Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.
We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.
A series of wild-type and mutant rafgenes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine-and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.The raffamily of proto-oncogenes consists of three active members: A-raf-1, B-raf, and c-raf-1 (2,4,5,20,22,23). The three proteins show the greatest stretch of homology in the carboxy-terminal half, which, in the case of c-raf-1, has been shown to have serine-and threonine-specific protein kinase activity (33, 48). Physiologically, Raf protein kinases function as information shuttles that communicate between the cell surface and nucleus. From Ki-ras revertant and anti-ras antibody microinjection studies, it is known that c-raf-1 acts independently of and perhaps downstream from Ras in signal transduction (2,22,36,41,42,52). In addition, we recently showed that Raf is hyperphosphorylated and enzymatically activated in cells which are transformed by src, fms, or ras, and in cells which have been treated with platelet-derived growth factor or 12-0-tetradecanoylphorbol-13-acetate (TPA) (35). In response to platelet-derived growth factor and TPA, Raf protein is also translocated from the cytoplasm to the perinuclear space, as is evident from immune fluorescence and cell fractionation studies (44). Ultimately, rafis thought to exert its effect by modulation of transcription factor activity via phosphorylation (42). Evidence for this comes from experiments in which cells transfected with activated raf genes showed increased transcriptional activity from an APl-dependent promoter in transient transfection assays, suggesting that Raf is involved in the regulation of transcription factors of the jun-AP1 gene family (56). Raf function is essential for normal rates of cell proliferation, as demonstrated by the lethal effect of mutations in the Drosophila melanogaster D-raf-1 locus (37).c-raf-1, the most-studied member of the family, encodes a * Corresponding auth...
A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3-azido-3-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37°C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.
APOBEC3 proteins are cytidine deaminases which help defend cells against retroviral infections. One antiviral mechanism involves deaminating dC residues in minus-strand DNA during reverse transcription, resulting in G-to-A mutations in the coding strand. We investigated the effects of mouse APOBEC3 (mA3) and human APOBEC3G (hA3G) upon Moloney murine leukemia virus (MLV). We find that mA3 inactivates MLV but is significantly less effective against MLV than is hA3G. In contrast, mA3 is as potent against human immunodeficiency virus type 1 (HIV-1, lacking the protective Vif protein) as is hA3G. The two APOBEC3 proteins are packaged to similar extents in MLV particles. Dose-response profiles imply that a single APOBEC3 molecule (or oligomer) is sufficient to inactivate an MLV particle. The inactivation of MLV by mA3 and hA3G is accompanied by relatively small reductions in the amount of viral DNA in infected cells. Although hA3G induces significant levels of G-to-A mutations in both MLV and HIV DNAs, and mA3 induces these mutations in HIV DNA, no such mutations were detected in DNA synthesized by MLV inactivated by mA3. Thus, MLV has apparently evolved to partially resist the antiviral effects of mA3 and to totally resist the ability of mA3 to induce G-to-A mutation in viral DNA. Unlike the resistance of HIV-1 and human T-cell leukemia virus type 1 to hA3G, the resistance of MLV to mA3 is not mediated by the exclusion of APOBEC from the virus particle. The nature of its resistance and the mechanism of inactivation of MLV by mA3 are completely unknown.Mammalian cells contain a number of mechanisms for protection against retroviral infection and retrotransposition. One of these mechanisms involves members of the APOBEC3 (APO) family of cytidine deaminases (19,45). The existence of this mechanism was first discovered through experiments on the human immunodeficiency virus type 1 (HIV-1) accessory protein Vif; it has become clear in recent years that the intracellular binding of Vif to human APOBEC3G (hA3G) results in proteasomal degradation of the APO protein, thus preventing its incorporation into virions (35,52). While the antiretroviral activity of hA3G was initially revealed in studies on HIV-1, it has recently become clear that hA3G is only one member of a family of cytidine deaminases (26) and that several APOBEC3 family members are able to inhibit infections by a remarkable variety of retroviruses. Indeed, it appears that endogenous retroviruses and retrotransposons, as well as exogenous retroviruses, are sensitive to the effects of these cellular proteins (6,11,12,38). Interestingly, another retrovirus, human T-cell leukemia virus type 1 (HTLV-1), resembles HIV-1 in that it excludes hA3G from virions, but this exclusion involves an unusual acidic region in the nucleocapsid domain of its Gag protein (9) rather than a nonstructural protein like Vif.Although this system has been studied intensively for several years, there are a number of outstanding questions that remain to be resolved. One of these is how APO pr...
