TRIM2, a novel member of the antiviral family, limits New World arenavirus entry.

Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot-Marie-Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.

siRNA screen for genes that affect Junín virus entry uncovers voltage-gated calcium channels as a therapeutic target.

New World hemorrhagic fever arenavirus infection results in 15 to 30% mortality in humans. We performed a high-throughput small interfering RNA screen with Junín virus glycoprotein-pseudotyped viruses to find potential host therapeutic targets. Voltage-gated calcium channel (VGCC) subunits, for which there are Food and Drug Administration (FDA)-approved drugs, were identified in the screen. Knockdown of VGCC subunits or treatment with channel blockers diminished Junín virus-cell fusion and entry into cells and thereby decreased infection. Gabapentin, an FDA-approved drug used to treat neuropathic pain that targets the α2δ2 subunit, inhibited infection of mice by the Candid 1 vaccine strain of the virus. These findings demonstrate that VGCCs play a role in virus infection and have the potential to lead to therapeutic intervention of New World arenavirus infection.

Junin virus infects mouse cells and induces innate immune responses.

Junín virus is the causative agent for Argentine hemorrhagic fever, and its natural host is the New World rodent Calomys musculinus. The virus is transmitted to humans by aerosolization, and it is believed that many of the clinical symptoms are caused by cytokines produced by sentinel cells of the immune system. Here we used the Junín virus vaccine strain Candid 1 to determine whether mouse cells could be used to study virus entry and antiviral innate immune responses. We show that Candid 1 can infect and propagate in different mouse-derived cell lines through a low-pH-dependent, transferrin receptor 1-independent mechanism, suggesting that there is a second entry receptor. In addition, Candid 1 induced expression of the antiviral cytokines tumor necrosis factor alpha and beta interferon in macrophages, and this induction was independent of viral replication. Using Candid 1, as well as virus-like particles bearing the viral glycoprotein, to infect different primary cells and established macrophage cell lines with deletions in the Toll-like receptor (TLR) pathway, we show that TLR2 is a cellular sensor of both the Parodi and Candid 1 viral glycoproteins. Because Junín virus is highly lethal in humans, the use of an experimentally tractable model system, such as the mouse, could provide a better understanding of the antiviral innate cellular responses to Junín virus and the role of these responses in pathogenesis.

Regional characterization of energy metabolism in the brain of normal and MPTP-intoxicated mice using new markers of glucose and phosphate transport.

The gibbon ape leukemia virus (GALV), the amphotropic murine leukemia virus (AMLV) and the human T-cell leukemia virus (HTLV) are retroviruses that specifically bind nutrient transporters with their envelope glycoproteins (Env) when entering host cells. Here, we used tagged ligands derived from GALV, AMLV, and HTLV Env to monitor the distribution of their cognate receptors, the inorganic phosphate transporters PiT1 and PiT2, and the glucose transporter GLUT1, respectively, in basal conditions and after acute energy deficiency. For this purpose, we monitored changes in the distribution of PiT1, PiT2 and GLUT1 in the cerebellum, the frontal cortex, the corpus callosum, the striatum and the substantia nigra (SN) of C57/BL6 mice after administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridinium (MPTP), a mitochondrial complex I inhibitor which induces neuronal degeneration in the striato-nigral network.The PiT1 ligand stained oligodendrocytes in the corpus callosum and showed a reticular pattern in the SN. The PiT2 ligand stained particularly the cerebellar Purkinje cells, while GLUT1 labelling was mainly observed throughout the cortex, basal ganglia and cerebellar gray matter. Interestingly, unlike GLUT1 and PiT2 distributions which did not appear to be modified by MPTP intoxication, PiT1 immunostaining seemed to be more extended in the SN. The plausible reasons for this change following acute energy stress are discussed.These new ligands therefore constitute new metabolic markers which should help to unravel cellular adaptations to a wide variety of normal and pathologic conditions and to determine the role of specific nutrient transporters in tissue homeostasis.

Host range and cellular tropism of the human exogenous gammaretrovirus XMRV.

