Paul E. Kennedy

HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.

A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated “fusin,” is a putative G protein-coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target cells. Fusin messenger RNA levels correlated with HIV-1 permissiveness in diverse human cell types. Fusin acted preferentially for T cell line-tropic isolates, in comparison to its activity with macrophage-tropic HIV-1 isolates.

CC CKR5: A RANTES, MIP-1α, MIP-1β Receptor as a Fusion Cofactor for Macrophage-Tropic HIV-1

Human immunodeficiency virus-type 1 (HIV-1) entry requires fusion cofactors on the CD4+ target cell. Fusin, a heterotrimeric GTP-binding protein (G protein)-coupled receptor, serves as a cofactor for T cell line-tropic isolates. The chemokines RANTES, MIP-1α, and MIP-1β, which suppress infection by macrophage-tropic isolates, selectively inhibited cell fusion mediated by the corresponding envelope glycoproteins (Envs). Recombinant CC CKR5, a G protein-coupled receptor for these chemokines, rendered CD4-expressing nonhuman cells fusion-competent preferentially with macrophage-tropic Envs. CC CKR5 messenger RNA was detected selectively in cell types susceptible to macrophage-tropic isolates. CC CKR5 is thus a fusion cofactor for macrophage-tropic HIV-1 strains.

CD46 Is a Cellular Receptor for Human Herpesvirus 6

Human herpesvirus 6 (HHV-6) is the etiologic agent of exanthema subitum, causes opportunistic infections in immunocompromised patients, and has been implicated in multiple sclerosis and in the progression of AIDS. Here, we show that the two major HHV-6 subgroups (A and B) use human CD46 as a cellular receptor. Downregulation of surface CD46 was documented during the course of HHV-6 infection. Both acute infection and cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Nonhuman cells that were resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. The use of a ubiquitous immunoregulatory receptor opens novel perspectives for understanding the tropism and pathogenicity of HHV-6.

Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors

Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.

Anti‐HIV‐1 immunotoxin 3B3(Fv)‐PE38: enhanced potency against clinical isolates in human PBMCs and macrophages, and negligible hepatotoxicity in macaques

Highly active antiretroviral therapy (HAART) against human immunodeficiency virus type 1 (HIV‐1) infection dramatically suppresses viral load, leading to marked reductions in HIV‐1 associated morbidity and mortality. However, infected cell reservoirs and low‐level replication persist in the face of suppressive HAART, leading invariably to viral rebound upon cessation of treatment. Toxins engineered to target the Env glycoprotein on the surface of productively infected cells represent a complementary strategy to deplete these reservoirs. We described previously highly selective killing of Env‐expressing cell lines by CD4(178)‐PE40 and 3B3(Fv)‐PE38, recombinant derivatives of Pseudomonas aeruginosa exotoxin A containing distinct targeting moieties against gp120. In the present report, we compare the in vitro potency and breadth of these chimeric toxins against multiple clinical HIV‐1 isolates, replicating in biologically relevant primary human target cell types. In PBMCs, 3B3(Fv)‐PE38 blocked spreading infection by all isolates examined, with greater potency than CD4(178)‐PE40. 3B3(Fv)‐PE38 also potently inhibited spreading HIV‐1 infection in primary macrophages. Control experiments demonstrated that in both target cell types, most of the 3B3(Fv)‐PE38 activity was due to selective killing of infected cells, and not merely to neutralization by the antibody moiety of the chimeric toxin. High‐dose treatment of rhesus macaques with 3B3(Fv)‐PE38 did not induce liver toxicity, whereas equivalent dosage of CD4(178)‐PE40 induced mild hepatotoxicity. These findings highlight the potential use of 3B3(Fv)‐PE38 for depleting HIV‐infected cell reservoirs persisting in the face of HAART.

A Human Immunodeficiency Virus–Transgenic Mouse Model for Assessing Interventions that Block Microbial-Induced Proviral Expression

A human immunodeficiency virus (HIV) type 1-transgenic mouse line (166) that previously showed up-regulated expression of viral proteins and infectious particles after infection with pathogenic agents was tested as a model for screening the in vitro and in vivo efficacy of inhibitors of HIV-1 immune activation. Two types of interventions were assessed: use of either the immunosuppressive drug prednisolone or an HIV-1 envelope-targeted toxin (sCD4-PE40). Both agents inhibited lipopolysaccharide-induced p24 expression by splenocytes in vitro and, when administered to transgenic mice, suppressed the induction of plasma p24, as well as the ex vivo production of p24 and infectious virus stimulated by in vivo infection with Mycobacterium avium. Moreover, HIV-1 mRNA levels in the spleen were greatly reduced in mice treated with either agent. Because HIV-1 expression cannot be induced in T lymphocytes from line 166 mice, this model may be of particular advantage for testing interventions that target virus production by non-T cell virus reservoirs.