Rebeca Geffin

Functional and Structural Defects in HIV Type 1 nef Genes Derived from Pediatric Long-Term Survivors

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

A longitudinal assessment of autologous neutralizing antibodies in children perinatally infected with human immunodeficiency virus type 1

The evolution of autologous neutralizing antibodies to sequential human immunodeficiency virus type 1 (HIV-1) isolates was studied in a population of 16 children who were perinatally infected with human immunodeficiency virus type 1. The cohort included seven children with rapid disease progression (RP) and nine who had nonrapid disease progression (NRP). Four of the NRP after 6 months of age harbored viruses that could be neutralized by antibodies found in autologous contemporaneous plasma (titers up to 1:640) while the majority of longitudinally collected viruses from five NRP were resistant to neutralization with contemporaneous plasma. Because of their shorter survival, only five of the RP had studies after 6 months of age; three of the five had neutralizing antibodies to contemporaneous virus isolates and the highest titers were 1:20. The highest titers in RP (up to 1:160) occurred in specimens obtained prior to 6 months of age but these were most likely of maternal origin. Most isolates that were not neutralized by contemporaneous plasma could be neutralized using noncontemporaneous plasma obtained months to years after the virus isolates. These autologous noncontemporaneous neutralizing antibodies persisted for years, had titers that were higher to viruses isolated at younger ages, and were generally more potent in children with NRP than RP. Demonstration of neutralizing antibodies to viruses previously resistant to neutralization by contemporaneous plasma suggests a continuous evolution of virus variants in vivo that are able to escape the effect of neutralizing antibodies.

Maturing neurons are selectively sensitive to human immunodeficiency virus type 1 exposure in differentiating human neuroepithelial progenitor cell cultures.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neuronal injury manifested by dendritic pruning, aberrant neurofilament metabolism, and decreased synaptic density. The central nervous system (CNS) responds to neuronal injury by differentiating new neurons and astrocytes from resident populations of multipotent neuroepithelial progenitor cells (NEP) located in regions such as the subventricular zone or hippocampus. In vitro studies have demonstrated that the HIV-1 virion or envelope glycoprotein gp120 can injure differentiated human neurons and astrocytes, suggesting that HIV-1 proteins could similarly injure NEP or NEP-derived glial and neuronal lineage-committed precursor cells. To answer this question, human fetal brain-derived “neurospheres” containing NEP and NEP-derived precursor cells were cultured in low serum differentiation medium containing lymphotropic HIV-1(SF2), macrophage-tropic HIV-1(SF128A), or recombinant gp120SF2 from HIV-1(SF2). These experiments indicate that exposure to HIV-1 does not affect the ability of the NEP to differentiate into cells expressing either astrocyte-specific or neuron-specific cytoskeletal antigens. However prolonged exposure to HIV-1 does selectively decrease expression of neuronal antigens (microtubule β-III-tubulin and intermediate filament neurofilament-L) but not astrocyte antigens (intermediate filament glial fibrillary acidic protein). The effects of continuous exposure to HIV-1 or gp120 may result from injury to developing neurons and/or impairment of the neuronal developmental process itself. By depressing neuronal microtubule and neurofilament protein expression, HIV-1 and gp120 exposure compromise the potential for postmitotic neuronal dendrite and axon development.

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequence-specific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4+ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4+ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4+ cells expressing the RNase P-associated U5 EGS (560) molecule.

Apolipoprotein E-Dependent Differences in Innate Immune Responses of Maturing Human Neuroepithelial Progenitor Cells Exposed to HIV-1

HIV enters the brain early during infection and induces a chronic inflammatory state that can result in neurological abnormalities in a subset of infected individuals. To investigate the effects of HIV exposure on neurogenesis and neuronal survival in the brain, we have used a model system consisting of human neuroepithelial progenitor (NEP) cells that undergo directed differentiation into astrocytes and neurons in vitro. Changes in gene expression in NEP cultures as a result of HIV exposure were investigated using gene expression microarrays with the Illumina HT-12 V4_0_R1 platform array. Through this approach, we identified a group of genes specifically upregulated by exposure to virus that are strongly related to interferon induced responses and antigen presentation. When the data were stratified by their apolipoprotein genotype, this innate immune response was more robust in the apolipoprotein E3/E3 genotype cultures than in the apolipoprotein E3/E4 counterparts. Biological processes as defined by the gene ontology (GO) program were also differently affected upon virus exposure in cultures of the two genotypes, particularly those related to antigen presentation and the actions of interferons. Differences occurred in both in numbers of genes affected and their significance in the GO processes in which they participate, with apoE3/E3 > apoE3/E4. These data suggest that maturing NEP cultures recognize HIV and respond to it by mounting an innate immune response with a vigor that is influenced by the apolipoprotein E genotype of the cells.

Depressed neurofilament expression associates with apolipoprotein E3/E4 genotype in maturing human fetal neurons exposed to HIV-1

Exposure of differentiating human neural progenitor cells (NEP) to HIV-1 results in a neuronal “failure to thrive” phenotype characterized by a relative decrease in neurofilament-light (NF-L) expression. However, when NEP were segregated by their apolipoprotein E genotype, differentiating apolipoprotein E3/E4 cells showed reduced NF-L expression upon HIV-1 exposure, but differentiating apolipoprotein E3/E3 or apolipoprotein E4/E4 cells did not. These data suggest that apolipoprotein E genotype is a host factor that could affect the development of neurocognitive dysfunction in HIV-1 infected individuals.