Recently, the first human infection with an exogenous gammaretrovirus (XMRV) was reported. In its initial description, XMRV was confined to prostate stromal fibroblasts, although subsequent reports demonstrated XMRV protein expression in prostate epithelial cells. Most recently, XMRV has been detected in blood cells of patients with chronic fatigue syndrome. The aim of this study was to elucidate the transmission routes and tissue tropism of XMRV by comparing its host range, receptor usage and LTR functionality with other MLV isolates. We demonstrate using pseudotype experiments that XMRV Env mediates efficient infection of cells from different species. We show that replication competent XMRV infects various human cell types, including hematopoietic cell lines and prostate stromal fibroblasts. XMRV-LTR activity is significantly higher in the prostate cancer cell line LNCaP and in prostate stromal fibroblasts, compared to other cell types tested and could be one factor contributing to efficient viral spread in prostate tissue.

Cell surface expression of the bovine leukemia virus-binding receptor on B and T lymphocytes is induced by receptor engagement.

Bovine leukemia virus (BLV), one of the most common infectious viruses of cattle, is endemic in many herds. Approximately 30-40% of adult cows in the United States are infected by this oncogenic C-type retrovirus and 1-5% of animals will eventually develop a malignant lymphoma. BLV, like the human and simian T cell leukemia viruses, is a deltaretrovirus but, in contrast with the latter, the BLV receptor remains unidentified. In this study, we demonstrate that the amino-terminal 182 residues of the BLV envelope glycoprotein surface unit encompasses the receptor-binding domain. A bona fide interaction of this receptor-binding domain with the BLV receptor was demonstrated by specific interference with BLV, but not human T cell leukemia virus, envelope glycoprotein-mediated binding. We generated a rabbit Ig Fc-tagged BLV receptor-binding domain construct and ascertained that the ligand binds the BLV receptor on target cells from multiple species. Using this tool, we determined that the BLV-binding receptor is expressed on differentiating pro/pre-B cells in mouse bone marrow. However, the receptor was not detected on mature/quiescent B cells but was induced upon B cell activation. Activation of human B and T lymphocytes also induced surface BLV-binding receptor expression and required de novo protein synthesis. Receptor levels were down-regulated as activated lymphocytes returned to quiescence. In the human thymus, BLV-binding receptor expression was specifically detected on thymocytes responding to the IL-7 cytokine. Thus, expression of the BLV-binding receptor is a marker of enhanced metabolic activity in B cells, T cells, and thymocytes.

Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells.

BACKGROUND: We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding. RESULTS: As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell. CONCLUSION: Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes.

Intrahost variations in the envelope receptor-binding domain (RBD) of HTLV-1 and STLV-1 primary isolates.

Four primate (PTLV), human (HTLV) and simian (STLV) T-cell leukemia virus types, have been characterized thus far, with evidence of a simian zoonotic origin for HTLV-1, HTLV-2 and HTLV-3 in Africa. The PTLV envelope glycoprotein surface component (SUgp46) comprises a receptor-binding domain (RBD) that alternates hypervariable and highly conserved sequences. To further delineate highly conserved motifs in PTLV RBDs, we investigated the intrahost variability of HTLV-1 and STLV-1 by generating and sequencing libraries of DNA fragments amplified within the RBD of the SUgp46 env gene. Using new and highly cross-reactive env primer pairs, we observed the presence of Env quasispecies in HTLV-1 infected individuals and STLV-1 naturally infected macaques, irrespective of the clinical status. These intrahost variants helped us to define highly conserved residues and motifs in the RBD. The new highly sensitive env PCR described here appears suitable for the screening of all known variants of the different PTLV types and should, therefore, be useful for the analysis of seroindeterminate samples.

HTLV envelopes and their receptor GLUT1, the ubiquitous glucose transporter: a new vision on HTLV infection?

We identified the ubiquitous glucose transporter GLUT1 as a receptor for Deltaretroviruses HTLV-1 and HTLV-2 envelopes (Env), mediating viral binding and entry. Here, we review the context and key observations that led us to this finding: functional modules of HTLV SU are similar to those of Gammaretrovirus Env which use multimembrane-spanning nutrient transporters as receptors; the HTLV Env receptor is an early marker of T lymphocyte activation; and HTLV Env inhibits glucose transport. We review several molecular, viral, cellular and physiological aspects of HTLV infection in relation to the in vivo and in vitro properties of GLUT1. Also, we examine the implications of HTLV-1 Env-GLUT1 interactions and altered glucose transport on the two major HTLV-1-induced diseases, adult T cell leukemia (ATL) and neurodegenerative tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM). Complementary to the classical models of disease progression, we propose new schemes that emphasize the potential metabolic alterations caused in different cellular compartments. Finally, we review the potential use of HTLV Env-derived constructs as tools for labeling GLUT1 in vivo and inhibiting GLUT1 transport in tumor cells.