Susan R. Ross

Murine retroviruses activate B cells via interaction with toll-like receptor 4

Although most retroviruses require activated cells as their targets for infection, it is not known how this is achieved in vivo. A candidate protein for the activation of B cells by either mouse mammary tumor virus (MMTV) or murine leukemia virus is the toll-like receptor 4 (TLR4), a component of the innate immune system. MMTV caused B cell activation in C3H/HeN mice but not in C3H/HeJ or BALB/c (C.C3H Tlr4lps-d) congenic mice, both of which have a mutant TLR4 gene. This activation was independent of viral gene expression, because it occurred after treatment of MMTV with ultraviolet light or 2,2′-dithiodipyridine and in azidothymidine-treated mice. Nuclear extracts prepared from the lymphocytes of MMTV-injected C3H/HeN but not C3H/HeJ mice showed increased nuclear factor κB activity. Additionally, the MMTV- and Moloney murine leukemia virus envelope proteins coimmunoprecipitated with TLR4 when expressed in 293T cells. The MMTV receptor failed to coimmunoprecipitate with TLR4, suggesting that MMTV/TLR4 interaction is independent of virus attachment and fusion. These results identify retroviral proteins that interact with a mammalian toll receptor and show that direct activation by such viruses may initiate in vivo infection pathways.

Troglitazone action is independent of adipose tissue.

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.

Transgenic Mouse Mammary Tumor Virus Superantigen Expression Prevents Viral Infection

Endogenous mouse mammary tumor virus (MMTV) proviruses have recently been shown to cosegregate genetically with the minor lymphocyte-stimulating loci, also termed self-superantigens. The antigenic activity has been localized to the open reading frame (ORF) protein encoded in the long terminal repeat of MMTV. We show here that unlike their nontransgenic littermates, transgenic mice expressing high levels of an ORF protein derived from the C3H exogenous MMTV specifically delete their V beta 14+ T cells and do not become infected with this virus when it is present in their mothers milk. Thus, it appears that MMTV utilizes cells of the immune system in its infection pathway, and mice that retain endogenous MMTVs should be immune to infection by exogenous virus. These results offer possible new approaches to anti-viral therapy or immunization.

APOBEC3 inhibits mouse mammary tumour virus replication in vivo

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3-/- mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.

Mouse transferrin receptor 1 is the cell entry receptor for mouse mammary tumor virus

Enveloped viruses enter cells by binding to their entry receptors and fusing with the membrane at the cell surface or after trafficking through acidic endosomal compartments. Species-specific virus tropism is usually determined by these entry receptors. Because mouse mammary tumor virus (MMTV) is unable to infect Chinese hamster cells, we used phenotypic screening of the T31 mouse/hamster radiation hybrid panel to map the MMTV cell entry receptor gene and subsequently found that it is transferrin receptor 1. MMTV-resistant human cells that expressed mouse transferrin receptor 1 became susceptible to MMTV infection, and treatment of mouse cells with a monoclonal antibody that down-regulated cell surface expression of the receptor blocked infection. MMTV, like vesicular stomatitis virus, depended on acid pH for infection. MMTV may use transferrin receptor 1, a membrane protein that is endocytosed via clathrin-coated pits and traffics through the acidic endosomes, to rapidly get to a compartment where acid pH triggers the conformational changes in envelope protein required for membrane fusion.

THE MATRIX ATTACHMENT REGION-BINDING PROTEIN SATB1 PARTICIPATES IN NEGATIVE REGULATION OF TISSUE-SPECIFIC GENE EXPRESSION

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

Toll-Like Receptor 4-Dependent Activation of Dendritic Cells by a Retrovirus

ABSTRACT Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyers patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.

Receptor determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses

Transferrin receptor 1 (TfR1) is a cellular receptor for the New World hemorrhagic fever arenaviruses Machupo (MACV), Junín (JUNV), and Guanarito (GTOV). Each of these viruses is specifically adapted to a distinct rodent host species, but all cause human disease. Here we compare the ability of these viruses to use various mammalian transferrin receptor 1 (TfR1) orthologs, including those of the South American rodents that serve as reservoirs for MACV, JUNV, and GTOV (Calomys callosus, Calomys musculinus, and Zygodontomys brevicauda, respectively). Retroviruses pseudotyped with MACV and JUNV but not GTOV glycoproteins (GPs) efficiently used C. callosus TfR1, whereas only JUNV GP could use C. musculinus TfR1. All three viruses efficiently used Z. brevicauda TfR1. TfR1 orthologs from related rodents, including house mouse (Mus musculus) and rat (Rattus norvegicus), did not support entry of these viruses. In contrast, these viruses efficiently used human and domestic cat TfR1 orthologs. We further show that a local region of the human TfR1 apical domain, including tyrosine 211, determined the efficiency with which MACV, JUNV, and GTOV used various TfR1 orthologs. Our data show that these New World arenaviruses are specifically adapted to the TfR1 orthologs of their respective rodent hosts and identify key commonalities between these orthologs and human TfR1 necessary for efficient transmission of these viruses to humans.

MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

Natural Resistance-Associated Macrophage Protein Is a Cellular Receptor for Sindbis Virus in Both Insect and Mammalian Hosts

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection.