The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

Identification of a cellular receptor for subgroup E avian leukosis virus

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.     

B and T cells are required for mouse mammary tumor virus spread within the mammary gland

Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, V beta 14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (mu-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors.     

Retroviral pseudo-virus carrying the envelope proteins of neurotropic Friend murine leukemia virus effectively transferred retroviral vector into glial cells

We isolated the neurotropic Friend murine leukemia virus, FrC6 and its molecular clone A8, which proliferated in rat glial cell lines in vitro and in the rat brain in vivo. To investigate the contribution of viral envelope proteins to the neurotropism of A8 virus, the retroviral pseudo-virus carrying the envelope proteins of A8 virus and Moloney murine leukemia virus (MoMLV) was produced by transfecting the env gene of A8 virus (A8env) in the MoMLV based packaging cell, psi CRE. The phenotypically mixed pseudo-virus infected the rat glial cell lines as well as NIH 3T3 cells, whereas the psi CRE-produced pure pseudo-virus without A8env expression infected the glial cells at lower efficiency. Furthermore, the psi CRE cells with A8env expression produced pseudo-virus at a higher titer than normal psi CRE cells. The infectivity of the phenotypically mixed pseudo-virus to the glial cells was abolished by a neutralizing antibody against A8 virus, which did not reduce the ability of the psi CRE-produced pure pseudo-virus to infect NIH 3T3 cells. These results indicated that the envelope protein of A8 virus is assembled into the pseudo-viral particles and that it contributes to glial cell infection by the A8 virus.     

A highly sensitive in vitro infection assay to explore early stages of mouse mammary tumor virus infection

Mouse mammary tumor virus (MMTV) infection of adult mice induces a strong response to superantigen (Sag) in their draining lymph nodes, which results from the presentation of Sag by MMTV-infected B cells to Sag-reactive T cells. To date, infection with physiologically relevant doses of MMTV can be detected in vivo only after several days of Sag-mediated T-cell-dependent amplification of infected B cells. Furthermore, no efficient in vitro system of detecting MMTV infection is available. Such a model would allow the dissection of the early phase of infection, the assessment of the contributions of different cell types, and the screening of large panels of molecules for their potential roles in infection and Sag response. For these reasons, we have established an in vitro model for detecting infection which is as sensitive and reproducible as the in vivo model. We found that the viral envelope (Env) protein is crucial for target cell infection but not for presentation of Sag. Furthermore, we show that infection of purified B cells with MMTV induces entry of Sag-responsive T cells into the cell cycle, while other professional antigen-presenting cells, such as dendritic cells, are much less efficient in inducing a response.     

Type D retrovirus capsid assembly and release are active events requiring ATP

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-D-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.     

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.     

Monitoring frequency of occurrence without awareness: evidence from patients with Alzheimer''s disease

Mouse mammary tumor virus (MMTV) is a type B retrovirus that is transmitted as an infectious milk-borne particle and that causes mammary carcinomas by insertional activation of cellular protooncogenes. Germ line infections result in endogenous Mtv proviruses integrated in the genome of most mouse strains. These endogenous proviruses have been integrated into the genomes of mice for only the past 3-5 million years. The open reading frame present in the 3' long terminal repeat (LTR) of the provirus encodes a superantigen (SAg) which is able to stimulate a large proportion of T cells sharing a common T-cell receptor beta chain variable domain (v beta). Expression of this SAg is critical to the MMTV life cycle. After expression of the SAg in B cells a significant number of T cells are recruited to respond to these MMTV infected cells. As a consequence both the T cells expressing the relevant TCR V beta domain and the infected B cells become activated and start dividing. This would facilitate integration of MMTV and amplify the number of virus infected lymphocytes. Most likely during lactation the mammary glands become receptive to viral infection. The presence of endogenous Mtvs induces an early clonal deletion of reactive T cells. For this reason it has been argued that the presence of these proviruses confers a selective advantage to the mouse population by protecting the host from infection with an exogenous MMTV coding for a cross-reactive SAg. However, recent results discussed herein suggest that Mtv proviruses may also be detrimental to the mouse population by participating in recombinations with exogenous MMTVs, giving rise to highly tumorigenic recombinant particles. These results are discussed in the light of recent reports suggesting the involvement of viral sequences with a high homology to MMTV in human mammary tumorigenesis.     

Syncytium-inhibiting monoclonal antibodies produced against human T-cell lymphotropic virus type 1-infected cells recognize class II major histocompatibility complex molecules and block by protein crowding

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).     

Cell-free HIV-1Zr6 vif mutants are defective in binding to peripheral blood mononuclear cells and in internalization

The vif gene of the human immunodeficiency virus (HIV-1) is required for productive virus infection of primary blood mononuclear cells (PBMCs) and macrophages in vitro. Replication of HIV-1 vif- mutants in T-lymphoid cell lines varies and is dependent on the cell line used for virus production. To further understand the role of Vif in HIV-1 infection, we constructed to vif deletion mutants from a molecular clone derived from an African patient (HIV-1Zr6). Cell-free Zr6 vif- virus pools made from transfected rhabdomyosarcoma (RD) cells do not replicate when added to cultures of stimulated PBMCs. However, vif mutants were able to spread from transfected RD cells to PBMCs if cell-to-cell contact was permitted. By Western blot analysis, viral structural proteins expressed after transfection of RD cells by wild-type or vif mutant proviruses were indistinguishable. However, binding of vif mutants to PBMCs or to purified CD4 and virus internalization were significantly reduced when compared with wild-type virus. The defects in cell-free infection, CD4 binding, and internalization were rescued by transcomplementation using a vif expression plasmid. Our results suggest a novel level at which the HIV-1 vif gene product acts to enhance cell-free infection and indicate that vif plays an important role in promoting HIV-1 binding and internalization. Combined with the previous reports of vif's effect at other steps in infection, this suggests that vif is a pleuripotent gene product that affects multiple stages of the infective process.     

The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins

The human cytomegalovirus (CMV) US28 gene encodes a functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the actual role of the protein in the CMV life cycle. Expression of US28 allows human immunodeficiency virus type 1 (HIV-1) entry into certain CD4(+) cells and their fusion with cells expressing HIV-1 envelope (Env) proteins. Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4, which behave as CD4-associated HIV-1 coreceptors. We found that coexpression of US28 and either CXCR4 or CCR5 in CD4(+) cells resulted in enhanced synctium formation with HIV-1 Env+ cells. This positive effect of US28 on cell fusion seems to be distinct from its HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also observed when US28 was expressed on the HIV-1 Env+ cells instead of an CD4(+) target cells. Furthermore, US28 could enhance cell fusion mediated by other viral proteins, in particular, the G protein of vesicular stomatitis virus (VSV-G). The HIV-1 coreceptor and fusion-enhancing activities could be affected by mutations in different domains of US28. The fusion-enhancing activity of US28 seems to be cell type dependent. Indeed, cells coexpressing VSV-G and US28 fused more efficiently with human, simian, or feline target cells, while US28 had no apparent effect on fusion with the three mouse or rat cell lines tested. The positive effect of US28 on cell fusion might therefore require its interaction with a cell-specific factor. We discuss a possible role for US28 in the fusion of the CMV envelope with target cells and CMV entry.     

The protein tyrosine kinase Fyn activates transcription from the HIV promoter via activation of NF kappa B-like DNA-binding proteins

Protein tyrosine kinase p59fyn (Fyn) associates with the TCR-CD3 complex, which suggests that Fyn plays a significant role in the signal transduction involving TCR complex. In addition to cellular genes, viral promoters such as the HIV long terminal repeat (LTR) are also activated upon T cell activation. To elucidate the functional significance of Fyn in the expression of viral promoters, we transfected a Fyn-expression vector together with a reporter plasmid containing the chloramphenicol acetyltransferase gene driven by HIV LTR into a human T cell line, Jurkat. In this assay, Fyn stimulated the promoter in HIV LTR when the transfected cells were treated with both concanavalin A and PMA as an antigen-mimic stimulation. This activation required the intact SH2 domain of Fyn. Mutational analysis of HIV LTR showed that the NF kappa B binding sites were responsible for this effect. Electrophoretic mobility shift assays and UV cross-linking experiments showed that activation of T cells by anti-CD3 antibody induced four kappa B-binding proteins (50, 60, 65 and 100 kDa) in Fyn-overexpressing cells more efficiently than in the parental cells. Our results suggested that Fyn was able to regulate expression of a subset of genes via kappa B-binding proteins upon T cell activation.     

Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus

Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV.     

Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments

Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.     

Characterization of T cell-expressed chimeric receptors with antibody-type specificity for the CD4 binding site of HIV-1 gp120

Chimeric T cell receptors (cTCR) with an antibody specificity have been proposed in several models as a combination of antibody and cellular immunotherapy without MHC restriction. Such a tool could be of a limited use in HIV infection because of the great variability of the virus. The human single-chain antibody (ScFv-b12) derives from the b12 antibody directed to the CD4 binding site of gp120, a potent neutralizer of different HIV-1 strains, including a large panel of primary isolates. A single-chain fragment variable (ScFv) bearing the VH Pro-->Glu mutation that improves b12 affinity 54-fold, called ScFv-b12E, was also constructed. The ScFv were linked to the signal-transducing y chain of the Fc(gamma)RIII, with or without spacer region, and expressed in the murine MD45 T cell line. The different cTCR formats behave similarly in terms of ScFv surface expression, but differ according to their activation threshold. T cell transfectants can be stimulated with immobilized gp120 derived from all HIV strains tested. BHK cells infected with Semliki forest virus (SFV) carrying an HIV-1 envelope gene (SFV-env) derived from either HIV-1 laboratory strains (LAI, MN12, HXB2) or field isolates (BX08, CHAR or 133) were used as targets for the transfectants. All gp120-expressing cells induced cTCR-specific activation. The latter result is contrasting with the lack of specific recognition of SFV-CHAR- or 133-infected cells by the native b12 antibody, as measured by cytofluorometric analysis. Finally, HeLa cells (which constitutively express the coreceptor CXCR4) are able to bind HIV-1 gp160 when transfected with the chimeric receptor ScFv-b12-gamma, but, importantly, do not become infected by the virus. Our results therefore suggest that cTCR with b12 specificity can confer to T cells broad anti-HIV reactivity without making them susceptible to HIV infection.     

The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells

We report on the functional cloning of a hitherto unknown member of the immunoglobulin (Ig) superfamily selected for its ability to confer susceptibility to herpes simplex virus (HSV) infection on a highly resistant cell line (J1.1-2 cells), derived by exposure of BHKtk- cells to a recombinant HSV-1 expressing tumor necrosis factor alpha (TNF-alpha). The sequence of herpesvirus Ig-like receptor (HIgR) predicts a transmembrane protein with an ectodomain consisting of three cysteine-bracketed domains, one V-like and two C-like. HIgR shares its ectodomain with and appears to be an alternative splice variant of the previously described protein PRR-1 (poliovirus receptor-related protein). Both HIgR and PRR-1 conferred on J1.1-2 cells susceptibility to HSV-1, HSV-2, and bovine herpesvirus 1. The viral ligand of HIgR and PRR-1 is glycoprotein D, a constituent of the virion envelope long known to mediate viral entry into cells through interaction with cellular receptor molecules. Recently, PRR-1, renamed HveC (herpesvirus entry mediator C), and the related PRR-2, renamed HveB, were reported to mediate the entry of HSV-1, HSV-2, and bovine herpesvirus 1, and the homologous poliovirus receptor was reported to mediate the entry of pseudorabies virus (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998; M. S. Warner, R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear, Virology 246:179-189, 1998). Here we further show that HIgR or PRR-1 proteins detected by using a monoclonal antibody to PRR-1 are widely distributed among human cell lines susceptible to HSV infection and commonly used for HSV studies. The monoclonal antibody neutralized virion infectivity in cells transfected with HIgR or PRR-1 cDNA, as well as in the human cell lines, indicating a direct interaction of virions with the receptor molecule, and preliminarily mapping this function to the ectodomain of HIgR and PRR-1. Northern blot analysis showed that HIgR or PRR-1 mRNAs were expressed in human tissues, with the highest expression being detected in nervous system samples. HIgR adds a novel member to the cluster of Ig superfamily members able to mediate the entry of alphaherpesviruses into cells. The wide distribution of HIgR or PRR-1 proteins among human cell lines susceptible to HSV infection, coupled with the neutralizing activity of the antibody in the same cells, provides direct demonstration of the actual use of this cluster of molecules as HSV-1 and HSV-2 entry receptors in human cell lines. The high level of expression in samples from nervous system makes the use of these proteins in human tissues very likely. This cluster of molecules may therefore be considered to constitute bona fide receptors for HSV-1 and HSV-2.