Innate immune responses to HIV infection in the central nervous system

Human immunodeficiency virus (HIV) invades the brain early during infection and generates a chronic inflammatory microenvironment that can eventually result in neurological disease, even in the absence of significant viral replication. Thus, HIV-1 infection of the brain has been characterized both as a neuroimmunological and neurodegenerative disorder. While the brain and central nervous system (CNS) have historically been regarded as immune privileged or immunologically quiescent, newer concepts of CNS immunity suggest an important if not defining role for innate immune responses generated by glial cells. Innate immunity may be the first line of defense against HIV infection of the brain and CNS, with multiple cellular elements providing responses that can be anti-viral and neuroprotective, but also potentially neurotoxic, impairing neurogenesis and promoting neuronal apoptosis. To investigate the effects of HIV exposure on neurogenesis and neuronal survival, we have studied the responses of human neuroepithelial progenitor (NEP) cells, which undergo directed differentiation into astrocytes and neurons in vitro. We identified a group of genes that were differentially expressed in NEP-derived cells during virus exposure. This included genes that are strongly related to interferon-induced responses and antigen presentation. Moreover, we observed that the host factor apolipoprotein E influences the innate immune response expressed by these cells, with a more robust response in the apolipoprotein E3/E3 genotype cultures compared to the apolipoprotein E3/E4 counterparts. Thus, neuroepithelial progenitors and their differentiated progeny recognize HIV and respond to it by mounting an innate immune response with a vigor that is influenced by the host factor apolipoprotein E.

A two-culture method for exposure of human brain organotypic slice cultures to replicating human immunodeficiency virus type 1.

To evaluate the effect of HIV-1 virus on neural cells, we have developed a method to culture human fetal organotypic brain slices in the presence of live virus. Brain slices were placed on semipermeable hydrophilic membrane inserts, resting on top of wells that contain cultured H9 T-cells chronically producing HIV-1. This system allows free exposure of the brain slices to HIV-1, HIV-1 proteins, and other molecules released by the infected T-cells. After specific lengths of time in culture, slices were stained for viability with Calcein-AM and propidium iodide, for neural cell markers such as GFAP, nestin and β-III-tubulin, tested for cell proliferation, and analyzed by fluorescent and confocal microscopy. When cultured in the presence of neural progenitor medium lacking serum, slices were viable and maintained active cell replication for at least 3 weeks in culture, without significant cell death. By comparison with slices co-cultured with uninfected T-cells or with medium alone, slices cultured in the presence of HIV-1 showed increased nestin and GFAP. Moreover, in slices exposed to HIV-1-producing H9 cells, regions of nestin stain were, over time in culture, replaced with GFAP stain. This suggested the process of gliosis often found in brains of HIV-1 infected individuals. This co-culture method can be used to model the dynamics and the microenvironment of brain tissue exposed to HIV-1 and can potentially be used to test therapies directed at preventing HIV-1-induced neural damage.

Fingolimod induces neuronal-specific gene expression with potential neuroprotective outcomes in maturing neuronal progenitor cells exposed to HIV

Fingolimod (FTY720), a structural analogue of sphingosine, targets sphingosine-1-phosphate receptor signaling and is currently an immunomodulatory therapy for multiple sclerosis. Fingolimod accesses the central nervous system (CNS) where its active metabolite, fingolimod phosphate (FTY720-P), has pleotropic neuroprotective effects in an inflammatory microenvironment. To investigate potential neuronal-specific mechanisms of fingolimod neuroprotection, we cultured the human neuronal progenitor cell line, hNP1, in differentiation medium supplemented with HIV- or Mock-infected supernatants, with or without FTY720-P. Gene expression was investigated using microarray and functional genomics. FTY720-P treatment increased differentially expressed (DE) neuronal genes by 33% in HIV-exposed and 40% in Mock-exposed cultures. FTY720-P treatment broadened the functional profile of DE genes in HIV-exposed versus Mock-exposed neurons, including not only immune responses but also transcriptional regulation and cell differentiation, among others. FTY720-P treatment downregulated the gene for follistatin, the antagonist of activin signaling, in all culture conditions. FTY720-P treatment differentially affected both glycolysis-related and immune response genes in Mock- or HIV-exposed cultures, significantly upregulating 11 glycolysis-related genes in HIV-exposed neurons. FTY720-P treatment also differentially upregulated genes related to innate immune responses and antigen presentation in Mock-exposed and more so in HIV-exposed neurons. However, in HIV-exposed neurons, FTY720-P depressed the magnitude of differential expression in almost half the genes, suggesting an anti-inflammatory potential. Moreover, in HIV-exposed neurons, FTY720-P reduced expression of the amyloid precursor protein (APP) gene, resulting in reduced expression of the APP protein. This study provides new evidence that fingolimod alters neuronal gene expression in inflammatory, viral-infected microenvironments, with the potential for neuroprotective effects.