HIV-1, three site-directed mutants (pMX) were constructed from a functional proviral HIV-2 plasmid clone (pSE). Transfection of COS-1 cells with all three mutants as well as pSE gave rise to equivalent amounts of virus. Each virus could be passaged in H9 and CEM lymphoid cell lines, peripheral blood lymphocytes, and monocytes with equal efficiency and demonstrated similar cytopathic effects. Hybridization data with DAN from the infected cells demonstrated the presence of similar levels of viral sequences and the mutations in each of the MX-infected cell lines. Immunoprecipitation analysis demonstrated a 16-kDa VPX protein in cells infected with SE virus, as well as in the virus particles, but not in cells infected with MX viruses or the particles themselves. However, equivalent levels of gag and env proteins were demonstrated in all infected cells and virion preparations. These data suggest that VPX is dispensable for virus replication and cytopathicity.

Rapid phenotypic drug susceptibility assay for HIV-1 with a CCR5 expressing indicator cell line

Phenotypic drug susceptibility assays of human immunodeficiency virus type 1 (HIV-1) isolates generally use time-consuming, expensive assays with peripheral blood mononuclear cells. A new HIV-1 indicator cell line, MAGI-CCR5, has been developed and applied for this purpose. This cell line expresses human CD4, the two major HIV-1 coreceptors, CCR5 and CXCR4, the reporter gene beta-galactosidase driven by the HIV-1 LTR, and quantitates infection within 48 h. A panel of reference strains and primary HIV-1 isolates were all found to infect this cell line. Susceptibility assays with a nucleoside (zidovudine, ZDV) and a non-nucleoside reverse transcriptase inhibitor (nevirapine, NVP) were performed with reference and primary isolates. The assay was modified into two steps for protease inhibitor (indivinavir, IDV and ritonavir, RTV) susceptibility assays. Primary isolates derived from drug naive patients displayed mean baseline 50% effective concentrations (EC50) of 0.14 microM for ZDV, 0.33 microM for NVP, and 0.02 microM for IDV. Isolates derived from patients under treatment displayed increased EC50 concentrations. The MAGI-CCR5 cell line offers a rapid, efficient, and reproducible method of testing a wide range of HIV-1 isolates for drug susceptibility.

Interaction of Human Immunodeficiency Virus Type 2 Vpx and Invariant Chain

ABSTRACT Vpx is a virion-associated protein of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency viruses. The yeast two-hybrid system was used to identify invariant chain (Ii) as a cellular protein that interacts with HIV-2 Vpx. Vpx-Ii interaction was confirmed in cell-free reactions using bacterially expressed glutathione S-transferase fusion proteins and by coimmunoprecipitation in transfected and infected cells. In chronically infected cells expressing Vpx, Ii levels were markedly decreased, presumably due to enhanced degradation. These findings suggest that Vpx may disrupt major histocompatibility complex class II antigen presentation.