Human T cell leukemia virus type 1 (HTLV-1) has evolved a remarkable strategy to thwart the antiviral effects of the cellular cytidine deaminase APOBEC3G (hA3G). HTLV-1 infects T lymphocytes in vivo, where, like HIV-1, it is likely to encounter hA3G. HIV-1 counteracts the innate antiviral activity of hA3G by producing an accessory protein, Vif, which hastens the degradation of hA3G. In contrast, HTLV-1 does not encode a Vif homologue; instead, HTLV-1 has evolved a cis-acting mechanism to prevent hA3G restriction. We demonstrate here that a peptide motif in the C terminus of the HTLV-1 nucleocapsid (NC) domain inhibits hA3G packaging into nascent virions. Mutation of amino acids within this region resulted in increased levels of hA3G incorporation into virions and increased susceptibility to hA3G restriction. Elements within the C-terminal extension of the NC domain are highly conserved among the primate T cell leukemia viruses, but this extension is absent in all other retroviral NC proteins.
Three late assembly domain consensus motifs, namely PTAP, PPPY, and LYPXL, have been identified in different retroviruses. They have been shown to interact with the cellular proteins TSG101, Nedd4, and AP2 or AIP, respectively. Human T-cell leukemia virus type 1 (HTLV-1) has a PPPY and a PTAP motif, separated by two amino acids, located at the end of MA, but only the PPPY motif is conserved in the deltaretrovirus group. Like other retroviral peptides carrying the late motif, MA is mono-or di-ubiquitinated. A mutational analysis showed that 90% of PPPY mutant particles were retained in the cell compared to 15% for the wild-type virus. Mutations of the PTAP motif resulted in a 20% decrease in particle release. In single-cycle infectivity assays, the infectious titers of late motif mutants correlated with the amounts of released virus, as determined by an enzyme-linked immunosorbent assay. We observed binding of MA to the WW domains of the Nedd4 family member WWP1 but not to the amino-terminal ubiquitin E2 variant domain of TSG101 in mammalian two-hybrid analyses. The binding to WWP1 was eliminated when the PPPY motif was mutated. However, MA showed binding to TSG101 in the yeast two-hybrid system that was dependent on an intact PTAP motif. A dominant-negative (DN) mutant of WWP1 could inhibit budding of the intact HTLV-1 virus. In contrast, DN TSG101 only affected the release of virus-like particles encoded by Gag expression plasmids. Electron and fluorescent microscopy showed that Gag accumulates in large patches in the membranes of cells expressing viruses with PPPY mutations. Very few tethered immature particles could be detected in these samples, suggesting that budding is impaired at an earlier step than in other retroviruses.
The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY ؊ mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly.Retroviruses recruit the multivesicular body (MVB) biogenesis machinery to accomplish particle release (for reviews, see references 7, 34, and 56). Peptide motifs found in retroviral Gag proteins serve as docking sites for components of the MVB pathway in order to divert the machinery to the site of particle assembly (43). MVBs are the penultimate endosomal compartment of the degradative pathway that delivers activated cell surface receptors to the lysosome. The signal for the recruitment of the receptor into the lysosomal degradation pathway is a low-level ubiquitination-usually one or two ubiquitin moieties are added at one to four lysine residues following phosphorylation of the receptor (16,20). Several E3-type ubiquitin ligases have been identified which perform this function, among them WWP1 and other members of the Nedd4 family (29). After internalization, the ubiquitinated receptor is recognized by the HRS/STAM complex (45), which hands it off to the ESCRT I complex, thus shunting it into the degradative rather than the recycling compartment of the early endosome (2). The ubiquitin modification serves as a tag on the receptor along the pathway consisting of the ESCRT II and III complexes. Finally, ubiquitin is removed from the receptor by a deubiquitinase and the vesicle buds into the MVB with the help of the VPS4 ATPase complex (1, 24).The region in retroviral Gag harboring the MVB machinery interaction motifs was designated the late domain (LD) (40), as it controls the late step of virus budding in the infectious cycle. Three motifs have been identified in retroviruses so far; they are PPXY (61), PT/SAP (12,21), and YPXL/LXXLF (44, 53) and connect to Nedd4 family E3 ubiquitin ligases (4,17,19,28,54,64), the TSG101 subunit of ESCRT I (11, 32, 59...
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