Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) persists despite a vigorous virus-specific host immune response, and causes adult T-cell leukemia and lymphoma in approximately 2% of infected individuals. Here we report that HTLV-1 has evolved a genetic function to restrict its own replication by a novel post-transcriptional mechanism. The HTLV-1-encoded p30(II) is a nuclear-resident protein that binds to, and retains in the nucleus, the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tex and Rex are positive regulators of viral gene expression, their inhibition by p30(II) reduces virion production. p30(II) inhibits virus expression by reducing Tax and Rex protein expression.

Retroviral integration into the host genome is not entirely random, and integration site preferences vary among different retroviruses. Human immunodeficiency virus (HIV) prefers to integrate within active genes, whereas murine leukemia virus (MLV) prefers to integrate near transcription start sites and CpG islands. On the other hand, integration of avian sarcoma-leukosis virus (ASLV) shows little preference either for genes, transcription start sites, or CpG islands. While host cellular factors play important roles in target site selection, the viral integrase is probably the major viral determinant. It is reasonable to hypothesize that retroviruses with similar integrases have similar preferences for target site selection. Although integration profiles are well defined for members of the lentivirus, spumaretrovirus, alpharetrovirus, and gammaretrovirus genera, no members of the deltaretroviruses, for example, human T-cell leukemia virus type 1 (HTLV-1), have been evaluated. We have mapped 541 HTLV-1 integration sites in human HeLa cells and show that HTLV-1, like ASLV, does not specifically target transcription units and transcription start sites. Comparing the integration sites of HTLV-1 with those of ASLV, HIV, simian immunodeficiency virus, MLV, and foamy virus, we show that global and local integration site preferences correlate with the sequence/structure of virus-encoded integrases, supporting the idea that integrase is the major determinant of retroviral integration site selection. Our results suggest that the global integration profiles of other retroviruses could be predicted from phylogenetic comparisons of the integrase proteins. Our results show that retroviruses that engender different insertional mutagenesis risks can have similar integration profiles.

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.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells ( March 20, 1995) ABSTRACT mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-a or IL-6 mAb. We asked, therefore, whether IFN-a-induced growth inhibition involved IL-6. IFN-a and mAb against IL-6, the IL-6R a-(gp8O) or fl-chain (gp13O) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-c-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-a drastically curtailed expression of the IL-6R a-and ,f-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-a, but not IL-6, greatly decreased gp8O and, to a lesser extent, gpl3O mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-a-induced IL-6 predominantly downregulated membrane-bound gpl3O. IFN-a-mediated decrease of gp8O levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-a inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R. (J. Clin.

Human T-cell leukemia virus type 1 (HTLV-1) infection profoundly alters T-cell gene expression, and the dysregulated synthesis of cytokines could influence the course and pathologic consequences of infection. In the process of screening T-cell lines for T helper 1 (Th1) and Th2 cytokine mRNAs, we observed that interleukin-13 (IL-13) mRNA was highly expressed in HTLV-1-infected, IL-2-dependent T-cell lines. IL-9 and interferon gamma (IFN-␥) mRNAs were also expressed at high levels in chronically in- IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects about 10 to 20 million people worldwide with endemic foci in Southern Japan, the Caribbean, Central and South Africa, and South America. Less than 5% of HTLV-1-infected individuals develop either adult T-cell leukemia (ATL) or a chronic inflammatory disease of the central nervous system termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 also has been implicated in a broader spectrum of diseases including uveitis, infective dermatitis, polymyositis, arthropathy, and Sjö gren syndrome. 1-3 HTLV-1-associated diseases may be accompanied by pulmonary disorders and immunosuppression, and it is noteworthy that patients with acute ATL generally succumb to opportunistic infections. 4 The majority of HTLV-1 proviruses are found in CD4 ϩ and in CD8 ϩ T cells, [5][6][7] and there is a clonal expansion of HTLV-1-infected CD4 ϩ T cells. 8 HTLV-1 infection of primary T cells in vitro results in the infrequent establishment of chronically infected T-cell lines that continuously proliferate in the absence of antigenic stimulation, but most require interleukin-2 (IL-2) for growth. [9][10][11][12] A small number of HTLV-1-infected T-cell lines also have been established that do not require IL-2, but these do not closely resemble patient-derived HTLV-1-infected T cells. 13 The viral trans-regulatory protein, Tax, is the primary effector of T-cell transformation. In addition to its role in activating virus transcription, Tax alters the expression or function of signal transduction proteins, cell cycle regulators, and transcription factors. 14 Targets of the Tax protein also include cytokine promoters, [15][16][17][18][19] and dysregulation of cytokine expression in HTLV-1-infected individuals may play an important role in the course of disease.Although expression patterns of a number of cytokines have been studied in HTLV-1-infected cells, IL-13 has not been examined in this context. Because IL-13 is a key mediator of various immune functions, [20][21][22] including the inhibition of tumor immunosurveillance 23,24 and the pathophysiology of asthma, 25,26 we asked whether its expression is altered in HTLV-1-infected T cells. IL-13 mRNA was expressed at high levels in 10 independent, chronically infected T-cell lines, but not uninfected controls. Transient transfections of Jurkat T cells revealed that the IL-13 promoter was transcriptionally activated by ectopic expression of the HTLV-1 Tax regulatory protein. Peripheral blo...

