Marvin S. Reitz

An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction

We report, to our knowledge, the first HIV type 1 (HIV-1) transgenic (Tg) rat. Expression of the transgene, consisting of an HIV-1 provirus with a functional deletion of gag and pol, is regulated by the viral long terminal repeat. Spliced and unspliced viral transcripts were expressed in lymph nodes, thymus, liver, kidney, and spleen, suggesting that Tat and Rev are functional. Viral proteins were identified in spleen tissue sections by immunohistochemistry and gp120 was present in splenic macrophages, T and B cells, and in serum. Clinical signs included wasting, mild to severe skin lesions, opaque cataracts, neurological signs, and respiratory difficulty. Histopathology included a selective loss of splenocytes within the periarterial lymphoid sheath, increased apoptosis of endothelial cells and splenocytes, follicular hyperplasia of the spleen, lymphocyte depletion of mesenteric lymph nodes, interstitial pneumonia, psoriatic skin lesions, and neurological, cardiac, and renal pathologies. Immunologically, delayed-type hypersensitivity response to keyhole limpet hemocyanin was diminished. By contrast, Ab titers and proliferative response to recall antigen (keyhole limpet hemocyanin) were normal. The HIV-1 Tg rat thus has many similarities to humans infected with HIV-1 in expression of viral genes, immune-response alterations, and pathologies resulting from infection. The HIV-1 Tg rat may provide a valuable model for some of the pathogenic manifestations of chronic HIV-1 diseases and could be useful in testing therapeutic regimens targeted to stages of viral replication subsequent to proviral integration.

Activation of NF-κB by the Human Herpesvirus 8 Chemokine Receptor ORF74: Evidence for a Paracrine Model of Kaposi's Sarcoma Pathogenesis

ABSTRACT Infection with human herpesvirus 8 (HHV-8), also known as Kaposis sarcoma (KS)-associated herpesvirus, is necessary for the development of KS. The HHV-8 lytic-phase gene ORF74 is related to G protein-coupled receptors, particularly interleukin-8 (IL-8) receptors. ORF74 activates the inositol phosphate/phospholipase C pathway and the downstream mitogen-activated protein kinases, JNK/SAPK and p38. We show here that ORF74 also activates NF-κB independent of ligand when expressed in KS-derived HHV-8-negative endothelial cells or primary vascular endothelial cells. NF-κB activation was enhanced by the chemokine GROα, but not by IL-8. Mutation of Val to Asp in the ORF74 second cytoplasmic loop did not affect ligand-independent signaling activity, but it greatly increased the response to GROα. ORF74 upregulated the expression of NF-κB-dependent inflammatory cytokines (RANTES, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Supernatants from transfected KS cells activated NF-κB signaling in untransfected cells and elicited the chemotaxis of monocytoid and T-lymphoid cells. Expression of ORF74 conferred on primary endothelial cells a morphology that was strikingly similar to that of spindle cells present in KS lesions. Taken together, these data, demonstrating that ORF74 activates NF-κB and induces the expression of proangiogenic and proinflammatory factors, suggest that expression of ORF74 in a minority of cells in KS lesions could influence uninfected cells or latently infected cells via autocrine and paracrine mechanisms, thereby contributing to KS pathogenesis.

Kaposi's Sarcoma-Like Tumors in a Human Herpesvirus 8 ORF74 Transgenic Mouse

ABSTRACT The product of human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is related structurally and functionally to cellular chemokine receptors. ORF74 activates several cellular signaling pathways in the absence of added ligands, and NIH 3T3 cells expressing ORF74 are tumorigenic in nude mice. We have generated a line of transgenic (Tg) mice with ORF74 driven by the simian virus 40 early promoter. A minority (approximately 30%) of the Tg mice, including the founder, developed tumors resembling Kaposis sarcoma (KS) lesions, which occurred most typically on the tail or legs. The tumors were highly vascularized, had a spindle cell component, expressed VEGF-C mRNA, and contained a majority of CD31+ cells. CD31 and VEGF-C are typically expressed in KS. Tumors generally (but not always) occurred at single sites and most were relatively indolent, although several mice developed large visceral tumors. ORF74 was expressed in a minority of cells in the Tg tumors and in a few other tissues of mice with tumors; mice without tumors did not express detectable ORF74 in any tissues tested. Cell lines established from tumors expressed ORF74 in a majority of cells, expressed VEGF-C mRNA, and were tumorigenic in nude mice. The resultant tumors grew rapidly, metastasized, and continued to express ORF74. Cell lines established from these secondary tumors also expressed ORF74 and were tumorigenic. These data strongly suggest that ORF74 plays a role in the pathology of KS and confirm and extend previous findings on the tumorigenic potential of ORF74.

Expression and Characterization of a Single-Chain Polypeptide Analogue of the Human Immunodeficiency Virus Type 1 gp120-CD4 Receptor Complex

ABSTRACT The infection of CD4+ host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.

Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro

We have tested a novel strategy of intracellular immunization to block human immunodeficiency virus (HIV) infection. The expression of a specific antibody within a cell was achieved by transduction of genes that encode for immunoglobulin chains with specificity to viral reverse transcriptase. We demonstrated that inhibition of this enzyme makes cells resistant to HIV infection by blocking an early stage of viral replication. If high efficiency transduction with a stable vector into lymphohaematopoietic stem cells or mature lymphocytes can be achieved, gene transfer-mediated intracellular immunization might be a feasible treatment strategy in AIDS.

Human Herpesvirus 8-Encoded vGPCR Activates Nuclear Factor of Activated T Cells and Collaborates with Human Immunodeficiency Virus Type 1 Tat

ABSTRACT Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposis sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-κB, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNFα). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-κB activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.

Human mesenchymal stem cells inhibit vascular permeability by modulating vascular endothelial cadherin/β-catenin signaling.

The barrier formed by endothelial cells (ECs) plays an important role in tissue homeostasis by restricting passage of circulating molecules and inflammatory cells. Disruption of the endothelial barrier in pathologic conditions often leads to uncontrolled inflammation and tissue damage. An important component of this barrier is adherens junctions, which restrict paracellular permeability. The transmembrane protein vascular endothelial (VE)-cadherin and its cytoplasmic binding partner β-catenin are major components of functional adherens junctions. We show that mesenchymal stem cells (MSCs) significantly reduce endothelial permeability in cocultured human umbilical vascular endothelial cells (HUVECs) and following exposure to vascular endothelial growth factor, a potent barrier permeability-enhancing agent. When grown in cocultures with HUVECs, MSCs increased VE-cadherin levels and enhanced recruitment of both VE-cadherin and β-catenin to the plasma membrane. Enhanced membrane localization of β-catenin was associated with a decrease in β-catenin-driven gene transcription. Disruption of the VE-cadherin/β-catenin interaction by overexpressing a truncated VE-cadherin lacking the β-catenin interacting domain blocked the permeability-stabilizing effect of MSCs. Interestingly, a conditioned medium from HUVEC-MSC cocultures, but not from HUVEC or MSC cells cultured alone, significantly reduced endothelial permeability. In addition, intravenous administration of MSCs to brain-injured rodents reduced injury-induced enhanced blood-brain barrier permeability. Similar to the effect on in vitro cultures, this stabilizing effect on blood-brain barrier function was associated with increased expression of VE-cadherin. Taken together, these results identify a putative mechanism by which MSCs can modulate vascular EC permeability. Further, our results suggest that the mediator(s) of these vascular protective effects is a secreted factor(s) released as a result of direct MSC-EC interaction.

Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER)

Depending on the nature of the costimulation of T lymphocytes, expression of regulatory cytokines and chemokines is either susceptibleor resistant to cyclic AMP (cAMP)‐mediated inhibition. Our data showthat cAMP‐mediated inhibition of endogenously expressed cytokines, which is characteristic for T helper (Th) 1‐ and Th 2‐like phenotypes, correlates with the induction of a potent transcriptional repressor, inducible cAMP early repressor (ICER), in both subsets of T cellsactivated under conditions of suboptimal interleukin‐2 (IL‐2)expression. Importantly, Th‐specific expression of certain chemokinesis also susceptible to cAMP‐mediated transcriptional attenuation. Todetermine whether ICER per se, rather than forskolin‐mediated elevationof intracellular cAMP, is responsible for the observed inhibitoryeffect, we generated transgenic mice expressing ICER under the controlof a lymphocyte‐specific lck promoter. On stimulation, transgenic thymocytes overexpressing ICER exhibited reduced levels of IL‐2 and interferon (IFN)‐γ and failed to express the macrophageinflammatory protein (MIP)‐1α and MIP‐1β genes. Splenic T cellsfrom ICER‐transgenic mice showed a defect in proliferation and lacked amixed lymphocyte reaction response, implying that ICER‐mediatedinhibition of cytokine and chemokine expression might play an importantrole in T‐cell inactivation.

Tumorigenesis by Human Herpesvirus 8 vGPCR Is Accelerated by Human Immuodeficiency Virus Type 1 Tat

ABSTRACT Human herpesvirus 8 (HHV-8), also called Kaposis sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-κB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1 tat (but not a transactivation-defective mutant) into these tumor cells increases NF-κB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.

A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice

The rational design of new therapies against HIV-1 necessitates an improved understanding of the mechanisms underlying the production of ineffective immune responses to HIV-1 in most infected individuals. This report shows that the CD8+ T cell responses to gp120 were greatly diminished in mice vaccinated with a bicistronic gp120-Tat DNA vaccine, compared with those induced by a DNA vaccine encoding gp120 alone. The CD8+ T cell responses induced by the latter included strong gp120-specific IFN-γ secretion and protective antiviral immunity against challenge by a vaccinia-env pseudotype. The degree to which Tat influenced CD8+ T cell responses depended on the bioactivity of Tat. Thus, a bicistronic DNA vaccine that expresses gp120 and a truncated Tat defective for LTR activation elicited strong IFN-γ -secreting CD8+ T cell responses to gp120 but conferred only marginal protection against the vaccinia-env challenge. The effect of Tat was completely blocked, however, by immunization with inactivated Tat protein before vaccination with the bicistronic gp120-Tat DNA vaccine.