Microsomal triglyceride transfer protein (MTP) plays a central role in the assembly and secretion of apoB-containing lipoproteins. In this study, we investigated the effect of ethanol on the expression of the large subunit of MTP in a human liver hepatoma cell line, the HepG2 cells. Exposure of HepG2 cells to low concentrations of ethanol reduced MTP mRNA levels in a concentration- and time-dependent manner. The level of MTP mRNA decreased significantly (P<0.05, -26% relative to pretreatment control) when the concentration of ethanol in the culture medium was 50 ppm (0.005%, v/v). Maximal suppression (-50%) was observed at 100 ppm ethanol; the MTP mRNA levels remained at 50% of control when the ethanol concentration was raised to 10,000 ppm. Furthermore, a 10-day ethanol treatment caused a significant 50% decrease in the MTP activity and apoB secretion rate in HepG2 cells. To investigate the molecular mechanisms underlying this phenomenon, we examined the effect of ethanol on the promoter activity of the MTP gene. Transient transfection analysis of human MTP promoter-driven luciferase gene expression showed that ethanol down-regulates MTP promoter activity in a manner parallel to that observed for mRNA levels. Deletion analysis suggested that the MTP promoter sequence contains a negative ethanol response element -612 to -142 bp upstream of the transcription start site. To evaluate the in vivo relevance of the effect of ethanol on MTP mRNA levels, rats were given a single oral dose of ethanol, with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats receiving 1 or 3 g/kg of ethanol exhibited substantially lower hepatic and intestinal MTP mRNA levels. Taken together, these results strongly suggest that ethanol can modulate the secretion of apoB-containing lipoproteins by down-regulating the expression of MTP large subunit, primarily through inhibiting the transcription of the MTP gene.

It is well established that cell-free infection with human T-cell leukemia virus type 1 (HTLV-1) is less efficient than that with other retroviruses, though the specific infectivities of only a limited number of HTLV-1 isolates have been quantified. Earlier work indicated that a postentry step in the infectious cycle accounted for the poor cell-free infectivity of HTLV-1. To determine whether variations in the pol gene sequence correlated with virus infectivity, we sequenced and phenotypically tested pol genes from a variety of HTLV-1 isolates derived from primary sources, transformed cell lines, and molecular clones. The pol genes and deduced amino acid sequences from 23 proviruses were sequenced and compared with 14 previously published sequences, revealing a limited number of amino acid variations among isolates. The variations appeared to be randomly dispersed among primary isolates and proviruses from cell lines and molecular clones. In addition, there was no correlation between reverse transcriptase sequence and the disease phenotype of the original source of the virus isolate. HTLV-1 pol gene fragments encoding reverse transcriptase were amplified from a variety of isolates and were subcloned into HTLV-1 vectors for both single-cycle infection and spreading-infection assays. Vectors carrying pol genes that matched the consensus sequence had the highest titers, and those with the largest number of variations from the consensus had the lowest titers. The molecular clone from CS-1 cells had four amino acid differences from the consensus sequence and yielded infectious titers that were approximately eight times lower than those of vectors encoding a consensus reverse transcriptase.